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https://openalex.org/W3201277051
https://raco.cat/index.php/da/article/download/v10-n2-gimenez-candela/446198
Spanish; Castilian
null
Animals in the Spanish Civil Code: A Reform Interrupted
Derecho animal
2,019
cc-by
5,339
Cita recomendada. GIMÉNEZ-CANDELA, M., Animales en el Código civil español: una reforma interrumpida, dA. Derecho Animal (Forum of Animal Law Studies) 10/2 (2019) - DOI https://doi.org/10.5565/rev/da.438 Cita recomendada. GIMÉNEZ-CANDELA, M., Animales en el Código civil español: una reforma interrumpida, dA. Derecho Animal (Forum of Animal Law Studies) 10/2 (2019) - DOI https://doi.org/10.5565/rev/da.438 Cita recomendada. GIMÉNEZ-CANDELA, M., Animales en el Código civil español: una reforma interrumpida, dA. Derecho Animal (Forum of Animal Law Studies) 10/2 (2019) - DOI https://doi.org/10.5565/rev/da.438 Cita recomendada. GIMÉNEZ-CANDELA, M., Animales en el Código civil español: una reforma interrumpida, dA. Derecho Animal (Forum of Animal Law Studies) 10/2 (2019) - DOI https://doi.org/10.5565/rev/da.438 Resumen El 14 de febrero de 2017 se dio inició en el Parlamento español al proceso de reforma del estatuto jurídico de los animales en el CC, la Ley Hipotecaria y la Ley de Enjuiciamiento Civil, destinada a transformar la condición de los animales de cosas a seres vivos dotados de sensibilidad. A falta de pocos trámites para su aprobación, la convocatoria de Elecciones Generales el 28 de abril de 2019, ha interrumpido esta necesaria Descosificación de los animales en el ámbito civil y ha abierto una etapa de incógnitas y de expectativas para una reforma que sigue siendo necesaria. Palabras clave: Código civil español; codificación; estatuto jurídico de los animales; descosificación; seres sentientes; art. 13 TFUE; propiedad; sentiencia. dA.Derecho Animal (Forum of Animal Law Studies) 2019, vol. 10/2 7-12 dA.Derecho Animal (Forum of Animal Law Studies) 2019, vol. 10/2 7-12 * Este trabajo forma parte del Proyecto de investigación MINECO DER2015-69314-P «Estatuto jurídico de los animales: origen, desarrollo y políticas» (2015-2019), del que es IP la autora del mismo y del que forman parte otros investigadores nacionales e internacionales. Animales en el Código civil español: una reforma interrumpida * Marita Giménez-Candela Fundadora y Editora-Jefe Orcid:0000-002-0755-5928 Recepción: Abril 2019 Aceptación: Abril 2019 1. Análisis de la reforma El 1 de marzo de 2019 se publicó, en el Boletín Oficial de las Cortes Generales, el Informe de la Ponencia1 que había conducido la tramitación de la “Proposición de Ley de modificación del Código civil, la Ley Hipotecaria y la Ley de Enjuiciamiento civil, sobre el régimen jurídico de los Animales”2. En siete escuetas páginas se sustancian dos años de intenso trabajo visible en pro del cambio de estatuto jurídico de los animales (2017-2019)3 y muchos años de trabajo no-visible (desde el 2006 en adelante) en pro de la misma finalidad4. Urge, por tanto un análisis de lo que la reforma del régimen jurídico de los animales en el CC pretendía, los resultados que se alcanzaron así como las dificultades para alcanzarlos y, por último, las perspectivas de futuro que esta necesaria reforma, ahora interrumpida, plantea. Es claro que Código civil español estaba5 (y está) necesitado de una reforma que actualice el estatuto jurídico de los animales y lo adapte a lo que demanda la legislación Europea de Bienestar Animal y las reformas emprendidas por los países de nuestro entorno más próximo (tanto geográfica como jurídicamente), en el mismo ámbito que se planteó nuestra reforma que no es otro que el de la naturaleza de la propiedad de los animales y los corolarios que de ello se siguen. En este sentido se expresa la Ponencia en la Exposición de motivos, que recoge -aunque de forma un tanto imprecisa-, los fundamentos que dieron origen a la mencionada Proposición de Ley hoy interrumpida después de una lenta tramitación de dos años6. 1La Ponencia estuvo integrada por los siguientes Diputados: D. Avelino de Barrionuevo Gener (GP), D. Miguel Lorenzo Torres (GP), D. Francisco Molinero Hoyos (GP), D. Javier Antón Cacho (GS), D.ª María Dolores Galovart Carrera (GS), D.ª Sara Carreño Valero (GCUP-EC-EM), D. Juan Antonio López de Uralde Garmendia (GCUP-EC-EM), D. Guillermo Díaz Gómez (GCs), D.ª María Virginia Millán Salmerón (GCs), D. Joan Capdevila i Esteve (GER), D. Mikel Legarda Uriarte (GV-EAJ-PNV), D. Enric Bataller i Ruiz (GMx) y D.ª Lourdes Ciuró i Buldó (GMx). ( ) y ( ) 2BOCG de 1 de marzo de 2019 (122/000134) Proposición de Ley de modificación del Código Civil, la Ley Hipotecaria y la Ley de Enjuiciamiento Civil, sobre el régimen jurídico de los animales. Sumario 1. Análisis de la reforma 2. Resultados y obstáculos Abstract - Animals in the Spanish Civil Code: A Reform Interrupted On the 14th February 2017 the process of reforming the legal status of animals in the Civil Code began in the Spanish Parliament. This also included corresponding amendments in the Mortgage Law and in the Civil Procedure Rules, which aimed at changing the consideration of animals as living beings endowed with sensibility instead of as things. Before the formalities of approving the reform were completed, it was interrupted by the call for General Elections on the 28th April 2019, causing a halt to the necessary de- objectification of animals in the civil field and is now in a period of uncertainty and expectations for a reform that still remains necessary. Keywords: Spanish civil code; codification; legal status of animals; de-objectification; sentient beings; art. 13 TFUE; property; sentience. * Este trabajo forma parte del Proyecto de investigación MINECO DER2015-69314-P «Estatuto jurídico de los animales: origen, desarrollo y políticas» (2015-2019), del que es IP la autora del mismo y del que forman parte otros investigadores nacionales e internacionales. https://doi.org/10.5565/rev/da.438 ISSN 2462-7518 ISSN 2462-7518 ISSN 2462-7518 Animales en el código civil español: una reforma interrumpida Marita Giménez-Candela 1. Análisis de la reforma j , g j http://www.congreso.es/public_oficiales/L12/CONG/BOCG/B/BOCG-12-B-167-1.PDF#page=1 3La propuesta de reforma se dio a conocer de manera oficial y pública el 14 de febrero de 2017, en que se aprobó por unanimidad la Propuesta no de ley presentada por el Grupo Parlamentario Ciudadanos. Vid. la crónica de CODINA, J.I., Unanimidad en el Congreso de los Diputados para instar la reforma del Código civil español y reconocer a los animales como seres dotados de sensibilidad, en dA. 2/2017 https://derechoanimal.info/es/actividades/2017/unanimidad-en-el-congreso-de-los-diputados-para-instar- la-reforma-del-codigo-civil . La actividad del Observatorio de Justicia y Defensa Animal, con la recogida de 350.000 firmas de la campaña “Animales-no-son-cosas”, en apoyo de la reforma y, coetáneamente, la labor impulsora de la Fundación Affinity, pueden calificarse de imprescindibles para la visibilidad y el avance de la misma. p p y 4La reforma del estatuto jurídico de los animales, ha sido el motor de la investigación del ICALP, liderado por quien suscribe este artículo, a través de iniciativas que han abarcado los Proyectos de investigación I+D “Retos de la Investigación”, concedidos por el MINECO en dos convocatorias competitivas sucesivas: DER 2010-2013,” Animales, Derecho y Sociedad: desde el Derecho Romano a la Sociedad Global” y DER 2015-2019 (DER2015-69314-P) “Estatuto jurídico de los animales: origen, desarrollo, políticas”, que han dado como resultado publicaciones (libros, artículos, revista jurídica), tesis doctorales, ponencias en Congresos que, además de otras acciones que se han detallado en la sede correspondiente, han justificado la intervención de los investigadores de ambos Proyectos en el proceso de preparación de la reforma ahora en fase de interrupción técnica. 5Para una visión asentada en los principios tradicionales de la consideración de los animales como cosas, ROGEL VIDE, C., Los animales en el Código Civil (Madrid 2017); ídem, Personas, Animales y Derechos (Madrid-México 2018); vid. la recensión a este último libro de HUI, M., Amending Classic Civil Codes, en JAL & IAWS 1 (2018) 337-351. 5Para una visión asentada en los principios tradicionales de la consideración de los animales como cosas, ROGEL VIDE, C., Los animales en el Código Civil (Madrid 2017); ídem, Personas, Animales y Derechos (Madrid-México 2018); vid. la recensión a este último libro de HUI, M., Amending Classic Civil Codes, en JAL & IAWS 1 (2018) 337-351. g ( ) 6GIMÉNEZ-CANDELA, M., La Descosificación de los animales en el Código Civil español, en dA. Derecho Animal (Forum of Animal Law Studies) 9/3 (2018) 7-47 https://doi.org/10.5565/rev/da.361. 8 Derecho Animal. Forum of Animal Law Studies, vol. 10/2 1. Análisis de la reforma Dicha unanimidad se expresó por primera vez, cuando el texto de la enmienda transaccional, presentada por el Grupo Ciudadanos, fue objeto de votación el 14 de febrero de 20177, posteriormente también cuando, siendo presentada por el Grupo Popular, se admitió a trámite la Proposición de ley de modificación del Código Civil, la Ley Hipotecaria y la Ley de Enjuiciamiento Civil sobre el régimen jurídico de los animales el 13 de octubre de 20178 La proyectada reforma del Código civil español9, que pivotaba sobre el reconocimiento de la sentiencia de todos los animales (y no sólo de los animales de compañía), se articulaba en torno a tres ejes:  la redefinición del marco jurídico sobre el que se asienta la propiedad sobre los animales  la propiedad misma sobre los animales considerados como seres vivos dotados de sens la propiedad misma sobre los animales considerados como seres vivos dotados de sensibilidad  las leyes de acompañamiento, para hacer efectivo el reconocimiento de los animales com sintientes  las leyes de acompañamiento, para hacer efectivo el reconocimiento de los animales como seres sintientes 1. Análisis de la reforma En el mismo volumen 9/3 (2018) se publicaron también en esta revista, como Dossier temático, cuatro artículos sobre el proyecto de reforma del Código civil, obra de los siguientes autores: ALÁEZ CORRAL, Algunas claves de la futura reforma del Estatuto Jurídico Civil del animal en España, en dA. Derecho Animal (Forum of Animal Law Studies) 9/3 (2018) https://doi.org/10.5565/rev/da.342; MENÉNDEZ DE LLANO, N., La modernización del estatuto del animal en la legislación civil española, en dA. Derecho Animal (Forum of Animal Law Studies) 9/3 (2018) https://doi.org/10.5565/rev/da.343; LELANCHON, L., La reforma del estatuto jurídico civil de los animales en el Derecho francés, en dA. Derecho Animal (Forum of Animal Law Studies) 9/3 (2018) https://doi.org/10.5565/rev/da.344; REIS MOREIRA, 8 Derecho Animal. Forum of Animal Law Studies, vol. 10/2 Animales enel código civil español: una reforma interrumpida Marita Giménez-Candela Durante este tiempo ha quedado palmariamente demostrada la voluntad expresada de forma unánime por la Cámara de los Diputados -en todas y cada una de las secuencias en las que el texto tuvo que ser sometido a votación en el Congreso-, de dotar a los animales de un estatuto jurídico más acorde con los siguientes elementos:  lo que la sociedad demanda,  lo que la ciencia ha demostrado,  lo que establece la legislación de Bienestar Animal de la Unión Europea, con especial referen dispuesto en el art. 13 TFUE,  lo que establece la legislación de Bienestar Animal de la Unión Europea, con especial refe dispuesto en el art. 13 TFUE,  lo que el giro animal, plasmado en la Descosificación de los animales, ya ha logrado en otros países europeos y también en el entorno global.  lo que el giro animal, plasmado en la Descosificación de los animales, ya ha logrado en otros países europeos y también en el entorno global. A., La reforma del Código Civil portugués respecto al estatuto del animal, en dA. Derecho Animal (Forum of Animal Law Studies) 9/3 (2018) https://doi.org/10.5565/rev/da.345 7Cfr. Diario de Sesiones del Congreso de los Diputados, pleno y diputación permanente, Año 2017, Nº 29, XII Legislatura, sesión plenaria Nº 27, martes, 14 de febrero de 2017, págs. 43-50. http://www.congreso.es/public_oficiales/L12/CONG/DS/PL/DSCD-12- PL-29.PDF; 8BOGC de 13 de Octubre de 2017 (122/00034) Proposición de Ley de modificación del Código Civil, la Ley Hipotecaria y la Ley de Enjuiciamiento Civil sobre el régimen jurídico de los animales http://www.congreso.es/public_oficiales/L12/CONG/BOCG/B/BOCG-12-B-167-1.PDF#page=1 9ARRIBAS ATIENZA, P., El nuevo tratamiento civil de los animales. Diario La Ley, Nº 9136, Sección Tribuna, 9 de Febrero de 2018 10Vid. ampliamente, con un cronograma de las reformas del estatuto civil de los animales en Austria, Alemania, Suiza, Cataluña, Portugal, Francia, Colombia, Chequia, Luxemburgo y otras iniciativas en el mismo sentido, GIMÉNEZ-CANDELA, M., Dignidad, sentiencia, personalidad: relación jurídica humano-animal, en dA, Derecho Animal (Forum of Animal Law Studies) 9/2 (2018) https://doi.org/10.5565/rev/da.346 11GIMÉNEZ-CANDELA, M., Persona y Animal. Una aproximación sin prejuicios, en dA, Derecho Animal (Forum of Animal Law Studies) 9/1 (2019) 8-20 https://doi org/10 5565/rev/da 417 ( ) p g 7Cfr. Diario de Sesiones del Congreso de los Diputados, pleno y diputación permanente, Año 2017, Nº 29, XII Legislatura, sesión plenaria Nº 27, martes, 14 de febrero de 2017, págs. 43-50. http://www.congreso.es/public_oficiales/L12/CONG/DS/PL/DSCD-12- PL-29.PDF; A., La reforma del Código Civil portugués respecto al estatuto del animal, en dA. Derecho Animal (Forum of Animal Law Studies) 9/3 (2018) https://doi.org/10.5565/rev/da.345 PL 29.PDF; 8BOGC de 13 de Octubre de 2017 (122/00034) Proposición de Ley de modificación del Código Civil, la Ley Hipotecaria y la Ley de Enjuiciamiento Civil sobre el régimen jurídico de los animales http://www.congreso.es/public_oficiales/L12/CONG/BOCG/B/BOCG-12-B-167-1.PDF#page=1 9ARRIBAS ATIENZA, P., El nuevo tratamiento civil de los animales. Diario La Ley, Nº 9136, Sección Tribuna, 9 de Febrero de 2018 10Vid. ampliamente, con un cronograma de las reformas del estatuto civil de los animales en Austria, Alemania, Suiza, Cataluña, Port gal Francia Colombia Cheq ia L emb rgo otras iniciati as en el mismo sentido GIMÉNEZ CANDELA M Dignidad 2. Resultados y obstáculos Así mismo, también existe una amplia normativa autonómica de protección animal que es aplicable en el ámbito al que se refiere el Código civil, que no es otro que el del Derecho Privado. El texto propuesto consta de cuatro párrafos (art. 333): 1. Los animales son seres vivos dotados de sensibilidad. Solo les será aplicable el régimen jurídico de los bienes en la medida en que sea compatible con su naturaleza y con las disposiciones destinadas a su protección. 2. El propietario de un animal puede disfrutar y disponer de él respetando su cualidad de ser dotado de sensibilidad, asegurando su bienestar conforme a las características de cada especie. El derecho de uso no ampara el maltrato. El derecho de disponer del animal no incluye el de abandonarlo o sacrificarlo salvo en los casos establecidos en las normas legales o reglamentarias. 2. El propietario de un animal puede disfrutar y disponer de él respetando su cualidad de ser dotado de sensibilidad, asegurando su bienestar conforme a las características de cada especie. El derecho de uso no ampara el maltrato. El derecho de disponer del animal no incluye el de abandonarlo o sacrificarlo salvo en los casos establecidos en las normas legales o reglamentarias. 3. Los gastos destinados a la curación de un animal herido por un tercero son recuperables por su propietario en la medida en que hayan sido proporcionados y aun cuando hayan sido superiores al valor del animal. 4. Sin perjuicio de la indemnización debida según las normas generales de responsabilidad civil, en el caso de que la lesión de un animal de compañía, causada por un tercero, haya provocado su muerte, la privación de un miembro o un órgano importante, o una afectación grave o permanente de su capacidad de locomoción, su propietario y quienes convivan con el animal tienen derecho a una indemnización, que será fijada equitativamente por el tribunal, por el sufrimiento moral sufrido.” Una consecuencia de la redefinición de los animales, como categoría a se, o categoría sui generis dentro de la propiedad, es la afirmación de que el uso no ampara el maltrato y que la disposición excluye el abandono y el sacrificio sin una normativa que lo determine. Es claro que aquí se hace referencia al abandono de los animales que constituye el delito del art. 2. Resultados y obstáculos Con esta perspectiva la reforma del régimen civil de los animales, que la Comisión de Justicia, con poderes plenos, aceptó después de la fase de enmiendas, afectaba en primer término a la denominación del Título segundo que quedaba configurado como sigue: “De los animales, los bienes, de la propiedad y de sus modificaciones”. Esta modificación es de la mayor importancia y, probablemente, la que justifica de forma sustancial el sentido de la reforma, que no se limitaba a cambiar la denominación de los animales: de cosas a seres sentientes, sino que les otorgaba una categoría diferenciada de las cosas inertes. La clave de entendimiento de esta nueva categoría, estriba en reconocer que los animales deben tener un régimen jurídico adaptado a su condición de seres sentientes, lo que les aparta de la consideración de bienes muebles como hasta ahora lo había establecido el Código civil de 1889, conforme a la aquilatada tradición romana que, sin solución de continuidad se incorporó, tras la promulgación del Código Napoleón, a la mayoría de los regímenes jurídicos occidentales y, por ende, a todos los Códigos civiles europeos y latinoaméricanos, así como a los Códigos Civiles de Japón y Turquía, modelados según el BGB alemán10. Apartarse de la conocida summa divisio gaiana (personas-cosas) es difícil para quien desconoce la verdadera proyección de tal distinción11. Sigue, pues, resultando arduo hacer entendible a la mayoría de los juristas, que tratar de forma diferenciada a los animales-porque tienen una naturaleza propia que los hace Derecho Animal. Forum of Animal Law Studies, vol. 10/2 9 Animales en el código civil español: una reforma interrumpida Marita Giménez-Candela merecedores de tal trato jurídico diferenciado12-, no supone ninguna amenaza para el Derecho sino un enriquecimiento merecedores de tal trato jurídico diferenciado12-, no supone ninguna amenaza para el Derecho sino un enriquecimiento En este sentido, la reforma del art. 333 CC, es la muestra de cómo se concibe el tratamiento de los animales de acuerdo a su condición de “seres vivos dotados de sensibilidad”, que deberá ser acorde con su naturaleza y con las disposiciones destinadas a su protección. Esta precisión del párrafo 1, remite a la amplísima legislación europea de protección animal que tiene como enseña el Bienestar. Normativa que se incorpora de forma mandatoria para los Estados miembros, de conformidad con lo dispuesto por el art. 13 TFUE13. 12Sugerente en este sentido, desde postulados científicos, la reflexión de BINDER, R., Die Würde des Tieres is antastbar, en Rechtswissenschaft. Zeitschrift für rechtswissenschaftliche Forschung 3 (2016) 497ss. 13Art. 13 TFUE https://www.boe.es/doue/2007/306/Z00001-00271.pdf “Al formular y aplicar las políticas de la Unión en materia de agricultura, pesca, transporte, mercado interior, investigación y desarrollo tecnológico y espacio, la Unión y los Estados miembros tendrán plenamente en cuenta las exigencias en materia de bienestar de los animales como seres sensibles, respetando al mismo tiempo las disposiciones legales o administrativas y las costumbres de los Estados miembros relativas, en particular, a ritos religiosos, tradiciones culturales y patrimonio regional. 14CP art. 337 bis; cfr. REQUEJO CONDE, C., El delito de maltrato a los animales tras la reforma del código penal por la Ley Orgánica 1/2015, de 30 de marzo, en dA. Derecho Animal (Forum of Animal Law Studies) 6/2 (2015) https://doi.org/10.5565/rev/da.77 15 L 32/2007 d 7 d i b l id d d l i l l ió i ió ifi i 15 Ley 32/2007, de 7 de noviembre para el cuidado de los animales en su explotación, transporte, experimentación y sacrificio https://www.boe.es/buscar/act.php?id=BOE-A-2007-19321 16 i i d l l did d i l di l id l i d l i encia por daños morales por la pérdida de un animal, se dictó en Cataluña en 2007; vid. el comentario del propio entencia, ARIAS, G., Sentencia 466/07, de dieciséis de mayo, Juzgado de primera Instancia num. 32 de Barcelona, 10 Derecho Animal. Forum of Animal Law Studies, vol. 10/2 16La primera sentencia por daños morales por la pérdida de un animal, se dictó en Cataluña en 2007; vid. el come juez que dictó la sentencia, ARIAS, G., Sentencia 466/07, de dieciséis de mayo, Juzgado de primera Instancia num. p p aplicar las políticas de la Unión en materia de agricultura, pesca, transporte, mercado interior, investigación y gico y espacio, la Unión y los Estados miembros tendrán plenamente en cuenta las exigencias en materia de animales como seres sensibles, respetando al mismo tiempo las disposiciones legales o administrativas y las Estados miembros relativas, en particular, a ritos religiosos, tradiciones culturales y patrimonio regional. 2. Resultados y obstáculos 337 bis del Código Penal, según se estableció en la reforma del mismo del año 201514 así como al maltrato que causa la muerte del animal y al sacrificio que no esté de acuerdo con la normativa prevista, que no es otra que la de la ley 32/2007, de 7 de noviembre para el cuidado de los animales en su explotación, transporte, experimentación y sacrificio15. Como es obvio, la reforma del CC no se refería sólo a los a los animales de compañía, sino -como ya se ha señalado- a todos los animales. Otra consecuencia innovadora en el nuevo régimen jurídico de los animales, es una nueva consideración del valor del animal, que ya no es estrictamente el valor de mercado, como lo es una cosa en propiedad, sino el que deriva de su sentiencia y del vínculo humano-animal que reconocidamente se establece por ello. La proyección de este nuevo reconocimiento, se refiere a los gastos de curación de un animal herido por un tercero, que son recuperables por el propietario también si han sido superiores al valor del animal, así como al reconocimiento de los daños morales por la muerte y lesiones causadas por un tercero. En este sentido ya se había pronunciado la Jurisprudencia que, en tema de reconocimiento de la singularidad de las relaciones humano-animal, ha jugado un papel innovador muy importante en los últimos años16. 10 Derecho Animal. Forum of Animal Law Studies, vol. 10/2 Marita Giménez-Candela Animales enel código civil español: una reforma interrumpida Un aspecto innovador de la frustrada reforma se refería a la introducción del criterio de la custodia compartida que pueda ser aplicado por el juez en caso de separación o divorcio, teniendo en cuenta el interés de los miembros de la familia y el bienestar del animal (art. 90.1.c; art 94bis; 103,2). Que la proyectada reforma del CC lo reconociera, no hace más que consolidar y reconocer una práctica que se presenta como habitual entre los abogados de familia y que carece de una regulación, que es muy necesaria dados los cambios que el modelo de familia ha experimentado en los últimos años en nuestro país17. El reconocimiento de la sentiencia animal también ha propiciado la introducción en la Ley Hipotecaria (art. en dA, Derecho Animal (Forum of Animal Law Studies) https://doi.org/10.5565/rev/da.236 17CASAS DÍAZ, L. / CAMPS I VIDELLET, X., Las crisis matrimoniales y los animales de compañía: una aproximación práctica desde el ejercicio de la abogacía, en dA, Derecho Animal (Forum of Animal Law Studies) 9/1 (2019) https://doi.org/10.5565/rev/da.397 18BOCG de 1 de marzo de 2019 (122/000134) Proposición de Ley de modificación del Código Civil, la Ley Hipotecaria y la Ley de Enjuiciamiento Civil, sobre el régimen jurídico de los animales. http://www.congreso.es/public_oficiales/L12/CONG/BOCG/B/BOCG-12-B-167-1.PDF#page=1 2. Resultados y obstáculos 111) de la prohibición de extender la hipoteca a los animales que formen parte de una finca ganadera, industrial o de recreo, así como también la prohibición de extender el pacto de hipoteca a los animales de compañía. Por lo demás, la reforma se proyectaba también en la prohibición del embargo de los animales de compañía, mediante la modificación del art. 605 de la ley de Enjuiciamiento Civil (Ley 1/2000, de 7 de enero). La Ponencia hacía, para concluir, una reflexión que sorprende por la claridad y por lo inusual del empleo de expresiones en las que el legislador se haga eco de un deseo de mejora, que no resulta posible en el trámite que se expone en el texto de la Proposición de Ley. En sustancia, se reconocía que la reforma del régimen jurídico de los animales no es suficiente, pues deja a los animales parcialmente sometidos al régimen de las cosas, siempre que no haya normas específicas destinadas a regular las relaciones jurídicas en las que intervengan los animales, por lo que sería “deseable” de lege ferenda que el “régimen protector vaya extendiéndose progresivamente a los distintos ámbitos en que intervienen los animales y se vaya restringiendo con ello la aplicación supletoria del régimen jurídico de las cosas”18. Que la reforma del estatuto jurídico de los animales en el Código civil no haya culminado con su aprobación, es en mi opinión, una etapa más de un recorrido que será difícil que no se retome con el nuevo Parlamento surgido de las urnas el 28 de abril de 2018. Los elementos que la propiciaron siguen vigentes. La sociedad ha cambiado su actitud respecto a los animales, la ciencia cada vez ofrece resultados más consolidados en afirmación de la sentiencia animal, el movimiento descosificador es una realidad global perceptible en muchos países y, por último, la legislación del Bienestar animal sigue vigente y vincula a España como estado Miembro que no puede desoir sus obligaciones. en dA, Derecho Animal https://doi.org/10.5565/rev/da.346  GIMÉNEZ-CANDELA, M., La Descosificación de los animales en el CC español, en dA. Derecho Animal (Forum of Animal Law Studies) 9/3 (2018) https://doi.org/10.5565/rev/da.361  GIMÉNEZ-CANDELA, M., La Descosificación de los animales en el CC español, en dA. Derecho Animal (Forum of Animal Law Studies) 9/3 (2018) https://doi.org/10.5565/rev/da.361 É  GIMÉNEZ-CANDELA, M., Persona y Animal. Una aproximación sin prejuicios, en dA, Derecho Animal (Forum of Animal Law Studies) 9/1 (2019) https://doi.org/10.5565/rev/da.417  HUI, M., Amending Classic Civil Codes, rec. a ROGEL VIDE, C., Personas, Animales y Derechos (Madrid-México 2018), en JAL & IAWS, nº 1 (2018) 337ss.  LELANCHON, L., La reforma del estatuto jurídico civil de los animales en el Derecho francés, en dA. Derecho Animal (Forum of Animal Law Studies) 9/3 (2018) https://doi.org/10.5565/rev/da.344  MENÉNDEZ DE LLANO, N., La modernización del estatuto del animal en la legislación civil española, en dA. Derecho Animal (Forum of Animal Law Studies) 9/3 (2018) https://doi.org/10.5565/rev/da.343  REIS MOREIRA, A., La reforma del Código Civil portugués respecto al estatuto del animal, en dA Derecho Animal (Forum of Animal Law Studies) 9/3 (2018) https://doi.org/10.5565/rev/da.345  REQUEJO CONDE, C., El delito de maltrato a los animales tras la reforma del código penal por la Ley Orgánica 1/2015, de 30 de marzo, en dA. Derecho Animal (Forum of Animal Law Studies) 6/2 (2015) https://doi.org/10.5565/rev/da.77  REQUEJO CONDE, C., El delito de maltrato a los animales tras la reforma del código penal por la Ley Orgánica 1/2015, de 30 de marzo, en dA. Derecho Animal (Forum of Animal Law Studies) 6/2 (2015) https://doi.org/10.5565/rev/da.77 ( ) p g  ROGEL VIDE, C., Los animales en el Código Civil (Madrid 2017) ROGEL VIDE, C., Personas, Animales y Derechos (Madrid-México 2018)  ROGEL VIDE, C., Personas, Animales y Derechos (Madrid-México 2018) Referencias a ROGEL VIDE, C., Personas, Animales y Derechos (Madrid-México 2018), en JAL & IAWS, nº 1 (2018) 337ss.  LELANCHON, L., La reforma del estatuto jurídico civil de los animales en el Derecho francés, en dA. Derecho Animal (Forum of Animal Law Studies) 9/3 (2018) https://doi.org/10.5565/rev/da.344  MENÉNDEZ DE LLANO, N., La modernización del estatuto del animal en la legislación civil española, en dA. Derecho Animal (Forum of Animal Law Studies) 9/3 (2018) https://doi.org/10.5565/rev/da.343  REIS MOREIRA, A., La reforma del Código Civil portugués respecto al estatuto del animal, en dA. Derecho Animal (Forum of Animal Law Studies) 9/3 (2018) https://doi.org/10.5565/rev/da.345  REQUEJO CONDE, C., El delito de maltrato a los animales tras la reforma del código penal por la Ley Orgánica 1/2015, de 30 de marzo, en dA. Derecho Animal (Forum of Animal Law Studies) 6/2 (2015) https://doi.org/10.5565/rev/da.77  ROGEL VIDE, C., Los animales en el Código Civil (Madrid 2017)  ROGEL VIDE, C., Personas, Animales y Derechos (Madrid-México 2018) Referencias jurídicas  CP art. 337 bis, Ley Orgánica 1/2015, de 30 de marzo  Art. 13 TFUE https://www.boe.es/doue/2007/306/Z00001-00271.pdf  Diario de Sesiones del Congreso de los Diputados, pleno y diputación permanente, Año 2017, Nº 29, XII Legislatura, sesión plenaria Nº 27, martes, 14 de febrero de 2017, págs. 43-50. http://www.congreso.es/public_oficiales/L12/CONG/DS/PL/DSCD-12-PL-29.PDF  BOCG de 1 de marzo de 2019 (122/000134) Proposición de Ley de modificación del Código Civil, la Ley Hipotecaria y la Ley de Enjuiciamiento Civil, sobre el régimen jurídico de los animales. http://www.congreso.es/public_oficiales/L12/CONG/BOCG/B/BOCG-12-B-167-1.PDF#page=1  BOGC de 13 de Octubre de 2017 (122/00034) Proposición de Ley de modificación del Código Civil, la Ley Hipotecaria y la Ley de Enjuiciamiento Civil sobre el régimen jurídico de los animales http://www.congreso.es/public_oficiales/L12/CONG/BOCG/B/BOCG-12-B-167-1.PDF#page=1 en dA, Derecho Animal (Forum of Animal Law Studies) 9/2 (2018) https://doi.org/10.5565/rev/da.346 en dA, Derecho Animal (Forum of Animal Law Studies) 9/2 (2018) https://doi.org/10.5565/rev/da.346 12 Derecho Animal. Forum of Animal Law Studies, vol. 10/2 Referencias  ALÁEZ CORRAL, Algunas claves de la futura reforma del Estatuto Jurídico Civil del animal en España, en dA. Derecho Animal (Forum of Animal Law Studies) 9/3 (2018) https://doi.org/10.5565/rev/da.342  ARIAS, G., Sentencia 466/07, de dieciséis de mayo, Juzgado de primera Instancia num. 32 de Barcelona, en dA, Derecho Animal (Forum of Animal Law Studies) https://doi.org/10.5565/rev/da.236  ARRIBAS ATIENZA, P., El nuevo tratamiento civil de los animales. Diario La Ley, N Sección Tribuna, 9 de febrero de 2018  BINDER, R., Die Würde des Tieres is antastbar, en Rechtswissenschaft. Zeitsch rechtswissenschaftliche Forschung 3 (2016) 497ss.  CASAS DÍAZ, L. / CAMPS I VIDELLET, X., Las crisis matrimoniales y los animales de compañía: una aproximación práctica desde el ejercicio de la abogacía, en dA, Derecho Animal (Forum of Animal Law Studies) 9/1 (2019) https://doi.org/10.5565/rev/da.397  CASAS DÍAZ, L. / CAMPS I VIDELLET, X., Las crisis matrimoniales y los animales de compañía: una aproximación práctica desde el ejercicio de la abogacía, en dA, Derecho Animal (Forum of Animal Law Studies) 9/1 (2019) https://doi.org/10.5565/rev/da.397  CODINA, J.I., Unanimidad en el Congreso de los Diputados para instar la reforma del Código civil español y reconocer a los animales como seres dotados de sensibilidad, en dA. 2/2017 https://derechoanimal.info/es/actividades/2017/unanimidad-en-el-congreso-de-los-diputados-para- instar-la-reforma-del-codigo-civil  CODINA, J.I., Unanimidad en el Congreso de los Diputados para instar la reforma del Código civil español y reconocer a los animales como seres dotados de sensibilidad, en dA. 2/2017 https://derechoanimal.info/es/actividades/2017/unanimidad-en-el-congreso-de-los-diputados-para- i l f d l di i il CO N , J. ., U d d e e Co g eso de os pu dos p s e o de Cód go civil español y reconocer a los animales como seres dotados de sensibilidad, en dA. 2/2017 https://derechoanimal.info/es/actividades/2017/unanimidad-en-el-congreso-de-los-diputados-para- instar-la-reforma-del-codigo-civil https://derechoanimal.info/es/actividades/2017/unanimidad en el congreso de los diputados para instar-la-reforma-del-codigo-civil instar-la-reforma-del-codigo-civil , Derecho Animal (Forum of Animal Law Studies) https://doi.org/10.5565/rev/da.236 Derecho Animal. Forum of Animal Law Studies, vol. 10/2 11 Animales en el código civil español: una reforma interrumpida Marita Giménez-Candela en dA, Derecho Animal (Forum of Animal Law Studies) 9/2 (2018) https://doi.org/10.5565/rev/da.346  GIMÉNEZ-CANDELA, M., La Descosificación de los animales en el CC español, en dA. Derecho Animal (Forum of Animal Law Studies) 9/3 (2018) https://doi.org/10.5565/rev/da.361  GIMÉNEZ-CANDELA, M., Persona y Animal. Una aproximación sin prejuicios, en dA, Derecho Animal (Forum of Animal Law Studies) 9/1 (2019) https://doi.org/10.5565/rev/da.417  HUI, M., Amending Classic Civil Codes, rec. Referencias jurídicas  CP art. 337 bis, Ley Orgánica 1/2015, de 30 de marzo  Art. 13 TFUE https://www.boe.es/doue/2007/306/Z00001-00271.pdf  Diario de Sesiones del Congreso de los Diputados, pleno y diputación permanente, Año 2017, Nº 29, XII Legislatura, sesión plenaria Nº 27, martes, 14 de febrero de 2017, págs. 43-50. http://www.congreso.es/public_oficiales/L12/CONG/DS/PL/DSCD-12-PL-29.PDF  BOCG de 1 de marzo de 2019 (122/000134) Proposición de Ley de modificación del Código Civil, la Ley Hipotecaria y la Ley de Enjuiciamiento Civil, sobre el régimen jurídico de los animales. http://www.congreso.es/public_oficiales/L12/CONG/BOCG/B/BOCG-12-B-167-1.PDF#page=1  BOGC de 13 de Octubre de 2017 (122/00034) Proposición de Ley de modificación del Código Civil, la Ley Hipotecaria y la Ley de Enjuiciamiento Civil sobre el régimen jurídico de los animales http://www.congreso.es/public_oficiales/L12/CONG/BOCG/B/BOCG-12-B-167-1.PDF#page=1  CP art. 337 bis, Ley Orgánica 1/2015, de 30 de marzo  Art. 13 TFUE https://www.boe.es/doue/2007/306/Z00001-00271.pdf  Diario de Sesiones del Congreso de los Diputados, pleno y diputación permanente, Año 2017, Nº 29, XII Legislatura, sesión plenaria Nº 27, martes, 14 de febrero de 2017, págs. 43-50. http://www.congreso.es/public_oficiales/L12/CONG/DS/PL/DSCD-12-PL-29.PDF  BOCG de 1 de marzo de 2019 (122/000134) Proposición de Ley de modificación del Código Civil, la Ley Hipotecaria y la Ley de Enjuiciamiento Civil, sobre el régimen jurídico de los animales. http://www.congreso.es/public_oficiales/L12/CONG/BOCG/B/BOCG-12-B-167-1.PDF#page=1  BOGC de 13 de Octubre de 2017 (122/00034) Proposición de Ley de modificación del Código Civil, la Ley Hipotecaria y la Ley de Enjuiciamiento Civil sobre el régimen jurídico de los animales http://www.congreso.es/public_oficiales/L12/CONG/BOCG/B/BOCG-12-B-167-1.PDF#page=1  BOGC de 13 de Octubre de 2017 (122/00034) Proposición de Ley de modificación del Código Civil, la Ley Hipotecaria y la Ley de Enjuiciamiento Civil sobre el régimen jurídico de los animales http://www.congreso.es/public_oficiales/L12/CONG/BOCG/B/BOCG-12-B-167-1.PDF#page=1 12 Derecho Animal. Forum of Animal Law Studies, vol. 10/2
https://openalex.org/W4390170278
https://jurnal.relawantik.or.id/index.php/abdimas/article/download/31/23
Indonesian
null
Pelatihan Perakitan Komputer Untuk Meningkatkan Keterampilan Bagi Santri di Ponpes Al Mutmainnah
Jurnal Pengabdian Literasi Digital Indonesia
2,023
cc-by-sa
2,469
Jurnal Pengabdian Literasi Digital Indonesia E-ISSN. 2961-9440 Volume: 2, Nomor 2, Desember, 2023, Hal: 49-54 Abstract The role of computers in everyday life is increasingly important. Computers can be used as a tool for running a business and doing all office, industrial, construction and lecture tasks. Computers have many varieties of both types and types, ranging from standard types, medium to high-tech computers. With the large role of computers at this time, the Al Mutmainnah Islamic Boarding School in the Kediri sub-district, West Lombok Regency, collaborated with the community service team at Qamarul Huda Badaruddin University to provide computer assembly training to students. This training is intended to equip students to have special skills in computer assembly and later it is hoped that they will be able to open their own jobs from this computer assembly training. The material provided can be received, digested, and understood by the participants well. The large number of participants in proportion to the number of devotees made this training more conducive. The activity went smoothly, on time and as expected and the students were able to communicate well with the speakers and other students. This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License. Copyright: © 2023by the authors. Keywords : computers, assembling, Islamic boarding school, students Pelatihan Perakitan Komputer Untuk Meningkatkan Keterampilan Bagi Santri di Ponpes Al Mutmainnah Valian Yoga Pudya Ardhana1*, M. Dermawan Mulyodiputro1 1Universitas Qamarul Huda Badaruddin valianypa81@gmail.com, m.dermawan.m@gmail.com 1Universitas Qamarul Huda Badaruddin valianypa81@gmail.com, m.dermawan.m@gmail.com Abstrak Peranan komputer dalam kehidupan sehari-hari semakin terasa penting. Komputer dapat dijadikan sebagai alat bantu dalam menjalankan usaha dan mengerjakan semua tugas kantor, industri, konstruksi maupun perkuliahan. Komputer memiliki banyak ragam baik tipe maupun jenis, mulai dari tipe standar, menengah sampai komputer dengan teknologi tinggi. Dengan besarnya peranan komputer pada saat ini, Ponpes Al Mutmainnah yang berada di kecamatan Kediri Kabupaten Lombok Barat bekerjasama dengan tim pengabdian kepada masyarakat Universitas Qamarul Huda Badaruddin untuk memberikan pelatihan perakitan komputer kepada santri. Pelatihan ini dimaksud untuk membekali para santri agar mempunyai keterampilan khusus dalam perakitan komputer dan nantinya diharapkan mereka mampu untuk membuka lapangan kerja sendiri dari pelatihan perakitan komputer ini. Materi yang diberikan dapat diterima, dicerna, dan dipahami peserta dengan baik. Jumlah peserta yang banyak sebanding dengan jumlah pengabdi menjadikan pelatihan ini menjadi lebih kondusif. Kegiatan berlangsung lancar, tepat waktu dan sesuai dengan yang diharapkan dan para santri dapat berkomunikasi dengan para pembicara dan santri lain dengan baik. DOI: https://doi.org/10.57119/abdimas.v2i2.31 *Correspondensi: Valian Yoga Pudya Ardhana Email: valianypa81@gmail.com DOI: https://doi.org/10.57119/abdimas.v2i2.31 *Correspondensi: Valian Yoga Pudya Ardhana Email: valianypa81@gmail.com Received: 02-06-2023 Accepted: 10-08-2023 Published: 17-08-2023 This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License. Copyright: © 2023by the authors. DOI: https://doi.org/10.57119/abdimas.v2i2.31 *Correspondensi: Valian Yoga Pudya Ardhana Email: valianypa81@gmail.com Kata Kunci: komputer, perakitan, ponpes, santri I. PENDAHULUAN Seiring dengan kemajuan dan perkembangan teknologi pada saat sekarang ini khususnya di bidang komputer sudah semakin maju (Ardhana et al., 2023). Tahapan perubahan diharapkan mampu membawa Hal 49 dari 54 Hal 49 dari 54 Jurnal Pengabdian Literasi Digital Indonesia E-ISSN. 2961-9440 Volume: 2, Nomor 2, Desember, 2023, Hal: 49-54 bangsa ke arah kemajuan peradaban yang lebih tinggi dan meresap secara utuh sebagai jati diri bangsa tersebut (Ardhana et al., 2018). Tingkat penguasaan ilmu dan teknologi merupakan bukti nyata keberhasilan pembangunan (Ardhana et al., 2021). Peningkatan penguasaan ilmu dan teknologi tidak hanya dilakukan pada pendidikan formal, namun juga dapat dilakukan melalui pendidikan nonformal (Ardhana et al., 2021). Termasuk juga di Pulau Lombok sudah banyak berbagai spesifikasi dan jenis komputer yang dipasarkan dengan beragam harga. Industri pembuatan komponen hardware komputer pun saling bersaing dalam memuaskan para konsumen maupun pelanggannya (Ardhana et al., 2022). Untuk itu kita sebagai konsumen harus berhati-hati dalam memilih dan membeli komponen hardware tersebut. Sehingga kita tidak tertipu dengan harga murah yang ditawarkan tetapi harus mengetahui spesifikasi dan kualitas barang yang ditawarkan (Ardhana et al. 2021). Agar kita tidak kecewa, ada baiknya kita memiliki pengetahuan sedikit tentang komponen –komponen hardware tersebut. Merakit komputer merupakan suatu tahapan kegiatan menyatukan komponen-komponen yang dibutuhkan agar komputer dapat berjalan dengan baik. Untuk memahami proses perakitan komputer yang benar, dibutuhkan pemahaman terhadap perangkat keras komputer baik secara logical dan physical (Yuliati et al., 2022). Sementara itu agar komputer dapat dioperasikan sesuai dengan fungsinya perlu dilakukan instalasi. Instalasi komputer yaitu memasang program (perangkat lunak) ke dalam komputer semua perangkat lunak (misalnya, Microsoft Windows, Microsoft Office, dan perangkat lunak lainnya). Selain itu, instalasi juga berfungsi untuk menyesuaikan program dengan alatalat terpasang pada komputer dan menguraikan file-file yang dipadatkan (Ardhana et al., 2023). Keahlian merakit dan instalasi laptop/komputer mampu menjadikan ladang usaha ekonomi kreatif berbasis teknologi Informasi (Wahyu et al., 2020). Peranan komputer dalam kehidupan sehari-hari semakin terasa penting. Komputer dapat dijadikan sebagai alat bantu dalam menjalankan usaha dan mengerjakan semua tugas kantor, industri, konstruksi maupun perkuliahan (Ardhana et al., 2022). Komputer memiliki banyak ragam baik tipe maupun jenis, mulai dari tipe standar, menengah sampai komputer dengan teknologi tinggi (Ginting et al., 2022). Ada 2 jenis komputer , yaitu komputer rakitan dan komputer branded (built up). Keuntungan dengan komputer rakitan selain komponen hardwarenya banyak pilihan dan bisa berlainan vendor (merk pabrik pembuatannya), komponennya juga bisa dikurangi ataupun ditambah jika memang dibutuhkan. I. PENDAHULUAN Sedangkan untuk komputer branded spesifikasi hardwarenya sudah ditetapkan dari pihak vendor tersebut seperti HP,Lenovo,Dell dan lain-lainnya. Komponen-komponen komputer yang harus disediakan untuk merakit atau memasang pada komputer adalah : prosesor, mainboard (motherboard), memory (RAM), harddisk, kartu garfis (VGA card), kartu audio (soundcard), casing, cd drive, monitor, keyboard, mouse, cd/dvd room, kabel dan komponen pelengkap lainnya. Sementara itu perangkat komputer dapat dibagi menjadi 3 yaitu sebagai berikut : hardware (perangkat keras), software (perangkat lunak), dan brainware (pengguna) (Alfirman et al., 2019). Dengan besarnya peranan komputer pada saat ini, Ponpes Al Mutmainnah yang berada di kecamatan Kediri Kabupaten Lombok Barat bekerjasama dengan tim pengabdian kepada masyarakat Universitas Qamarul Huda Badaruddin untuk memberikan pelatihan perakitan komputer kepada santri. Pelatihan ini Hal 50 dari 54 Hal 50 dari 54 Jurnal Pengabdian Literasi Digital Indonesia E-ISSN. 2961-9440 Volume: 2, Nomor 2, Desember, 2023, Hal: 49-54 dimaksud untuk membekali para santri agar mempunyai keterampilan khusus dalam perakitan komputer dan nantinya diharapkan mereka mampu untuk membuka lapangan kerja sendiri dari pelatihan perakitan komputer ini. Pelatihan yang dilakukan berupa: memberikan materi pengetahuan dasar tentang perakitan komputer, melakukan praktek langsung bagaimana cara merakit komputer dan Setelah praktek dilaksanakan, kemudian melakukan tes/ujian langsung kepada para santri untuk mengetahui seberapa paham santri tentang materi yang telah diberikan. Manfaat yang diharapkan dalam kegiatan pengabdian ini adalah: Adanya peningkatan pengetahuan dan keterampilan para santri tentang perakitan dasar komputer, dan santri dapat menggunakan computer dengan baik dan berjalan lancar sehingga penggunaan komputer dapat lebih optimal, serta mempunyai keterampilan khusus. II. METODE Metode pelaksanaan kegiatan pengabdian kepada masyarakat ini dilaksanakan melalui pelatihan dengan pemanfaatan modul yang telah disediakan yaitu dengan metode teori pembelajaran dan praktik langsung di lokasi mitra pengabdian kepada masyarakat. Kegiatan pelatihan dilaksanakan selama 2 hari dimana setiap hari dimulai pukul 08.00 sampai pukul 16.00 Selain itu, mereka juga diberikan contoh dan kasus serta simulasi cara merakit laptop/komputer langkah demi langkahnya dan instalasi beberapa software. Melalui kegiatan pengabdian kepada masyarakat, pelatihan kepada peserta para santri Pondok Pesantren Al Mutmainnah dilakukan oleh beberapa instruktur profesional dibidangnya dengan materi pelatihan sebagai berikut: 1. Pengenalan dan Persiapan Komponen Komputer 2. Peralatan Merakit Komputer 3. Praktek Merakit Komputer 4. Pengujian Hasil Merakit Komputer 5. Instalasi Aplikasi dan Pengelolaan 6. Pengujian dan Implementasi Komputer III. HASIL DAN PEMBAHASAN III. HASIL DAN PEMBAHASAN Hasil kegiatan pengabdian kepada masyarakat ini untuk membantu meningkatkan keterampilan para santri Ponpes Al Mutmainnah Kediri. Kegiatan pelatihan ini diikuti dengan tertib selama kegiatan pelatihan berlangsung. Materi berfokus pada nama-nama komponen komputer, fungsi setiap komponen komputer, perakitan komputer serta peran penting komputer. Selanjutnya, narasumber menjelaskan fungsi setiap komponen yang ada pada komputer dan bagaimana cara merakitnya, sehingga para santri tidak salah membeli komputer. Sepanjang hasil diskusi, para peserta tampak semangat dalam bertanya dan merakit komputernya. Hal ini kelihatan dari antusiasnya bertanya tentang fungsi pada setiap komponen yang ada pada komputer. Di akhir sesi, para peserta memberikan umpan balik bahwa pada umumnya para peserta memahami materi disampaikan dan dapat menambah pengetahuan mereka dalam menentukan komputer Hal 51 dari 54 Jurnal Pengabdian Literasi Digital Indonesia E-ISSN. 2961-9440 Volume: 2, Nomor 2, Desember, 2023, Hal: 49-54 yang seperti apa yang cocok digunakan. Untuk selanjutnya, peserta mengharapkan kegiatan pelatihan ini dapat dilanjutkan dengan membahas topik media cetak seperti printer mana yang baik dan bagus. Untuk menunjang pelatihan yang dilakukan tersebut supaya berjalan sukses dan lancar maka dapat disebutkan beberapa peralatan yang antara lain adalah sebagai berikut : 1 PC Jurnal Pengabdian Literasi Digital Indonesia E-ISSN. 2961-9440 Volume: 2, Nomor 2, Desember, 2023, Hal: 49-54 yang seperti apa yang cocok digunakan. Untuk selanjutnya, peserta mengharapkan kegiatan pelatihan ini dapat dilanjutkan dengan membahas topik media cetak seperti printer mana yang baik dan bagus. Jurnal Pengabdian Literasi Digital Indonesia E-ISSN. 2961-9440 Volume: 2, Nomor 2, Desember, 2023, Hal: 49-54 yang seperti apa yang cocok digunakan Untuk selanjutnya peserta mengharapkan kegiatan pelatihan ini Jurnal Pengabdian Literasi Digital Indonesia E-ISSN. 2961-9440 Volume: 2, Nomor 2, Desember, 2023, Hal: 49-54 yang seperti apa yang cocok digunakan. Untuk selanjutnya, peserta mengharapkan kegiatan pelatihan ini dapat dilanjutkan dengan membahas topik media cetak seperti printer mana yang baik dan bagus. Untuk menunjang pelatihan yang dilakukan tersebut supaya berjalan sukses dan lancar maka d di b k b b l l i d l h b i b ik yang seperti apa yang cocok digunakan. Untuk selanjutnya, peserta mengharapkan kegiatan pelatihan ini dapat dilanjutkan dengan membahas topik media cetak seperti printer mana yang baik dan bagus. dapat dilanjutkan dengan membahas topik media cetak seperti printer mana yang baik dan bagus. IV. KESIMPULAN Pelatihan ini memberikan beberapa materi yang terkait dengan pemahaman dalam merakit dan instalasi laptop/komputer untuk menambah keterampilan dan kompetensi para santri agar memiliki keterampilan dalam bidang hardware dan software laptop/komputer. Materi yang diberikan dapat diterima, dicerna, dan dipahami peserta dengan baik. Jumlah peserta yang banyak sebanding dengan jumlah pengabdi menjadikan pelatihan ini menjadi lebih kondusif. Kegiatan berlangsung lancar, tepat waktu dan sesuai dengan yang diharapkan dan para santri dapat berkomunikasi dengan para pembicara dan santri lain dengan baik. III. HASIL DAN PEMBAHASAN Untuk menunjang pelatihan yang dilakukan tersebut supaya berjalan sukses dan lancar maka dapat disebutkan beberapa peralatan yang antara lain adalah sebagai berikut : Untuk menunjang pelatihan yang dilakukan tersebut supaya berjalan sukses dan lancar maka dapat disebutkan beberapa peralatan yang antara lain adalah sebagai berikut : 1. PC 2.LCD Proyektor 3.Slide Materi Perakitan Komputer 3.Slide Materi Perakitan Komputer 4.Diktat 4.Diktat 5.Spidol Snowman 6.Komponen-komponen komputer 7.Tools untuk merakit komputer Gambar 1. Peserta Pelatihan Gambar 1 diatas memperlihatkan para peserta pelatihan antusias mengikuti kegiatan pengabdian kepada masyarakat. 5.Spidol Snowman 6.Komponen-komponen komputer 7.Tools untuk merakit komputer Gambar 1. Peserta Pelatihan Gambar 1. Peserta Pelatihan Gambar 1 diatas memperlihatkan para peserta pelatihan antusias mengikuti kegiatan pengabdian kepada masyarakat. Gambar 1 diatas memperlihatkan para peserta pelatihan antusias mengikuti kegiatan pengabdian kepada masyarakat. Gambar 1 diatas memperlihatkan para peserta pelatihan antusias mengikuti kegiatan pengabdian k d k atas memperlihatkan para peserta pelatihan antusias mengikuti kegiatan pengabdian p y Gambar 2. Perakitan oleh Narasumber Gambar 2 juga menunjukkan narasumber semangat melakukan kegiatan ini. Gambar 2. Perakitan oleh Narasumber Gambar 2. Perakitan oleh Narasumber Gambar 2 juga menunjukkan narasumber semangat melakukan kegiatan ini Hal 52 dari 54 Hal 52 dari 54 Jurnal Pengabdian Literasi Digital Indonesia E-ISSN. 2961-9440 Volume: 2, Nomor 2, Desember, 2023, Hal: 49-54 Jurnal Pengabdian Literasi Digital Indonesia SSN. 2961-9440 ume: 2, Nomor 2, Desember, 2023, Hal: 49-54 Gambar 3. Perakitan oleh Peserta Gambar 3 tampak para peserta melakukan perakitan komputer yang dalam hal ini laptop. Gambar 3. Perakitan oleh Peserta Gambar 3 tampak para peserta melakukan perakitan komputer yang dalam hal ini Gambar 3. Perakitan oleh Peserta Gambar 3 tampak para peserta melakukan perakitan komputer yang dalam hal ini laptop UCAPAN TERIMA KASIH Kami ucapkan banyak terimakasih kepada pengurus Ponpes Al Mutmainnah yang telah memberikan dukungan terhadap kegiatan pengadian kepada masyarakat ini. DAFTAR PUSTAKA Alfirman. Sukamto. and Bahri, Z (2019) “Perakitan dan Perbaikan Komputer Untuk Menaikkan Kualitas Remaja di Desa Pandau Jaya Kecamatan Siak”, Unri Conference Series, 1, pp. 08-21 Alfirman. Sukamto. and Bahri, Z (2019) “Perakitan dan Perbaikan Komputer Untuk Menaikkan Kualitas Remaja di Desa Pandau Jaya Kecamatan Siak”, Unri Conference Series, 1, pp. 08-21 Alfirman. Sukamto. and Bahri, Z (2019) “Perakitan dan Perbaikan Komputer Untuk Menaikkan Kualitas Remaja di Desa Pandau Jaya Kecamatan Siak”, Unri Conference Series, 1, pp. 08-21 Ardhana, V. Y. P. and Mulyodiputro, M. D. (2023) “Pelatihan E-Commerce dan Marketplace Bagi Masyarakat Muda Desa Dasan Baru Kediri”, Jurnal Pengabdian Literasi Digital Indonesia, 2(1), pp. 1–6. doi: 10.57119/abdimas.v2i1.19. Ardhana, V. Y. P. and Mulyodiputro, M. D. (2023) “Pelatihan E-Commerce dan Marketplace Bagi Masyarakat Muda Desa Dasan Baru Kediri”, Jurnal Pengabdian Literasi Digital Indonesia, 2(1), pp. 1–6. doi: 10.57119/abdimas.v2i1.19. Ardhana, V. Y. P. (2022). Evaluasi Usability E-Learning Universitas Qamarul Huda Menggunakan System Usability Scale (SUS). Resolusi: Rekayasa Teknik Informatika dan Informasi, 3(1), 1-5. Ardhana, V. Y. P. (2022). Evaluasi Usability E-Learning Universitas Qamarul Huda Menggunakan System Usability Scale (SUS). Resolusi: Rekayasa Teknik Informatika dan Informasi, 3(1), 1-5. Hal 53 dari 54 Hal 53 dari 54 Jurnal Pengabdian Literasi Digital Indonesia E-ISSN. 2961-9440 Volume: 2, Nomor 2, Desember, 2023, Hal: 49-54 Jurnal Pengabdian Literasi Digital Indonesia E-ISSN. 2961-9440 Ardhana, V. Y. P., & Mulyodiputro, M. D. (2022). Pelatihan Pengenalan Internet Dan Microsoft Office Bagi Siswa SMP Al Mutmainnah. Bakti Sekawan: Jurnal Pengabdian Masyarakat, 2(1), 71-74. Ardhana, V. Y. P. and Sapi’i, M. (2021) “Perancangan Aplikasi Keuangan Kampus Berbasis Web”, SainsTech Innovation Journal, 4(2), pp. 130–133. doi: 10.37824/sij.v4i2.2021.304. Ardhana, V. Y. P. (2021) “Analisa Quality of Service (QoS) Jaringan Internet di SMP Al Mutmainnah”, SainsTech Innovation Journal, 4(2), pp. 139–143. doi: 10.37824/sij.v4i2.2021.299. Ardhana, V. Y. P., Sapi’i, M. and Mulyodiputro, M. D. (2021) “Sistem Informasi E-Learning Berbasis Web Pada Universitas Qamarul Huda Badaruddin”, SainsTech Innovation Journal, 4(1), pp. 115–119. doi: 10.37824/sij.v4i1.2021.298. Ardhana, V. Y. P., Sapi’i, M. and Mulyodiputro, M. D. (2021) “Web Based UCloud Application Using CodeIgniter Framework”, SainsTech Innovation Journal, 4(1), pp. 126–129. doi: 10.37824/sij.v4i1.2021.296. Ardhana, V. Y. P., & Yulianto, A. W. (2018). Analisis Perbandingan Quality of Service (QoS) Wifi Universitas Qamarul Huda Badaruddin Terhadap Hotspot 4G XL. SainsTech Innovation Journal, 1(1), 1-5. Ardhana, V. Y. P., & Mulyodiputro, M. D. (2023). Analisis Quality of Service (QoS) Jaringan Internet Universitas Menggunakan Metode Hierarchical Token Bucket (HTB). DAFTAR PUSTAKA Journal of Informatics Management and Information Technology, 3(2), 70-76. Ginting, J.N. Jamry. and Ronny. (2022) “Pelatihan Perakitan Komputer Sebagai Dasar Pengenalan Teknologi Bagi Calon Pelaku UMKM”, Jurnal Abdimas Hawari, 2(1) Wahyu, T. and Noor, A.F. (2020) “Pelatihan Merakit dan Instalasi Komputer Pada Santri Yayasan Pendidikan Islam Pondok Pesanren Wali Songo Banjarbaru”, Jurnal Ilmiah Pengabdian Kepada Masyarakat, 5(4), pp. 383-387. Yuliati. Handayani,T. and Saputra, J. (2022) “Pelatihan dan Pembelajaran Untuk Meningkatkan Kualitas Pendidikan Siswa Tentang Perakitan Komputer di SMA PGRI Dumai”, Community Development Journal, 3(1), pp. 961-967 Hal 54 dari 54
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Equations of heat conduction with slow combustion
Bulletin of the Australian Mathematical Society
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EOUATIONS OF }{EAT COIVDUCTION !fiTTH by Graeme C. :/AI{E, I'i.Sc. (Vict. ) A Thesis subnitted for the degree of Doctor of Philosophy in Victoria University of l{el-1-ington Department of i',iathematics January ,L96?. January ,L96?. G) oomErus 'Ohapter J.i Ohap'ter 2. Chapter 3". :Chapter 4. 0hapter 5. Chapter 6. r0baBter f. Introducti,ou Ebd.eteace apdl lftr5.queneB6: of Solutior Contrmer5;son BeErll.te and tbefu ABpLLca,tS.ou to Spec5.a-l FrobJ.ene Bouu<lary Con0iti.ous anil the Bouad,ary Layer |[,he Space-AveragLag ProseEE eud Crl,tl.,cal. Etatee Uhg 0ut-off .!tob].ea Conelugj.one Appeadl.x Bibliograph.y 1 ? a6 4l 54 67 g5 8J 9X 'Ohapter J.i Ohap'ter 2. Chapter 3". :Chapter 4. 0hapter 5. Chapter 6. r0baBter f. Introducti,ou Ebd.eteace apdl lftr5.queneB6: of Solutior Contrmer5;son BeErll.te and tbefu ABpLLca,tS.ou to Spec5.a-l FrobJ.ene Bouu<lary Con0iti.ous anil the Bouad,ary Layer |[,he Space-AveragLag ProseEE eud Crl,tl.,cal. Etatee Uhg 0ut-off .!tob].ea Conelugj.one Appeadl.x Bibliograph.y 1 ? a6 4l 54 67 g5 8J 9X ER.RATA Ttre following are noted. ER.RATA Ttre following are noted. p.22, Line five. A better Proof can be given: p.22, Line five. A better Proof can be given: "suppose a> br then "suppose a> br then "suppose a> br then =|i{1 = tfi *B-ld*' s EB-l ror sone t e (b'a)' where h - a - b.. I'hen the expreeeion is lesa than or equal ,*B-1.r' where h - a - b.. I'hen the expreeeion is lesa than or equal ,*B-1.r' ,*B-1.r' p.23 . Line four from bottom. Reverse inequality' p,30 p.31 . Line nine, ehould read ... "For t large and negative' p'(ttt "' Figure 3.2. ltre eingularity ie stable, sd thus the arrowa s'hould be directed towarde the origin and not €retay from it' p.44. eline three from bottors' Refere to a note on p'5 and not p'6' P.45 Line eix. A negative eign instead, of a poaitive sign should be ineerted before the last terre in GZ. i p.47. P.48. i t Equation (4.8). omit tt8'r, also from u "-!t 18nlr' -'"tt' Equation (4.9), should read'r 6 = 1(4zlnl - 2'2 - z'3lznl."')"' p 61-2 . Equatione (5.L2)r(5.14). Omit ttat' fron both equationa' .p.61 2 p.77. Equation (6.18). The first terrn on the right hand side ahould be ,, 1 1 -3 rt not rt lrn . 2, p.79 . Line fourteen, ehould read ... tbe chosen ae the poeitive alternativet which ...tt i fi i i P.79 . Bottom of page. The eecond contour choeen cLearly gatiefies ttre eriterion iryoeed. Ttre for:n given for the correEPonding aolution ie that obtained by Letting the radius of the circle about the originr 3ay e' tend to zero' Ttris does give a solution of the differential equation (6'25) since the eecond derivative of G is unifornly convergent as e + or for each e ) o' the detaile have been ornitted. -2- -2- Line two from bcttom. Insert the curly bracket before e-r8 p.7g. Line two from bcttom. Insert the curly bracket before e-r8 and not after in the integrand' p.7g. Line two from bcttom. Insert the curly bracket before e-r8 t ft i th integrand' p.7g. and not after in the integrand' and not after in the integrand' p.SB.Linefivefrombottom.Thisehou]'dread...''thenobviously lo 1is an equicontinuous set cf functions".'t' Lnr p.SB.Linefivefrombottom.Thisehou]'dread...''thenobviously lo 1is an equicontinuous set cf functions".'t' Lnr p.SB (u) AsStlrAo: A study is:ade of the equations of heat coaduction wlth sl-ow combustion. A nathematical nod,el- is established from an interpreta- tion of the physical raodel, r+ith a few sinpJ-ifying assumptions. ftris gives rise to a coupled pair of partial d.ifferentiaL eguatious which are the d.irect concern of this thesisr fhe depend.ent variables being the tenperature and reactant concentration as functLons of position and. tine. The modeL is shown to possess a unique solution for which some properties, such as Lipschitz conclitj-ons etc. , are establishedl. An j-nvestigation into the use of a conparieon theorem j-s givenr in which it is shcwn that no ilirect comparison theorem is i,oesible for this and related systerirs. However, it is also shown tbat it is possible to obtain upper and. loirer estimates by appeal-ing to tbe physical model. .il, d,iscussion of the bound.ary layer is given and this is fol.l-owedl by a cletail ed d.iscussion of stabiJ-ity. fhe latter has been one of the main concerns of earLier authors oa this systenr. Iheir use of a space-averagiag process to establish a criterion for stability i6 al-so discussed, Frobably one of the nost interesting features of this system ie the subclass of problems for which the reactaat is exhausted in a finite tirne, These hare been named the 'Icut-offtf problems and. they can be tikened to the free bound.ary problens in fluict ctynarulcs. R discussion of the cut-off prob}em is given with particuLar exampl-ee cbosen to il-lustrate the nain features. (ri^l,) ghis theeis, contailo ao na,teri.af. ubi.ch has been aeceSrtoil f,or the .award of any other degree or ct5.pIoaa ia any Ualversity and to the be,et sf st bowledge aad, bettre-f , the the.s1s contel.as ao naterlal previouel,y pub-ltehedl or rrritten by' anotber pereoa, except rbere ,ilua refertence is nade j.:c. the tert of lihe thesfls. $.&,'Fj'Ln. G.C. WaJEe,. $.&,'Fj'Ln. $ , j G.C. WaJEe,. Chapter 1 Chapter 1 1. ACn_rourrDqF{$rfg I't ls a Bleasune to thaflk qr srlper&igora, FFofessor A..G. X{ae&1e, asw of the Uatverel,ty Of $lanylgad, ,a'11dL.!ft. A. Mc$abb, Appltefl Matle- natics, Diviei.bn, Departneu,t of Scieatific aad IsduetrJ.al Reaearo-h, I{err Zea1aud' f,-or thel,r Encoirragqagntn hel.pfu]. advicel and orLtl,sd.en theSr have g$se-B fihrougl,out tbe dluratf.oa of, thle wOF,k. I grate,f,ully ac&nowledge tbe ouBpont of, VictorLa UrLver'e{.ty' of k{sxlington f.sl ao sw.and unden thetr lleaoh1qg F,e].lelrshlp Sch,eme, G.0. Walse. INTRODUCTION This thesis j-s concernect wlth the cliffusion of beat 3-n a given naterial which is undergoing internaL combustion. The nateriaL con- tains reactant which is consumed by this conbuetion, and heat is produced. ft is assumed that both the thernal conductivity and cap- acity of the material as a whore are unchanged by this proces6. Tbe heat thus produced, is conducted through the material and so gives rise to a corresponding tenperature increase and. .Gnhances the react- ion rate. It is the intention of this thesj.s to set up a mathematical model for this phenomenon and to show that there exists a unique solutioa' which does in fact represent the temperature aud reactant concentration. Considerabte attention wilL be 6iven to particular examples which ilrustrate many of the qualitative a.speete of the problen. Recently the problem has received considerable attention and an extensive literature on this and related problens has accr:mulated. A comprehensive account of some of the earlier work is given in Frank-Kamenetskii 1. This work is confined to reactions in whieh the rate at which the reaction proceed.s is independent of how much reactant is present at any time (such reactions a.re carLed zero order rea.ctions). This problem, by no means a triviar- one, is nonetheless a much easier one to solve. For there is now just one dependent variablet that of the tenperature which is obtained as a fuactioa of position and tine. However in the general case, in which the 2. reaction rate does depend on the reactant concentration (say to the nth power), there is a coupling effect in the differential equations. That is, there are two dependent variables, tbat of the tenperature and reactant concentration, both of which appear in eacb tlifferentj-al- equation. Frank-Kanenetskii derlves the steady-state solution for a zero order reaction in one di-nension and examines the condi-tions under which it is possible to obtain such a sol-ution. The extreme conditions under which this is possibre are ca1Ied the crltical cond.itions. chanbre 2 coatains a similar treatment for two dimensional problems. Later work 314 u"r been generarised. to a nth order reaction, i.e. one in which the reaction rate d.epeads on the ath power of the reactant concentration. Both of these authors have used space-e.v€raged. tenperatures, i.e. have converted. what were partial differential equations into ordinary differential equations J-n time. The author lras introduced. I Reference to equatlons chapter number associated in the same chapter wi-th the nunber of will not include the the equation. INTRODUCTION to this problen in connection with the self-heating of woor-. This has been a subject of study for a number of years in New Zealand, and the prevention of spontaneous combustion in wool has an obvious relevance to the export trade of this country. Many experinents have been caried out to d.eternine the tenperature rises that actually do occur under various conditions. The results of these and nany assocj-ated calculations are avaj-lable in walke" et "1 5'6: Different shapes are used., for exanple cylindrical, spherical etc. The difficurty of obtaining solutions for these shapes generally increases with the geometry of the material. This is seen to be so, for in the case of a aero order reaction, such as that discussed by Frank-Kanenetskii and Chambre, no equivalent exact solution can be obtained in three dimensions. rn wake aad walk et ? , use is mad^e of the numericaL soJ-uti-on of the problem for a sphere to calculate the central tenperature rises in geometries which are mathematicalJ-y less tractable than that bf the former The approach presented here wilL be more qualitative, though many examples will be given. After setting up the model, further conments will be made on the space-averaging process. ( See Chapter S). I.1 Definltion of the Mod.el The rate at whicb the reaction proceeds is measured, by the rate at which the reactant is consuned.. rf I denotes the reactant concentration as a function of posj_tion (x) and time (t), then *i - - g(x'e'\)' . . . , (1.1) . . . , (1.1) where o denotes the tenperature. The fact that this rate ls aleo a function of position reflects the possibirity of a catalyst being present within the material. An nth order reacti-on is one in which T a term It occurs in the ri-ght-hand side of equation (1) . rn much of the literature use is made of the Anhenius equation .N -BA - AA € , 0l= at h A dB .... (t.z) .N -BA - AA € , 0l= at A dB .... (t.z) .... (t.z) constants. whereAandBare constants. whereAandBare constants. whereAandBare I, The rate at which heat is produced per unit volune likewise can be written a = F(xrorl). a ( ) ( ) This quantity Q is d.ireetly proportional to 9+ . INTRODUCTION The factor of UL proportionality is, in general, a function of temperature, since the amount of heat produced by a given amount of reactant depends on the temperature at which it is consumed. However, the authors previously rneatiorr"dJ'4 h"rr" implied that e is in fact equal to a constant nultiple of + . Therefore it nust be recognizecl that OE in writing in writing a = -"*f ..,. (L.4) a = -"*f ..,. (L. a = -"*f ..,. (L.4) ..,. (L.4) where a is a constant, that an approxiraation has been made. where a is a constant, that an approxiraation has been made. To be preciee i-t is assumed. that this proportionality faetor can be regard.ed as constant for the temperature range concerned, This assumption will- be nade with many of the examples which are discussed. herein, but it is to be rememberetl that there i-s this restriction to the fittlng of the analysis to any particular problen. The neclium is assumed to be of q dirnensions, that J-s, x denotes a vector in the cartesian space of that dimension. The heat produced by the conbustj-on of reactant di-ffuses through the nedium according to the equation . . . . (r.5) kao + e = cft, kao + e = cft, . . . . (r.5) wb'ere k and c are the thermal cond.uctivity and capacity of the nnaterial respectively, both being constants. a is the r,aplacian in q dinensions, i.e. + ao = 1 d20 fid*+"'...(L.6) + ao = 1 d20 fid*+"'. 5, Equation (5) can be wrLtten Equation (5) can be wrLtten Equation (5) can be wrLtten ].Ae = tc 0t f(x,orI) t . . ' . (t.7) AE wbere !- = k/C, is the thermal diffusitivtty of the material. Tbus there is the coupled pair of partial differential equatione (f) and Q) to be solved with prescribecl initial and bound.ary coaditions. Obviously the initial reactant concentration and temperature varla- tion can be specified. Further a boundary condition on the tenpera- ture and its first derivatives with respect to x is gj-ven. The latter corresponds to whatever physical condition is imposed on the surface of the naterial. The most usual of these are the following: . . ' . (t.7) of time. l. Prescribed surface temperature, which nay be a function of time. l. Prescribed surface temperature, which nay be a function 2. INTRODUCTION Thermal insulation ae which means ffi' = O denotes differentlation i.e. zero heat fl-ux on the surfacet on aII points of the surface. 0/0N in the direction of the outward normal- to the surface. 3, Radiation at the surface, where the flux across the surface is assuned to be proporti-onal to the temperature difference between the surface and the surrounding nedium, i.e. 3, Radiation at the surface, where the flux across the surface is assuned to be proporti-onal to the temperature difference between the surface and the surrounding nedium, i.e. .00 -, kEF + h(O - eo) = O on all points of the surface. The quantity h j-s called the coefficient of surface heat transfer. As h'+O this tends to the boundary condition 2, and as h+@ it tends to the condition L. 3, Radiation at the surface, where the flux across the surface is assuned to be proporti-onal to the temperature difference between the surface and the surrounding nedium, i.e. .00 -, kEF + h(O - eo) = O on all points of the surface. The quantity h j-s called the coefficient of surface heat transfer. .00 -, kEF + h(O - eo) = O on all points of the surface. The quantity h j-s called the coefficient of surface heat transfer. As h'+O this tends to the boundary condition 2, and as h+@ it tends to the condition L. If the rate of reaction does not d.epencl on the reactant con- centration then an immediate sinplification occurs. nquation (?) 6, becones a quasi-linear equation for the depend,ent variable O. fhis is the case investigated in Fraak-Kanenetskiil, with tbe rate of heat procluction given in the Arrhenius equatioa (Z) witb n = O. No attenpt was made in tbis work to dj-scuss the inpJ.icatione of the reactant be- coming exhausted.. The reason beiug of course, that Frank-Famenetskil- wae intereeted i-n the critical state and hence in tines long before nuch reactant was consumed.. Eowever, consid.eratlon will be given to this problem here (with general n) and it vtrtII be seen that a criterj.a for esbablishing the types of reactions for which this doee occur can be obtained. In the uext chapter, the exi-stence and uniqueness of the solut6on J-s proved. This is fol-l-owed. by a discussioa of comparison functione and reference is made to the boundary layer in chapter 4. INTRODUCTION An expran- ation l-s offerecl of the work of ThonasJ and nnfg4 (in whicb the idea of space-averaged temperatures waa exploited) iu cnapted 5, where a d'iscussLon of criticaL states is given arso, Final1y, a detal_Led discussi-on of a particuLar cut-off problen is given in chapter 6. 7. Chapter 2 EXISTENCE AND IINIQUENESS OF SOLUTION 7. Chapter 2 Chapter 2 EXISTENCE AND IINIQUENESS OF SOLUTION Not all- differential equations have solutious. If a neaningful physical- problen has been comectly fornuLated nathenatically as a differential equation, then i-t should have a solution. Thus the question of exi-stence arises. Further if a solution iloes existr wbat of other possible solutj-ons? And so the question of uniqueness arises a16o. It ts intend.ed. to establish the existence and uniquenes€ of the solutions under conditions to be prescribed, and. deduce certain prop- erties of these solutions. The equations to be discussed are, of course, those of (t'L) and (1.7) but it is intended to write these more general].y so as to include more general situations, There are bowever, certain requirenents which are suggested by this particuLar problem, as described in Chapter J.. The functions f(xror\) ana g(xrOr\) are required to be bounded functions of their argunents over any finite range. They wiLl, also be continuous functions of their arguments, with the exception of a zero reaction, if and when the reactant concentration (l) became z?too In this case f and g will have finite d.iscontinuities when )t = O, since obviously no heat can be prod.uced and no further reactant cao be con- sumed once thls has occurred.. The differential- equatioas wiJ-3- be written a€: I q, r(o) = f ",r(x,t)3.1q Lf, ' oxroxt IC/ ti(*,t)oo d dxi B(x, t rgr\ ) + c(xrt)o - ae 0t aaa f (xrt ror\ ) . (2.1) OI 0t . . . . (Z,Z) 8. The d.onain of the space varj-ables wiLl- be denoted by B. This describes the volume in q d.inensions occupied by the nedium in which the equations (2.1) and (Z.Z) apply. It can be seen that the above can be specialized to the problen as posed j.n Chapter 1 by writing lgt as t' -g for g1 l tti 6ij ll i'j The d.onain of the space varj-ables wiLl- be denoted by B. This describes the volume in q d.inensions occupied by the nedium in which the equations (2.1) and (Z.Z) apply. EXISTENCE AND IINIQUENESS OF SOLUTION It can be seen that the above can be specialized to the problen as posed j.n Chapter 1 by writing lgt as t' -g for g1 "tj = 6ij , all i'j b+ - O r aJ-Li c=0i and supposing that f and g are independent of time, One reason that the equations are wrj-tten in thj-s nore general way, is that this r^rould be the first step to generalizing to a more realistic situation in which the thernal properties of the mediun depended on the temper- ature and reactant concentration. The source terrn (-f) genera1-J-y wiJ-1 be positive as the conbustion ie exothermic. AIso the source will- be a nond.ecreasing function of temperature, since the rate at which heat is produced increases with, or is unaffected by, temperature. Further the source w"iJ-J- be non- d.ecreasing with the reactant concentration, for it usual.J'y depends on )r lik" In where n)7 Q, SinilarJ.y g wil-l be aegative for the reactant concentration decreases as the reaction proceeds. Also the reaction rate (-g) is nondecreasing with tenperature and reactant concentration. Hence in this particular problenn, the functions f ".od g in equations r5 (f) and (20 wilL be nondecreasing in g and I. Thj.:s/a very usefuL fact for it is possible to generate a sequence of bounds for the quantities o and|. For, if an upper bound i.s inserted for \ :.n equation (1) (i,e. more reactant is supposed. to be present than there is in reality)1 then there is a greater source and hence an upper bound is obtained 9. for o. Likew"ise if a! upper bound io substituted for 0 in equation (2) (i.e. the temperature is supposed to be hotter tban it actually is), then there is a faster consumption of reactant and hence a lower bound is obtained for I This approach can be used to denonstrate the existence of solutions to the equatlons. However, though it is intencted to discuss this further, a mucb Iess restrictive proof can be given. A very sinilar system is exanined in ltcNabblO in which f was required to be nonincreasing in I and g nondecreasing in O. McNabb set up comparison theorems (which will be discussed later) and using these, he established the existence and uniqueness of solutions with these conditions. EXISTENCE AND IINIQUENESS OF SOLUTION An apparently more geaeral system is discussed in zeragi;alI, that is a system of n coupled parabolic partial differentiaL equations, t+t - Aui = fi(xrtrrr, ..., uil) r . . . . (2,3 0t for i = 1r ...1[1. Zeragija hae set up comparison theorems and d.educed^ the existence and uniqueness of the solutions of the equations (l). Eowever, the conditions which he has imposed on tbe functions f; , viz, lq(xrtrur, ...ru.) - f;(xrtr[rt , ...rul)l ( Klui - ufl , inpl.y that 1 is independent of each u. for i / j. Tbis meane that the system of equations are in effect uncouplecl and the results are nerely applicable to the equation 0u;i Au = f (x, t,u) . 0u;i Au = f (x, t,u) . 10. 2.1 Pre].l-ninarY Definitions Let B be a bounded q-ctirnensional domain of real variables ancl r. - 1 DT = f (xrt) : xCB, O<I<TJ . Further denote the boundary of B by dB and S* as the cylinder l(r,t) I x€aBr O<t(T] If u(x,t) is dpfined in a given domain D, then for 0 < a < 1 the followlng clefiaitions will be made: clefiaitions will be made: = Eup lu(xrt) I r (x't)eo unDto) = lu(P) - u(a)l , fae,ql]" + aot"l where l"ll l"ll sup PrQ€ o l"lf . . . . (2.4) ....(2,5) ....(2.6) . . . . (2.4) unDto) = lu(P) - u(a)l , fae,ql]" + aot"l here, l"ll sup PrQ€ o l"lf where, where, d(PrQ) = (lx-x, 12 * lt-t,l)L/z n if P= (xrt) and g= (xrrtr). d(PrQ) = (lx-x, 12 * lt-t,l)L/z n if P= (xrt) and g= (xrrtr). n n;-(u) is cal,led. the Edlaer coefficient ofuiaD. n n;-(u) is cal,led. the Edlaer coefficient ofuiaD. If u is <tifferentiabl-e, define If u is <tifferentiabl-e, define (z,z) . . . . (2.9) ....(z.g) l"lrfo Iolrfo l"ll where, r.D(u) - 6up lu(P) - u(Q) | PrQ€D lx-xf l t lt-tt; t = l"lD* + = l"l ,fo + = l"lf + # l:s; .{l*={" r,D(o), + l*tf ' nn r.D(u) - 6up lu(P) - u(Q) | PrQ€D lx-xf l t lt-tt; l t ""' r.D(u) - 6up lu(P) - u(Q) | PrQ€D lx-xf l t lt-tt; it is said that u is of class CP. rr lu rl "o".r, -;""' "p f.D(u) is the tipschitz coefficleat of u i:l D. rf ueCI ancl ou,/0q € cI for aLL i, it i id th t i f rr lu rl "o".r, -;""' "p f.D(u) is the tipschitz coefficleat of u i:l D. f I f i it is said that u is of class CP. tben it is sai-al that u GC?, Note evertrnrvhere clifferentlabl-e. rr lu rl "o".r, -;""' "p f.D(u) is the tipschitz coefficleat of u i:l D. rf ueCI ancl ou,/0q € cI for aLL i, ue Cl does not inpl.y tbat u is A surface S is saial to be of cLass CP tt S can be LocaLly repre- LL. LL. sented in the forn *i = X(xrr ..r xi-1, *i-I, .., xnrt) . . . . (z.rr) for some i and the functions X are of class CP. 2.1 Pre].l-ninarY Definitions Tbe surface S is covered by a finite nunber of neighbourhoods s- each of which has a fixed. global representation of the forn (ff) witU X of class gP. The quantity l"lf iu then interpreted as the maxinun of lul', -!i taken over all these neighbourhood's. The followi-ng assumptions are made on the partlal- differential operator L(9) of equation (t). I. L is of paraboli-c type in F*. That is, there exlsts a positive number Ho such that for a1l- (xrt) € il, and any rear- vector { , K^ii(i757, ,41r'. I. L is of paraboli-c type in F*. That is, there exlsts a positive number Ho such that for a1l- (xrt) € il, and any rear- vector { , K^ii(i757, ,41r'. ( , and in addition a. . are of class CI. r-J If. "ij, bi, and c are of class cc (for sone o(c(l-) in D-1, ( If. "ij, bi, and c are of class cc (for sone o(c(l-) in D-1, and in addition a. . are of class CI. r-J Tbe functions f and g will be required to satisfy the Lipscbitz condition for each (xrt) in 5r, ln(x,t,o1,It) - h(x,t,02,I2)l< M(lor - e2l + l\ - Iall.. . (2.r2) Further they wilJ- be required to satisfy the Lipschitz condition wilJ- be required to satisfy the Lipschitz condition Further they wilJ- be required to satisfy the Lipschitz condition Itr(x,t,gr\) - rr(x'ttrgr))l < K(lx - xrl + It - trl), . . . Q.:-l) for each fixecl value of O andl. In (fz) aaa (l-f), h E f or g. fhe initial and boundary conditions of the tenperature aad Itr(x,t,gr\) - rr(x'ttrgr))l < K(lx - xrl + It - trl), . . . Q.:-l) for each fixecl value of O andl. In (fz) aaa (l-f), h E f or g. fhe initial and boundary conditions of the tenperature aad reactant concentration are pree cribed. Siace tr satisfies a first order d.ifferential equation in t, the initial value of .\ ls specified when t = O. The temperature O, whieh satisfies a second order L2, partial differential equation, wil-l- need to be specifled oa 0B (t > O) as well as on E ioitir4y. These conditions, which physically are just what can be fixecl, a.re sufficient to shotil that there exists a unique sol-ution. It is assumed that there existe a function? 2.1 Pre].l-ninarY Definitions in D, whlch coincides w'ith the boundary conditions Oo(xrt) on S* and. E at t = 0. The inltial. concentration of rea.ctant is r*'ritten aE )o(xrO). Firstly it is intencted to show thaf,, provicled there does exlst a sol-ution of the systen (1) ana (2) with these boundary and initial conditions, then it is unique. 2.2 Uniqueness of the Solution This wtll be approached in the usual way of assuming the existence of two solutions O, r \, ana "Zt \Z and showi-ng that these are the s&[t€. are the s&[t€. Theorem I. Suppose Theorem I. Suppose (a) or_, trI, o2, )2 erist and are continuous in F1,r (b) their second order xi-derivatives and fi-rst order t-derivatives exist and. are uniformly bounded ia D1r (a) or_, trI, o2, )2 erist and are continuous in F1,r (b) their second order xi-derivatives and fi-rst order t-derivatives exist and. are uniformly bounded ia D1r (a) or_, trI, o2, )2 erist and are continuous in F1,r (c) t(oi) = f(xrtrgir\), uIi dt- = g(xrtreirli), of = oo(xrt) on sn and on E at t = or of = oo(xrt) on sn and on E at t = or \i = Io(x,o) onEatt=or \i = Io(x,o) onEatt=or for i = 1r2. \i = Io(x,o) onEatt=or L7. (A) f and g satisfy a Li-pschitz condition as j-n equation (12) i then O, = O, attd )r- = \r. then O, = O, attd )r- = \r. Define o, = 01 t remt and I" = ), + sent where rrs are arbitrary constants and m isrsorils number greater than 2M. (M is the Lipschitz constant defined. by (fZ) ). Then since 01 = O, and \f = \, when t = O, unless o, and Orr tr1 .oA \, coincide for t ) O, o, and \2 must intersect some members of the fanil-y of surfaces o,. and )u. Hence there exists a tr such that lO, - Orl ( lO" - orlr lI2 - trrl at (xtrtt). In the first case choo€e r> O and lsl = r. Then at the point (xtrtt) it can be showu by an argunent usiag a theorem of Paraf and Fej6r (as in B"ter"nlz), that r(or) < r,(o"). This neans trrat r(Or) ( i,(o.) - "rutt'. That is, Proof 3 rutt . T,(e2) - f(xr rt'ro2r 12) < M(lo2 - oll + lIz - \11) - "ru't' ( ( (tt(r + lsl) - rn)ent' i.e. t(92) - f(x'rtrr02r12) i.e. t(92) - f(x rtrr02r12) since n > 2M this contradicts the assumption that orrl, are sorutions of the equations. rn the second case choose s >o and lrl = 6. Then at the point (x'rtr)n since Ia ( tr. fo" t S trr alZ ) 0I. . ff-, F is contradicts the assumption that orrl, are sorutions of the equations. rn the second case choose s >o and lrl = 6. Then at the point (x'rtr)n since Ia ( tr. fo" t S trr alZ ) 0I. Theorem I. Suppose . ff-, F That ls, ru"tt t z 6trr . Eence, dt That ls, ru"tt t z 6trr . Eence, dt 0I., E - - g(x"t"o1r\1) ( e(*f 't',92r12) - g(>crt'no'r\) _ msemtl 14. 14. g(xtrt t, elrl1) ntl mse g(xtrt t, elrl1) ntl mse g(xtrt t, elrl1) ntl mse ( (n(lrl + s) -te)"tt' ( (2M -r)""ttt. Againt since sol-utions of argument can two sets nust n)lM this contraciicts the assumption that the equations. Eence no such tr exi"sts and be carried through for sufficiently sna1l. r coincide. That is gt = OZ and \1 = \2' O1r )1 are since the aad st the This conpletes the uniqueness theorem under the conditioas as stated. It should be noted. that the requirement (cl), viz' that f and g satisfy a lipschitz condition in O ancl \ , excludes the case of a reaction of order n where O(n<1. In this case the functions will not satisfy the Li-ps{4itz condition as \+ 0. For examplet f =g= X*e. However in such cases, this difficulty is resolved by red'efining f and g in the neighbourhood of I= Or say for \( € I so that they do in fact satisfy a Lipschitz condition in O and \ evertrnlhere' Then call the corresponding soluti-ons I, ana \g (which by the above will be unique) ancl d.efine O and \ "t the Linits as t+O . It has to be shown, of course, that these Ii-nits satisfy the differential equat- ions and, in fact, that they exist. This wil-l be shown in section 2.4, after the problen bf, existence has been investigated. 2.3 Existence of the Solution Before the existense proof can be given, three theoremsr which form the basis of the existence proof in this section, are required. 15. The first two of these are proved in Friedmanu. Theorem 2. Assume that the lateral boundary Sn of Dn ie both of classes C2 a.nd "2+u (o< q, <1), that L satisfiee the conditions I and rf, and that /(xrt) is loca11y Holder coutinuous in Dn. Assume finally, that thenexists a functio\f of class C2+e io Df which coincides w:tth the given boundary conditions Oo(xrt) on S, and on B at t=O. Then there exists a solution of the systen r(e) = 6(x,t) for (x rt ) in o, , g = go(xrt) on S* and' on E at t = o. Furthermore, tbe solution is of class cl*F io D, for any o<B<1 and of class c2+[ io il* for some f,>o. g = go(xrt) on S* and' on E at t = o. Furthermore, tbe solution is of class cl*F io D, for any o<B<1 and of class c2+[ io il* for some f,>o. Theorem J. Assume that Sn belongs to both C2 and. ,2+a' and that I satisfies both I and II. Let /(xrt) be a continuous functlon in =D* and 1et 9(xrt) Ue a solution of the system L(o) = /(xrt) in DT, Theorem J. Assume that Sn belongs to both C2 and. ,2+a' and that I satisfies both I and II. Let /(xrt) be a continuous functlon in =D* and 1et 9(xrt) Ue a solution of the system L(o) = /(xrt) in DT, =D* and 1et 9(xrt) Ue a solution of the system L(o) = /(xrt) in DT, O = ? onSl andonFatt=0. Then for any O ( 6( 1, there exists a constant P dependj-ng on3-y on 6, the operator L, and the donain Dn such that lulr.*u ( p|il lo t l?12) . O = ? onSl andonFatt=0. Then for any O ( 6( 1, there exists a constant P dependj-ng on3-y on 6, the operator L, and the donain Dn such that lulr.*u ( p|il lo t l?12) . Using these two results it is possible to prove the existence of solutions to t(o) = rlr(xrtrg) . .. . (2.r4) with the same boundary conlitions. In acid.ition to the requirements inplied fron Theorens 2 and ], the function l, is requlred to satisfy a I,ipschitz cond.ition in 0, i.€. 2.3 Existence of the Solution t(o) = rlr(xrtrg) i h with the same boundary conlitions. In acid.ition to the requirements inplied fron Theorens 2 and ], the function l, is requlred to satisfy a I,ipschitz cond.ition in 0, i.€. 3-6. I tl(*,t,er) -t(*,t,o2) | < ulor - 9el for al-I (xrt) in D1. Ancl further it is assumed that there exists two functions e and 6' which are continuous in Dr, and satisfy the inequalitiee r(g)-1,(xrtr9) tto>, t(6) t(*,t,d), ....(2,t5) Q(xrt) ( eo(xrt) ( d(xrt) on t1 ancl on B at t=o. r(g)-1,(xrtr9) tto>, t(6) t(*,t,d), ....(2,t5) Q(xrt) ( eo(xrt) ( d(xrt) on t1 ancl on B at t=o. The proof given in Append.ix I uses the nethod of successive approrimations. The sol-ution is shown to be of class Cl+p fot "oy O < P < I and of class C2+T for some T > O. The third of the theorems necessary to prove the existence for the system, involves the rate equation (2). That is the existence of a solution to the systen 0l * = f(x'tt))t \ = \o(xro) on E at t=o, is required. under certain conditions to be prescribed. Note that x is nerely a parameter as far as thJ-s system is concerned, The statement given here is due to Coddington and Lu.riosool4. Theorem 4. Asslr.ne I t. continuous in t and I . Then there exists a solution of the systen is required. under certain conditions to be prescribed. Note that x is nerely a parameter as far as thJ-s system is concerned, The statement given here is due to Coddington and Lu.riosool4. Theorem 4. Asslr.ne I t. continuous in t and I . Then there exists a solution of the systen OT 't = 1G't'))' \ = \o(x,o) oo g at t=or on Bome t i_nterval i such that | \(x,tr) - I(x,tz) I No nentioa is made of the behaviour of the solution with x. This is given in the foJ-lowing resul.t:- Corollary. If \(xrt) is a solution to the system ia Theoren 4n tben, L7. frovf&ng f t,s o.f sf.as6, Llpscbits eonctition) and i.s atr6o of ela-Es. gx ia +. tlonain. ca l,n x and X Iu(xnO) :is o'f, Frultber \ (i.e. 2.3 Existence of the Solution eati-eflee a 1 clase C- in x, I is b"ounded i.n the 3EE{,: fi:on Tbeorea 4, taking tbe sol-ution at enI two pointe x and x'r t it ean be seelr that $(x,t) at $(xf t) E f(*rtrI(xrt)), t t X f )) $(x,t) at $(xf ,t) 0t E f(*rtrI(xrt)), = 9 txrrt, Xxf rt)). Define h(t) = Xxrt) - ),(xt'rt). Then fron abov'e Define h(t) = laul h-Tl = i'€' I*tl Xxrt) - ),(xt'rt). Then fron abov'e l$tx,+rXx,t)) 9(o',1, Xx',t)) l, \1"-x'l + Mlhl Define h(t) = laul h-Tl = Xxrt) - ),(xt'rt). Then fron abov'e l$tx,+rXx,t)) 9(o',1, Xx',t)) l, laul h-Tl = l$tx,+rXx,t)) 9(o',1, Xx',t)) l, i € I*tl \1"-x'l + Mlhl wher',e l\ *d M a,re t-he Lipeoh:lts consta,ate for f(xrtr]). Eheu b;roonsidering n $ O oeparately aod J.nteg at,ilrg the laet resul-t, it caq be shown that lhl ( Mrfx - xrl. That is, ll(*rt) - I(x',tll{ utlN - xrlr for dus xrx-rl uhich:is the required restilt. The secoad resu.It ie obtaineil f,ron tahi.ng the LipechLts eoa.GlLtl,oa torf, f,$(xr1,11 - Al { Idlil , rtr}rene L og,(xrtro). Fe.DG€r l$il ( lnl + ul)rl . Asaiq, by corrsideit,nslf o sepanatery ar*t integrat!.ng, uppef ead lor,rer bourds afe sbtained fof l. It is now po€6ibLe to prove the aain resdlt of tbis sectioa. The proof whLch is given in Appendtx 2 was tsotivated by the :remrks nade erarlLer la this chapter. r[,n lt,eratj.ve ,BGheile, is, ,Fet utrl aad 18. it is shown that the sequences so obtained converge to the solutions of the system given tn (1) anct (2). A fundamental step in establiehlng the result is a theorem proved by Ascolj- i^rhich (as in Cod'dingtoo and Levinson) is stated here as a Lernrna. This result enablee us to say that there exists a linit to the sequences so obtained' Ascolirs Lerma. On uniformly boundedt a bounded domain D' tet O = le] ue an infiaite, equicontinuous set of functions. Then @ contains a sequence tUJr r = Lrzt.., which is unifornly con- vergent on D. one restriction that has been applied, a physical one taken from the real problen, is that the reactant concentration is con- fined between its initial concentration and zero. 2.3 Existence of the Solution Al1 this is saying is that the reactant is being consuned as time proceed.s and can only be consu:ned as long as the concentration is greater than zero, Hence I w'il-l be negative, and zero when \ it u""o. This requirement can be relaxed though it neans a further difficulty with obtaining uni-form bounds for the iterative solutions for \o. This i-s however, sinple to resolve, though this is not done in the proof given in Appendix 2. 2.4 The Cut-off Problen It will be shorun later that for a reaction of order less than unity, the reactant wi1-l be exhausted in a finite time. For such reactions it is clear from the examples given previously, that the functions f and g will not satisfy the Lipschitz condition in the L9. neighbourhoocl of \ = O. So it is necessary to show that there existE a unique solution when this condition is rel-axed i. €. whea the react- ion is of order less than unity. In a. reaction of zero order both f and g will be discontinuous on\= o. rt will be shor,ra that \ is of class cl as before, but g wiLl not be of cLass g2tc rrorfornly in Dr. Tbe reason for this linitation j-s the discoutinuity in f on ) = g onJ-y. It will be assumed here that heat is being l.lberated only, that is the reaction is exotherrnic. The physical- situatiou from which thie analysis arose did' in fact ensure this, but the results tn tbe previous sections can be applieil to endothernic reactions a].so. The problen wilL be approached by redefining the function f for \q€as foLlows: = f(xrtre)), \)€ fg(x,trer\) = 4rlCrrt, - \)f(xrtrer\), O.<\€ € = 0, I(O where €' is smalL. rhe variati-oa of fg wr.th\for any fixect (xrt) in D1 and any value of o io oonparecl with that of f ln Fig , z,L, 20. Then tl.ith the assumptions of the previous theoremsr define Then tl.ith the assumptions of the previous theoremsr define 0\ fr = g(xrtrdr\t), L(on) = fe"(x rt ro111\n) I S = g(xrtron-l,Xr), 0t with On = e6(xet) n7,Lr oB Sg and, on 17, L n = 2r7r.,. E at t {, and \j - \o(xrOf n77I1 on E at t d. Further (t.) l-e a noaotone decreasing seguence of positive numbers such that (tn)+O as n+e. For each a, there exists a Op(xrt) and )n(xrt) in D-1 6uch that 9r, is of class g2*Y (I>O) in D1 and lo(xrt) is continuous in d1. Since heat is being liberatecl only it follows that Oa) 06 and hence es will serve as a lower borrnd. 2.4 The Cut-off Problen Further, since fron the main result (in Appenclix 2) it was assumed that there exists a d(xrt) such that L(on) = fe"(x rt ro111\n) I 17, L S = g(xrtron-l,Xr), 0t with On = e6(xet) n7,Lr oB Sg and, on n = 2r7r.,. E at t {, and \j - \o(xrOf n77I1 on E at t d. Further (t.) l-e a noaotone decreasing seguence of positive numbers such that (tn)+O as n+e. For each a, there exists a Op(xrt) and )n(xrt) in D-1 6uch that 9r, is of class g2*Y (I>O) in D1 and lo(xrt) is continuous in d1. Since heat is being liberatecl only it follows that Oa) 06 and hence es will serve as a lower borrnd. Further, since fron the main result (in Appenclix 2) it was assumed that there exists a d(xrt) such that o 6', and as fe >. f , it follows that Cr, ( d in D-1'. That is the same e serves as a.n upper bound for the sequence of functlons (en). From the ostinates of Fried- man given in Theoren ], it al-so fol_lows that it follows that Cr, ( d in D-1'. That is the same e serves as a.n upper bound for the sequence of functlons (en). From the ostinates of Fried- man given in Theoren ], it al-so fol_lows that lonlr+o As both the gars and \ots are bound.ed in D1 (oo( oo( 6, o (\n(\o), then oo and. dorr/Oxi are HtiLder continuous of exponent 6 for a1l i. This means that the set of functions (orr) are equicontinuous on l-g and sot by Ascolits lemrnar there exists a li-nrit O defined on Dg whLch is Eolder continuous of exponent 6 in il1. This follows exactly as in the previous existence theorem, Sinil-arly AO/Oxt ie Edlcier continuoua 2L. of exponent 6 in D*. These estimates will apply for al-l types of reactions i.e. for any reaction order. It wil-I be shown beLow that in all reactions other thaa one special caser the estinatae can be improved. However, firet some properties must be established. for the function \o. From tbe equation olo iE- = g(xrtrgn-lrlo), g = \ah(=rtror\), (o 4 q' < r), tr> o olo iE- = g(xrtrgn-lrlo), write write where h is of class c+, and g = O g = O g = O wben I= O. wben I= O. wben I= O. 2.4 The Cut-off Problen 11 t Then it 1tl' =^ , where F = 1.1(f - e) it follor+s that aPo l-. R' Tf = U=h(x,t,on-l ,h'), (9 > L) andfn ) 0' 0n # = o fotltl- = o. fotltl- = o. ClearLylgr,(xrt) wiJ.l. be unifornl-y of cl-ass Cl in each region ancl uo flo is of cl-ass cl in the whole regioa Dr. This follows slnce lh i. contiauous on the comrnon bouadary of the separate regions lLoT O and rUo = 0. Hence ClearLylgr,(xrt) wiJ.l. be unifornl-y of cl-ass Cl in each region ancl uo flo is of cl-ass cl in the whole regioa Dr. This follows slnce lh i. contiauous on the comrnon bouadary of the separate regions lLoT O and .rUo = 0. Hence l/n(P) - /{/n(a) | < MIP-Ql tor any P,Q. Dr where lp-gl - lx-*'1 + lt-t'lifp=(xrt),Q=(x'rtt). lLoT O and .rUo = 0. Hence l/n(P) - /{/n(a) | < MIP-Ql tor any P,Q. Dr where lp-gl - lx-*'1 + lt-t'lifp=(xrt),Q=(x'rtt). ( ) { ( ) | where lp-gl - lx-*'1 + lt-t'lifp=(xrt),Q=(x'rtt). Fron this it is possible to deduce that ,)lo is of class Cl in D* as folJows. Consid,er lIo(r)-lo(e) t llnpce)-1trptell . lqo(p)/n(a)l -TFAT- = @ --TFAI- and note that the second. factor is unifornly bounded io 5'-. AIso 'I and note that the second. factor is unifornly bounded io 5'-. AIso 'I 22. since trrey',\'s are bounded on F, @ 1ln1 f\ot*,o)] Ur, and by noting the J'o1l-owing ).emma it fol-l-ows tbat the first factor is bounded on F-. A A Lemma: There exi-sts a upper bound for *l for o sarb( Rr 97 r. Lemma: There exi-sts a upper Proof : Suppose a2, FIA (aP-bY) -=;s- = bound for *l for o sarb b. Then the exoression can be written Lemma: There exi-sts a upper Proof : Suppose a2, FIA (aP-bY) -=;s- = bound for *l for o sarb( Rr 97 r. b. Then the exoression can be written : Suppose a2, b. Then the exoression can be written FIA (aP-bY) -=;s- = So I' is of class CI :-o o-*. Thus the set of functions ()rr) a"e therefore equicontinuous on 5', and so, once more by Ascolifs lenna, there exists a linit \ d"rio"a on il,r. Likewiee there exists a linit /.t which is the linit of the sequence of functions V) and VF = \ . 2.4 The Cut-off Problen As in Appendix 2 it is possibte to prove ,ft is of class Cl in 5', fron the e,luations satisfied by each lo, Then, from an argunent as above, it follows that \ is also of cLaes Cl in ilr. The quantities o and \ of class cl is sufficient to ensure the existence of a solutioa to gL = g(x,t,orl) , 0t gL = g(x,t,orl) , 0t X = \o(xro) ongatt=or with the j-nterpretation that g = O when \ = O. j." o-"r. Now )f is of class Cl *et X-^"1 = g(x,t,o,\) and I-Io=OonFatt=O. i. e. fuf )'-Iol < M( lo-orr_, | + g(x, t rgo_1r\rr) l\-Ll) where the *et X-^"1 = g(x,t,o,\) and I-Io=OonFatt=O. i. e. fuf )'-Iol < M( lo-orr_, | + g(x, t rgo_1r\rr) l\-Ll) where the right hand Eide and I Io=OonFatt=O. i. e. fuf )'-Iol < M( lo-orr_, | + l\-Ll) where the right hand Eide i. e. fuf )'-Iol < M( lo-orr_, | + l\-Ll) where the right hand Eide 2U, tends to zero as n tends to inftnity. Thus It coincides with \ ia Fg. And Eo a\r/at erists also. tends to zero as n tends to inftnity. Thus It coincides with \ ia Fg. And Eo a\r/at erists also. It can be shown that the eurface deflned by .\o(xrt)=Or i.e. t=Ca(x) , teads to that of \(xrt)=O r i.€. t=C(x), ual-formly in B a6 n tends to infinity. Conslder, as before, = !b(xrtroiln, ruol), p = !h(xrt ,artrq), gan at w 0t = !b(xrtroiln, ruol), p = !h(xrt ,artrq), gan at w 0t Then by definition ot / , it foJ-lows that la(xrt) --+1u(xrt) , Then by definition ot / , it foJ-lows that la(xrt) --+1u(xrt) , A/JOI + ap/at unifornnl-y in O g as n tende to inflnity I{ote th Then by definition ot / , it foJ-lows that la(xrt) --+1u(xrt) , A/JOI + ap/at unifornnl-y in O-g as n tende to inflnity. I{ote that tbe cut-off surface g:iven by /(xrt)= o ie tbat of t= c(x). Thenrlo(xr0(r) ) +4tt(xrC(x)) as a tende. Now the fuaction b caa be taken ae ind.ep- endent of / for t( c(x). Eence, aln/ldt ls less tb.an -a for arr n, where alO. Fi-A. 2.2 Consiiter, (i) Cn -( C. Ihen y't(x.cn) - lr (x,c) cnc i.€. 1u(xrar) - 7u(xra) ), a(c - co). 2.4 The Cut-off Problen And so as sicle tends to zerc as n teade, so Co- C a6 n+e. the rlght hand Fi-A. 2.2 (fl) cn )2 c, The arguneat folJ.owc aE above, wtth /-o*a/ interchanged, Hence it caa be concludecl that cs(x)-+c(x) a6 n +ao I 24. unlfornly in E. unlfornly in E. Al-so, by considering Al-so, by considering Al-so, by considering ah 6t = g(xrtro(xrt) rI(xrt)) - g(xt rtro(xt rt), Xxt rt)), where n(t) = Xxrt) - Xxtrt), it is possibte to show that the surface c(x) is of class cl in x, unifornly in B. This neaus that 0C/0x. is finite evertrn*here for all. i. -t- Now, excruding the case in which H- fe"(*rtrgr\) is dis- continuous at \=or it can be shown tbat o anct \ satisfy the systen (1) and (2). rt has already been shor^rn that\ d.oes satisfy tbe equations and the same result follows for c. This neans that o is of class cl*F io D, for aay o<B<1 and of class az+u in D, for some Y> o. rn particular, this means that tbe tempera- ture o, its first two d.erivatives in x., and its first d.erivatlve in trare conti-nuous on the surface t = C(x). However,in the case where f is discontinuous on I= 0 (tnie coincides with the idea of a zero-ord,er reaction but the discoa- tinuity in g d.oes not affect the anarysis), the two reg:ions \ > o and \ = o are coDsl-dered. independeutly. rn fact the problem is consid'ered as two separate problens which both have solutions by the results of section ji I. t(O) = f(x,t,O,I), 3-\ at g(xrtrOr)), I. t(O) = f(x,t,O,I), 3-\ at g(xrtrOr)), O= Oo(xrt) on S, and on E at t = O. O= Oo(xrt) on S, and on E at t = O. \= Io(x,O) onEat t -0, \= Io(x,O) onEat t -0, in the cJ.osure of the region in the cJ.osure of the region 25. II. t(o) = O= t(*,t) : xeB, (x,t)e5r, t < c(xf . f (xrt rO rO) , Oo(xrt) on S* t(*,t) : xeB, (x,t)e5r, t < c(xf . II. t(o) = f (xrt rO rO) , II. t(o) = f (xrt rO rO) , in the closure of tbe region in the closure of tbe region l- It*rt) : xeBr (xrt)tDrrt > c(x)J. It is assumed that the boundaries of these regions satisfy the conditions required by Friedman in Theorens 2 an'd 7. fhat is t they are both of classes C2 and C2+a for some &)O. Fi-A. 2.2 If thls is not so, tben remove a strip about t = C(g) so that tbe boundaries of E and F d.o satisfy this condition. These theorens imply that O is of claes C2*{ for some T>O in each of E ana F. Note that iu order to solve II use is nade of the estinate obtained early in this section, that is the 1+6 estinate. This neans that O antl dO/lx,, for all i, are continuous on t = C(x), and tbis wi1l, be all ' l-' the information that can be obtained for thls particular type of reaction. By considering the equation for O, equation (1) t it can be seen why no informatj-on is gained for AA/AL. In the special case C(x) is a constant, i.e. t-C(x), and this will be a character- istic of the equation L(O) = f, it fol.lows that the second deriva- tives of O with respect to x. ane coatinuous ancl 0Or/0t w:iI1 have a finite discontinuity oa t = C(x). The precise value of this dis- contiuuity will be in fact the value of the beat source just prior to the conplete exhaustion of the reactant. However, ia the genera case it is not possible to attribute the d.iscontinuity on t = C(x) whorly to either of ao/Ot or to the second derivatives of o with respect to the x.. This concludes the discussion of the cut off prob 26, Chapter J COMPARISON RESULTS AND TI{EIR APPLICATION TO SPECIAI PROBLEMS Many exj-stence proofs of systems such as those discussecl in this thesis, begj-n by estabLishing a conparj-son theoren which enables bounds to be obtained. for the iterations set up. l,lctlabtlO took this approach j-n proving the existence and uniqueness of the systern (e't) and (2'2) t when f is a nonincreasing function of \ antl g is a nondecreasing funct- ion of 6. His preliminary result wae used in Chapter 2 to establish the existence and uniqueness of t,he system therein defined. The result proved by McNabb is stated here as the following tbeorem. Theoren 5. (a) bt, 92, \f, )g exist and are continuous in DT. (U) Their second order xi-derivatives ancl fj-rst order t-derivatives exist and. are unifornly bounded in D1, satisfying there the inequalities r(0r) - T(xrtro1, \1) $r - s(xrtro1, \1) \< gI'z - g(x,trg2, \2), 0t 0t and where, Af /A\ 0' / A D O' 0t and where, (c) St(eZ on 51 and O1 (O2, trf < \2 on E at t=O. Then 01( o2 antl Ir ( \z io i1. 11 Zeragj-ja" likewise attenpted to establish a sinilar result for the system he described (see Chapter 2). In this chapter it w:ilJ. be shown that it is not possible to estab- li:h /a conparisou theorem in the general case without invoking requirenents on the boundary conditions. This will be fotrlowed by sone exampLes in 27, wbich it is possible to obtaiE upper and lower bounds oa the solution. wbich it is possible to obtaiE upper and lower bounds oa the solution. J.I' Counter-exanple In this section an exanple wil-l be given to il-lustrate the statement that a SeDeral comparison theorem is inpossible' This wtll be done by consi-d.ering the system (2.1) and (Z.Z), w'ith, at present! no requirenents on the functions f and g' It wi]-l be assumecl that: It wi]-l be assumecl that: f = a9+b\, g = a9+b\, f = a9+b\, f = a9+b\, where a and b are constants. For sinplicity, the geometry shall- be confined to Q = I and' lxl <fi/z. Thus tbe equations are o2g - oo = ao+br, ox2 ot 0\;t = ao .t, b\. ....(t.L) ..,. (3.2) ....(t.L) o2g - oo = ao+br, ox2 ot o2g - oo = ao+br, ox2 ot 0\;t = ao .t, b\. ....(t.L) ..,. (3.2) ....(t.L) ..,. (3.2) This problen will be posed with zero boundary aad initial values. Eence the solution of the above w:i1L be the trivial solution: ( ) e=O, h=0. . . . . (t,t) e=O, h=0. . . . . (t,t) This so$ution wil-1 be the knqqn solution for which conparison functions will be found.. These will be obtained simply by separ- ating the varj"ables, It will be shown that in general it is inpossible to find functions prq which renain of constant sign for all tri.e. which are consistent upper aad lower bounds for the solution (]). ?8. If one proceed-e 1ln the w.ay iad,ioatedl b1r lf-heoreu 5r ed attedpts to finel solntisns Otr}r rhieh setiaf;r d2O r _ AOf oxZ ot a\ldfi >/ a€ r + b,r['r d2O r _ AOf oxZ ot a\ldfi >/ a€ r + b,r['r a\ldfi >/ a€ r + b,r['r Lt wou1d, be exBected that, pnovided the inl.tl,al and boundary conditlo!.s ,are speo5.fted. co-rrectLy, Or, I a"e upper bo,ueds for the solutisns to equati.o,a6 (1) ana (e). I{ote that llheortn 5 appJ.J.es only to thc caee a ?t Q, b ( O. Xn tbe above tbe casc of equaLi t w.iil3-a be t'aken. Further, teke the bouncl,any condf,tioa fo,r Of :as Or = Q oa x =!n/Zr atriltny6r=p(!)cosx..f..(r,4) atriltny6r=p(!)cosx..f..(r,4) \t = q(t)cosx, ..,.(1.5\ \t = q(t)cosx, ..,.(1.5\ as a solutlon of the Broblen. Eence, oo substttuti.on Ln the as a solutlon of the Broblen. Eence, oo substttuti.on Ln the equati ons it fol-J'ows that prg nuet satisfy as a solutlon of the Broblen. J.I' Counter-exanple Eence, oo substttuti.on Ln the equati,ons, it fol-J'ows that prg nuet satisfy equati,ons, it fol-J'ows that prg nuet satisfy S E -(a+r)p-bq, ... r (r,6) q = ap*bq, .... (5,71 S E -(a+r)p-bq, ... r (r,6) q = ap*bq, .... (5,71 S E -(a+r)p-bq, where " = 4-. fhese two equfrtroui ane to be Eor.vedl ulth tbe dt i i f i l d iti t i (0) where " = 4-. fhese two equfrtroui ane to be Eor.vedl ulth tbe dt ini,f,ial- ooad,itiotoi p(0) = n, e(O) = e. Botb p aud q are thus s ol-uti oae of the eecond order equati oa where " = 4-. fhese two equfrtroui ane to be Eor.vedl ulth tbe dt ini,f,ial- ooad,itiotoi p(0) = n, e(O) = e ini,f,ial- ooad,itiotoi p(0) = n, e(O) = e. ini,f,ial- ooad,itiotoi p(0) = n, e(O) = e. p aud q are thus s,ol-uti.oae of the eecond order equati.oa Botb p aud q are thus s,ol-uti.oae of the eecond order equati.oa t+(,a*tr--b)$-tr = O, ),1 t.(g.g) t+(,a tr--b)$-tr = O, ),1 t the sol-utions of, whLch are act whcre er eatiaf,Lee the sol-utions of, whLch are act whcre er eatiaf,Lee cra+(a*l-b)c-b = O. f ... (3.g) 29, From (5) and (Z) it follows that From (5) and (Z) it follows that . . . . (r.1o) dp -(a+I)p - bq - = dq ap+bq . . . . (r.1o) dp -(a+I)p - bq - = dq ap+bq dp -(a+I)p - bq - = dq ap+bq . . . . (r.1o) y a solution of the forn F = ke. This means that Try a solution of the forn F = ke. This means that Try a solution of the forn F = ke. This aa2+(a+l-+b)t+l = o, ... | (r.ft) the dlscrininant of which is gi-ven by (1+a+b)2 - 4*ti tbe same as that of equation (9). CaI1 these roots k1' k2. By considering the p, q plane j-t can be seen that the solution nust l-ie between these Iines. For P = krgr p = kAQ are two possible solutions and no solution can cross another at a regular point. It will be aesumed that k2 is the greater of these (whenever the discrininant is positive). This area is shown in Fig. 3.1. Further, if the tangent to tbe the origin, then at this point solution at a point it follows that paseed througb k= -(a+l)k - b. ak +b 1.e. k = k1 or k2. k= -(a+l)k - b. ak +b 1.e. k = k1 or k2. Thus the ouly possibLe tangents to the solution the origin are the lines p = kLq, p = k2e, passing through Thus the ouly possibLe tangents to the solution the origin are the lines p = kLq, p = k2e, passing through 30. Now sinee tbe cliscriulnants of equations (8) anti (tf) are the sane, then the case in which \ and k, are real coincides witb the condition that prq vary "u "ot wbere a is rea1. So one obvious requirement for p and q to be of the same sign for alL t is that this discrim:Lnant i-s positive. That isn ) ) (a+b+I)z -4ab >/ o. .... (3,l'2) For if this were not so, the solutions for prq would oscillate about the origia in spirals or elJ.ipses (c'f. stokerl5). .... (3,l'2) For t snallr Fr9 vari-es like "Ott, and for t J-arge they vary t-+ 1-ike e^2' . Whether or not \ and k, are of the same sj-ga depend.s on the sign of b/a, To ascertain the form of the integral- curves the various cases are considered. In each case arrows are drawn along the integraL curve to indicate the direction of t increasing, Also it is assumed in a1I cases that the requirement (fe) fs satisfied. From Stoker, the forn of the singul-arity is elther a nodal- point or a saddle point depending solely on the sign of the deteruinant formecl fron equati-ons (5) and (7). That ie whether or not -ab+(a+I)b=tf o. I. a7 On b€O (the case covered by Theoren 5). Here \kr-( O and so krlt O, \_<0. Fron the crj.teri.a of Stoker, the singularity J-s a nodal point. See Fig. t,2 over]-eaf. ,X"iF' B'? ,X iF trI. aSOr blO. {,gain k } O, tI< O, but thie tlnie the s5-ngsl"ar{ty is a s,add]-e polnt. See Sl-9. 5'3. 72, Now ln oa6e,s I and tfr if the initlal valuts (QrP) are such that f,lq> O, theu eLearLy f,or t ) O the eo1uti,on remalrrs in the aana quaetrant and ,go p anct q 'remain of the sane sige. Thl,o neans that a oonparidon theoft[ ie possibJ-e. (Note that eince ab ( O, oonctLtlou (1a) :LE a].w,aJrs eatLefi.eA). III. a}on b7 O, Euch that (12) ls satl.sfl'ed, Eence h. rke are of the sane 6igu and tbe sioguJ.ar1ty Le a sacldJ.e poiat. Eence h. rke are of the sane 6igu and tbe sioguJ.ar1ty Le a sacldJ.e poiat. a sacldJ.e poiat. (i) trh both posttlvei tr.r) h,ka borx f,'lg. 9,4 (i) trh both posttlvei f,'lg. 9 4 tr.r) h,ka borx f,'lg. 9,4 39, fhen it is cleanr that for each of Fre to remai-n s;f ttre saae sLgat the inttl-aI values ntret satiefy hSp/g iu the fl-rst case, ald 0>p'/Q?\ i.l' the secqndl caE'o. lthat fe (qrP) i.s ip t-he reg5.,oa s,OB or DOG iln (i), ana al"eo fa (*L). f,his neana that a eonpa:ieoa the,oarea is lnpossible without restriatt-ng the initl,a]. eoad.itioas. IVr a{ O, b(O, s,uch that (12) ls Eatiefied. Eenee, onrLe nore h-nhZ are o.f th,e,earn:e sign'r but tho siaggtar!.ty Is aow a noclal point. Eenee, onrLe nore h-nhZ are o.f th,e,earn:e sign'r but tho siaggtar!.ty Is aow a noclal point. p (t) q,k, bo:tb (ff) larn, both aegatl.rre: positive: (t) q,k, bo:tb (ff) positive: (t) q,k, bo:tb positive: (ff) larn, both aegatl.rre: ,4, In these cases for prQ to remain of the same sign the initial values nust satisfy p/Qlk-r in case (i) ancl o7 P/Q>.k, in case (ii). That is, exactlY as in III. Note that if the discrininant in (12) i-s zero, the singularity is always a nodal Point. Hence it can be seen that, in general it is inpossible to state a comparison theoren applicable to all cases, without some extra conditions. Most of the examples disCussecl later are reactions in whicb f is nouincreasing in \ (tf o) and g is nonincreasing ln O (alo). That is, case fII. a Following the remarks made early in chapter 2, tbat isr by taking upper and lower bounds for tr r upper and lower bounds are obtained for O. In view of the comnents in the previous sectiont however, no direct comparison theoren is possible. Hence to obtain upper and lower estimates for O an equation of the form aor - # = f(xrtrotrX), .... (1.t3) is solved. If f is nonincreaslng in \, it follows that j-f \t is an upper (lower) bound tor \ then Or is an upper (lower) bound. for O. Sinilarly, estimates can be obtained for \ Uy using upper and Lower bounds for O. Eowever, it is possible for a certaiu class of reactione to uncouple the differential equations and so get a sol-utlon of the forn Ag + f@) = g, .... (5.t4) at" Ag + f@) = g, .... (5.t4) at" .... (5.t4) 7t5,, It sril1 be shsl{n that I bXr eonsiritering the solutione sf (14) and boundls f,o.r these soJ-utl,.orl6 r boqnds ean be obtained 'for the reaOtiurt eojnce,ntrat:i-on ( l) . Ooneider the sYetea 00at' ? l-A .h Y-+ + al-O = ^c ox E+ E -f,e \ I d .r..(v.tiJ ....(1.16) Ooneider the sYetea Ooneider the sYetea Ooneider the sYetea 00at' ? l-A .h Y-+ + al-O = ^c ox E+ E -f,e \ I d .r..(v.tiJ ....(1.16) 00at' ? l-A .h Y-+ + al-O = ^c ox E+ E -f,e 00at' .h + + al-O = .r..(v.tiJ /*tO l-n the semi-infirr:lte region x O whe:ee O = \= I whea t = O aud /* O=1,onr =O. e'is aoouetaat aqd,nf L. SqlatS-on (16) canthug he wrttten oltl-tr\ #f,*:1) = o, ""(3;7) oltl-tr\ #f,*:1) = o, heuce by def,ining gt = # ii,e,. o = *9, it followe that and heuce by def,ining gt = # ii,e,. o = *9, it followe that t #tgl +"(*-rf{"# = # "..(;'xB) t #tgl +"(*-rf{"# = # "..(;'xB) On iategrati-u,g aud ,noting tbat F =-1y'(1-n) t \g/AE = 1 wbaa :t = 0 equatl,on (18) gives ) ). {1 + 1+ a(]'-(ililgfl0-.^l = #, ...,(r.r9) ox ). {1 + 1+ a(]'-(ililgfl0-.^l = #, ox fhi-s is an equatJ-o.u of th.e fsrn l-nitLcatEd in (I4). $[ fhi-s is an equatJ-o.u of th.e fsrn l-nitLcatEd in (I4). $[hon n = 1 def,Lae / = -xog) l.€. \ = "4. a Then as above lt fs1lowE thet Ph ! 1 + a(I-e-l) = g. r . . . (r.ao) ]f + J. + atl-e-/ = ffir r.. ox fhi-s is an equatJ-o.u of th.e fsrn l-nitLcatEd in (I4). $[hon n = 1 def,Lae / = -xog) l.€. \ = "4. Then as above lt fs1lowE thet Ph ! 1 + a(I-e-l) = g. r . . . (r.ao) ]f + J. + atl-e-/ = ffir r.. ox sublec't to the boUnil-ar3r a[d isi,tial. cosditl.onst fi=Owheat=Or 6=E oB x=,,Or ^A AMt=*o as x*@ ' ,5, Agaix aE equetion of the fsrm srlggeeted by (14) reeults" of the fsrm srlggeeted by (14) reeults" Now to t1luetrate the Lrlea.e Lnvolvedl, this la.tter equatLon rxt11 be t'qken aer anx exanple, Uae cag Doil b'€ aacle of llheo'reo- 5 wbiob can be Etatedl: Tf 0tr6Z exi.st aad' satis:fXr Tf 0tr6Z exi.st aad' satis:fXr dL thea il, ( gz everJnrhere. dL thea il, ( gz everJnrhere. One eeLe,ste varioug |tcomparisoc functionsrr atrd, trakes either ily ot f,, ae the :reqr1rifed ssl.lttiou, l[hnee of the possdb5.llt'J-es are 4.'iecussedl be,Iow3 1. the solutiOn lntlepe,ndent of x. ThiE rriLl be the astrrnptot:Lc vaLue of the solutJ.oa of (eo) as x @. It can be eh,om by di.rect lntegnatlsn of 1 * a(r.-e#) = $g , 1 * a(r.-e#) = $g , w"ith the inltial- eonditioa of V = 0 when t = O, to be 1 * a(r.-e#) = $g , w"ith the inltial- eonditioa of V = 0 when t = O, to be a( e ) $g , w"ith the inltial- eonditioa of V = 0 when t = O, to be w"ith the inltial- eonditioa of V = 0 when t = O, to be g@ = r""(#t1. ..,.(r,zu) Th{s will b,e ran uppe? boun(l, Sakiag ilZ 4 that rlefLnedl in (el-) and no,te that ou x '=' o, 6L = fl = t and that @ = r""(#t1. ..,.(r,z g@ = r""(#t1. g@ = r""(#t1. ..,.(r,zu) Th{s will b,e ran uppe? boun(l, Sakiag ilZ 4 that rlefLnedl in (el-) and no,te that ou x '=' o, 6L = fl = t and that ..,.(r,zu) Th{s will b,e ran uppe? boun(l, Sakiag ilZ 4 that rlefLnedl in (el-) and no,te that ou x '=' o, 6L = fl = t and that t E t""(*#g) ror any a ) o. a Sence it fot1ows fhat the solutiqn of (ZO) ;satlsf,Les d 97' Lt ellou|dl be noteit at thls stage that the s.oluilLoa st lnf,toity for o andl \ eattrsfyiog (15) a&d (16) whea L = 1r 15 gc'rrea b5r' i.B'ao =| - and\- = ft;;r ..(3,23) l+ae-(I+a)t ere(J Lt ellou|dl be noteit at thls stage that the s.oluilLoa st lnf,toity for o andl \ eattrsfyiog (15) a&d (16) whea L = 1r 15 gc'rrea b5r' Lt ellou|dl be noteit at thls stage that the s.oluilLoa st lnf,toity for o andl \ eattrsfyiog (15) a&d (16) whea L = 1r 15 gc'rrea b5r' i.B'ao =| - and\- = ft;;r ..(3,23) l ae (I a)t ere(J i.B'ao =| - and\- = ft;;r ..(3,23) l+ae-(I+a)t ere(J Ia vj,erir of the fnegual-l' y in (aa) it fsllowe that \> 1**, , f,orall I'tlo' " * "(I+a)t \> 1**, , f,orall I'tlo' " * "(I+a)t 2,, A lower bounril nay be eor*stllueted. by taklng fiL = r(x1t)F6 1 for sone euitabS-e frrnction r. tf E$) = # - # *1+aO-a-d), for sone euitabS-e frrnction r. tf E$) = # - # *1+aO-a-d) and d, is to he a Lower borrnd,, thea r nuet be aucb that f(S z 0. Eeace Lt is nequirecl tbat # and d, is to he a Lower borrnd,, thea r nuet be aucb that f(S z 0. Eeace Lt is nequirecl tbat f(S z 0. Eeace Lt is nequirecl tbat # P. (# - *i) ; r# * I + a(I - .-re') ), o, i'e' ilo.(* fry 1 - tr + a(t - r'+ '*4t- '-1fl^o\ D ot P. (# - *i) ; r# * I + a(I - .-re') ), o, aud a1so, r+1 Ba x*@. fhat Lsr /n+ f,o. Ih.ea tellse r pat'isfYiag 62r ar -it=o' dx 'f = O QE x = 0.r r = I 911 l=0. sherefsre r(x"t) = er .k/z&) 1 and thue o d r'('tr, It renaias to show that tbe exlreeeion E E 1-r*""-dt- e-F/'ao V o 5-8. f,or fl*2, O aed O( n ( L. 6o'Ds1.dor E as a fuaetlon of F@ and, tr. OE = o,;rFo-e-9@) 6T. : and E = O on F* = O.. Therefore EZ O. a [huE t(flr)zaQ, i[1so when t = 0, 9t = ilz = Qi antl on x = or 9L = o { dz' H it f lL that the sol utl on of (20) satiEf les Henoe it f,olLorrs that the sol,utl-on of (20) satiEf,les Henoe it f,olLorrs that the sol,utl-on of (20) satiEf,les il >/ roe/" * "ffil| erfl r \ ' \I+a I \x,a) 7, A qic-b bette:r reeul-t, in th6 E€rse tbat it is a cJ'osar lorEr bstrnd can ;be constructeel, by f,incling a functd.oa which t-4bes the eame vaf.ues on the bounclary as that takel by the f,unctl'ou to b,e found. For thisr take Eh-atie, 6r=, on.x=0r ilU=Q oEt=0' ilU=Q oEt=0' Ae in the p:r'evious casa, it e'ao be ehoun that F(4) 7 O, and hence (t a)t \ / )( (if ) (t+a)t r \ t+err/+)('"u(ifu-) -r) vt_/ \ Ehen "*r ["'r(*Xi - r"s(i tl) - 'J ', ). . ilo obtaiu bounde fsr O take ar equati.on in the for,n gf.ven by (15). r'or if (15). r'or if u(o) = *-ffi+"\o E o, ox u(o) = *-ffi+"\o E o, ox ....(r.24) *-ffi+"\o E o, ox ....(r.24) 24) 19. and an upper bound is taken for L saX It, then a function eatisfying ) and an upper bound is taken for L saX It, then a function eatisfying ) ) M'(o) = . - €? + axo ox will be a bound for the solution of equatisa (24). This is shown as follows: M(e) = Mr(o) + a( \ - l')e for anY g' M(e) = Mr(o) + a( \ - l')e for anY g' and. so M(e') = Nr'(et) + a(tr-)t)et. Notin6; that \ - )' < O it follows that if a trial function el can be found satisfying M t (g t ) ( 0, then l'I(o' ) Of ), e on the bound.aries the same inequality, e' V 9r will hold' ever;rwhere. Likewise by taking a lower bound for ); if it ls poss- ible to find a function er satisfying Mt(Ot) > O, it foll-ows tbat Ol is a lower bound for the eolution O. By appropriate choices of )', bound's are fouad for E below: 1. consider )t = e-t. This is clearly an upper bound' for ) as it is in fact the value of \ on the boundary x = 0. a Now the solution of Mt (o) = o will be aa upper bound for the solution of M(O) = O. Eowever, it is not possible to obtain a. simple analytical solution for this ancl so a function 9r is sought such th.at Mr (e ' ) Mt(e) = O as x+ao, tends to u(t), where Mt(e) = O as x+ao, tends to u(t), where u(t) = expa(l -e-t) anci u(o) = 1. ....(1,25) Further u is actually an upper bound as required, but is very weak, for, as t+o,u tends to ea whereas O (g{ven by (a4)) tencls *O,. t-o L + a. Tbis euggpet€ that e c' oser bouad soul"d Fossi.blJ'be sbtai.nect b;rsetting gr = l- + u\rr ilhere -lt satLaf,l.ag , $+ae-t = *i, v=o onx=0, an{rhell tooc .. (}.a5} o.x t-o L + a. Tbis euggpet€ that e c' oser bouad soul"d Fossi.blJ'be sbtai.nect b;rsetting gr = l- + u\rr ilhere -lt satLaf,l.ag , $+ae-t = *i, v=o onx=0, an{rhell tooc .. (}.a5} o.x , $+ae-t = *i, v=o onx=0, an{rhell tooc .. (}.a5} o.x $+ae-t = *i, v=o onx=0, an{rhell tooc .. (}.a5} o.x fhat is o.x fhat is fhat is r = aer,rktsg) - #("-r=e'rcffi - -4+eltrerrc(.= .*1 -+ ....(5.27) Fhus by rleing thio or it f,ollows that r^Zv 0v\ 0u M'(e,) = "1[* - gg\ - ol* r "*-t(l+uv), \a*? - o-TJ - vEE ' r^Zv 0v\ 0u "1[* - gg\ - ol* r "*-t(l+uv), \a*? - o-TJ - vEE ' = -r"-to - "r-tuv + *r-t(t+uv), uslng (45) ana (46) 1, €, I{t (o t ) = **-t(L-o} Eoweve:r, Or = O (=1) on x = 0 aad t = O aUtl e-o by TheoreA 5i O(xrt) ( L * rnr ,vrhere u and v are glve'n Uy (25) aad (a?), respectLveLy, Tft.ie bountl. wil. provf.de the berst estinate f,or I for e4e1L x oxly1 fqr aE xror I * !ry+ f +,afi- - "-t)*oB(a(1-e-5). tr'or t suffioiently large this l-aet value ie g:reater given fn (at), whioh aj-eo :[e err uppet boundl for O. ialwtratedl bel.ow, th Oo :' fhls La FxI. 5,8. FxI. 5,8. FxI. 5,8. 41 . Hence to obtaia the best upper bound at a poj-nt (xrt), select the enaller of I+uv and O- at tbat point. 2, To obtain a lower bound for O a lower One such bound. Note the solution of this Last probLen is given by a is tr- siven in Ql), as above it is possible to show that lower bound, where y satisfies bound is selected for I. Then proceeding exactly er=1+(y-l)z isa ...r(3.29) dvffi= "l-y, y(o) - I i.e. is o.i ...r(3.29) dvffi= "l-y, y(o) - I i.e. is o.i ti fi a:td, z satisfies ^2oz ''-= ^1 0x 0z-. z = Oon x = Oand ot I when =0. . (t,29) Eense t=O O, the sol-ution of (a4) sutSect to I satisfies =Lonx= 0 and when e>/ t.a(t-"-(J-*a)t' 4 ' -TGFtf+o-t- vrrll;o VtzJ . . . . (j,to) e>/ t.a(t-"-(J-*a)t' 4 ' -TGFtf+o-t- vrrll;o VtzJ . . . . (j,to) e>/ t.a(t-"-(J-*a)t' 4 ' -TGFtf+o-t- vrrll;o VtzJ It is also of interest to consider the same problem in a fin1te domainr though stiLl taken for convenience, in one dinenslon. Hence suppose that o anct.\ satisfy the same equations, viz. (r:) ana (16) with n = 1, where o = I on x = o g'4 x = b as well as initially. Then consider examples exactly as in the seni-infinite problen. I. By taking X = "-t (an upper bound) it follows tbat Of = 1 $ uv is an upper bound for o, where u = exp(a(r-e-t)) as before, and v is the solution of lt} * ""-t = #, v = 0 on x = o, b and whea t = o. dx' Note the solution of this Last probLen is given by l+2. +u2* I -./ +\.'' n=O' -ter*tFl v(x,t) = "G;#=t)"-t- Bhen ltr(ot} = ae-t'(L-u) ( 0, as t7 L. Iloruever, Or = o on x = and when t = O and so it folLows that L+uv ) I everywhere' (2n+n ) '(en+r)eoz - b2, +u2* I -./ +\.'' n=O' -ter*tFl v(x,t) = "G;#=t)"-t- Bh ltr(ot} ae t'(L u) ( 0 as t7 L Iloruever Or = o on x = o (2n+n ) '(en+r)eoz - b2, +u2* I -./ +\.'' n=O' -ter*tFl v(x,t) = "G;#=t)"-t- (2n+n ) '(en+r)eoz - b2, +u2* I / +\.'' n=O' -ter*tFl v(x,t) = "G;#=t)"-t- (2n+n ) '(en+r)eoz - b2, Bhen ltr(ot} = ae-t'(L-u) ( 0, as t7 L. Iloruever, Or = o on x = orb and when t = O and so it folLows that L+uv ) I everywhere' Bhen ltr(ot} = ae-t'(L-u) ( 0, as t7 L. a Iloruever, Or = o on x = orb and when t = O and so it folLows that L+uv ) I everywhere' Bhen ltr(ot} = ae-t'(L-u) ( 0, as t7 L. Iloruever, Or = o on x = orb and when t = O and so it folLows that L+uv ) I everywhere' As ,in t-he seni-in,{i.nl,te probl.en., a lower bsund' ls qbt'a{aetl for O by talcing as a Lowe'r bour,d for \ the expnossioa ).' Then it is, pos.eib}e to shgw that 9f = I + (y -Llz is a lower bound wbere IrE are g:lven Uy (a8) and [29), but n'or u = o Qn E = o g! x r b' that i.s ' 2. z, = *e+*:+r*4"-@Pt' Iil=U F.or Mt(Or) = a r(;-'s)>/ O a.E z 1!, andl 6 = gt 6,n the boUniiari.e:6 x = Orb anil uheu t = O, Eeno.e e> f + (y-l)s eYerSrwbere. z, = *e+*:+r*4"-@Pt' Iil=U F.or Mt(Or) = a r(;-'s)>/ O a.E z 1!, andl 6 = gt 6,n the boUniiari.e:6 x = Orb anil uheu t = O, Eeno.e e> f + (y-l)s eYerSrwbere. z, = *e+*:+r*4"-@Pt' Iil=U llbus it can be s'ee1 tbat r al-though rro d.irest oouparieOn theorea ie possllble, upler aod lo:rer bounde oarl be obtaj:red for the tewperatirrc and reactant conc'ent'.rat-,ior. 45. Chapter 4 BOIIIIDARY COI\TDITTONS AND TEE BOIIIIDART I,ATER At thi-s stage it is neces'ary to investigate the physical inpric- ations wben the tenperature is Prescribed on the bonndary. rn reallty, it is not always possible to' ignore the therna]. propetles of the sur- rounding ned'iun. rn a recent paper elitiplS examined the heat cond.uct- ioa between a sphere and a sumound.ing necllum of dl-fferent thernral properties ' fhe sphere was assu.ued. to be at a higher temperature j-a- itially. Philip found the tenperature and heat flux on tbe surface of the spbere aE a function of tine. suppose tbat the inner sphere is of thernal conductivlty and dl-ff- ueitivity given by k and,K respectively. Lircewise the correepondi_ng quantities for the sumoundingl medium (supposed, to be infinlte ia er_ tent) are \ *u &f Then the boundary conditions oa the surface of the sphere (r=a1 are that (i) ["]t="-o = [u]"="*o i.€. the tenperature is continuou., t"ql laol (ii) el*l = k -[-Orrr=a_o -:LFJ"=r*o t.". tbe flux Ls contiauouer (i) ["]t="-o = [u]"="*o i.€. the tenperature is continuou., t"ql laol (ii) el*l = k -[-Orrr=a_o -:LFJ"=r*o t.". tbe flux Ls contiauouer (i) ["]t="-o = [u]"="*o i.€. the tenperature is continuou., t"ql laol (ii) el*l = k -[-Orrr=a_o -:LFJ"=r*o t.". tbe flux Ls contiauouer fi The first of these expresses the fact that there is no sudcten Junp in the temperature on the surface, while the eecond states that there is no aecunulation of energy at the surface. rn ord,er to ascertain what physical assunption is nade when the surround,ing nedi'm ls neglected, two separate problens w:ir-I be cousidered: one ie based, on a mod.e' sirnilar to that of ,phllipfs, and the other oa a nod,el, typi-cal of those usua1ly consid.ered. h4. 4.1. Different tsound'arv Conditions A conparison wilL be made for the two problems described' belowt by calculating the temperature and fl-ux in the seml--infinite region x) O, r"ri-th a point source p].aced at x = xo(>O) and rrcltched on instantaneously at t = O. The tenperature and' fl-r:x due to tbis source wil-I be found and the expressS-ons can be compared directl-y. (a) x>0, therrnal conductivity k, thernal diffusitivity-( ' x40, rr tt \., tt tt 1r-' The equation governing the diffusion of heat in the region can be written 0, therrnal conductivity k, thernal diffusitivity-( ' \., tt tt 1r-' The equation governing the diffusion of heat in the region can be written )o-Gl t aGr - -?(El[- ox - 6(x-xo)6(t), !,rith the boundary conditj-ons t Gl and fact/0x are continuous or x = o. In (r) r< and & are interpreted. r" \ and f" for x(01 and the right hand side is mereJ-y the two-dimeneional delta function. Iience Gr(xrt lxor0) w'i1l be the Greenrs functioa for this problem. It corresponds to tbe temperature distribution in a region, initS-atly at zero temperature, when at t = O a concea- trated heat source is appliecl suddenJ-y and instantaneouely at the point x = xoc (b) Consider just the region x) O, of tbermal constants k and tL Again the equation can be written as ia (t) with G, for Gtr but now the boundary condition is vritten lr"dGr/dx = EGZ on x = O (see note 3 on page 6). H is calIed the surface co6ling coeffl- cient, QZ is th.e Greenrs function for thls probl-emr differeat from that of (a) because of the different boundary conditione. 45. These problerns can be solved by talclng LapJ.ace transforns of equation (1) and solving the resul-ting ordinary differential equation in xrusing the appropriate boundary cond.itions. on inverting the transforns so obtained. it is fouacl that the sol-utions are g:iven by: 1 , (x-xJ" l-+ - * (r'x'l \ (a) c1 = +(#)t("'W + ffi e-?,T ), ror a) o' ' ' (4'2) " 1/ G:'c' "-Hs)*#"t"tf'H"r"Gffr . *m). 4.1. Different tsound'arv Conditions (b) Gz = ;(#)zk-TTr + (4.r) (b lrrow these two expressions can be seen to be equal if we put \- (a) and E = O in (b) ' For both are now equal- to =Oin (a) and E = O in (b) ' For both are now equal- to (a) and E = O in ( ) o q ,1 , (.-ro)t Lxrrof 1 *Cfrlz("-F + e-?F I . This was expected, of course, because these two conditioas mean that tbe boundary cond'ition at x = Or for both probJ-ems is 0G/0x = O. 'Ihat is, the surface x = 0 is tbermally insulated. fronl (a) tbe second. med.ium in the first caser ana (b) from its su*oundings in the second case. Also, if in (b)r B tends to infinity, the expreosioo G2 tends to the expression 1r^+ r.)r 1 e-@) i that is, the sane as G, when { Ls set tends to infinity corresponds to the caee in whicb G, 5-s prescribed on the boundary x = O (in ttrie case G, = O on x = O). Exactl_y the same happens wben the flux kdG/dx is consLdered in the two cases. Hence, it follows that if the temperature ie prescrlbed on the boundary, that this is eguilval.ent to neglecting the thermal dl.iffusiviti of the neighbouring necliun. . ) (x-r.l *tftlz("-z.zr equal to zero. iI ,1 , (.-ro)t Lxrrof 1 *Cfrlz("-F + e-?F I . This was expected, of course, because these two conditioas mean that tbe boundary cond'ition at x = Or for both probJ-ems is 0G/0x = O. 'Ihat is, the surface x = 0 is tbermally insulated. fronl (a) tbe second. med.ium in the first caser ana (b) from its su*oundings in the second case. Also, if in (b)r B tends to infinity, the expreosioo G2 tends to the expression This can be argued from physi-cal coneliderations al-so. For if jq i" negligible (serntr'ared to \), this corresponds to a meditrn wj-th a large thermal capacity C. Eence any heat which is transfemed acro6B 46, the bound,ary x = 0 w:i-ll not produce any significant change in tbe tenperature in the regioa x ( O. the bound,ary x = 0 w:i-ll not produce any significant change in tbe tenperature in the regioa x ( O. .... (4.5) 4.2. The tsoundary Layer fn the preceding section, consideration was given to the phys5.- cal reality of the boundary cond.itl-ons inposed. on tbe surface of the mediun. In any problem, for wh,ich it is desired to find the tenpera- ture at any poiat as a function of tlne, one of the first things of interest is the effect of the boundary aail the condltions irnposed tbere tn particular, how long iloes it take for these conclitione to have aa appreciabre affect oa the sorution at any given point? The equatl,6n for the temperature being paraboJ-ic, iopJ-ies that the effects due to the boundary are felt ionecliately throughout the med.iun. Eowever, in practice' one Looks for the points for which the bound.ary condltLoas have hail a significant effect on the solution there. Sucb poLnts are eaid to be urithin the boundary J-ayer. Outside the bouadary layer, the eolution bebaves as though the boundary is not there at all. For example, take the problen of a semi-iafinite region with a constant heat source and homogeneous boundary and initLal conditions, i. g. i. g. 2 0-o -+r ox subjectto€=O teki.ngi a Laplace ae t& onx=O transforn (4.4) and at t intso = O. The soLutioo is found by that 2 0-o -+r ox ae t& )td-c _E 2E- dx- 1 = -[ r )td-c _E 2E- dx- 1 = -[ r .... (4.5) 47, where 6(xrp) = jot"rt)e-ptdt. " " (4'5) On sol-ving (5) it follows that 5 = *, (1 - e-{ix) and so by inverting p- this transforn, using Erdelyf-rs tabl-esl7r where 6(xrp) = jot"rt)e-ptdt. " " (4'5) On sol-ving (5) it follows that 5 = *, (1 - e-{ix) and so by inverting p- this transforn, using Erdelyf-rs tabl-esl7r " " (4'5) + o(x1t) = t**t|)ue-*^/+r 6*1*2)ettck/ztt), .. .. (4'?) The expression in (?) is, of course, the complete solution' Eowever much is to be gained from 3-ooking at tbe soLution more closely' The solution 5-ndependlent of x is O = t, obtained fron (7) as the asynrptotlc value as x tends to infinity. At any given ti-ne, it is possible to obtain a measure of the behaviour of the solution as x becomes 1,arge. Thls is d.one by considering the asyllptotic expression for erfc(z) for z large. Hence, fron (7) 1 o(xrt) = t(]- -+-= "-^2.,. ), . '. ' (4'8) BrEz2 rshere z = x/Zt* and terms of Otf- tl bave been neglected. Then Zrt by cboosiag a rflevel of significancetr s, that isr for %.7, T, where "-Y26nty3 = u, the second term in (B) can be negl-ected, a measure of the vridth of tlre bouadary Layer is obtained.. In the xrt p1ane, the boundary layer will be marked by tbe parabola *2 = 4Y2t. This is illustrated in Fig. 4.1. The sorresponding soLution as a functLon of positioa, at any fixed time is j.ll-ustrated in Fi-g. 4.2. . '. ' (4'8) 4.1' 48. 48. The boundary Layer ie l-il3e a ldave advancing iato the nedium wlth 1 speed inverseLy proportionaL to (t)2. The solution ia (8) is, of courEie, on1-y valid fot z large and so is not appl-icable near the boundary x = o. outside the boundary layer the solution can be approxinatett by the sol-utioa indepenclent of xs i-.€. O = t. To obtain an equivalent form of the soLution (7) near tne bounclary x = Or use is nad.e of the asynptotic expaneion for erfc(z) rrhen z is small . Eence, 1^=1 t( 2z/fi2 - z1/2 * ztft\nz ... ).' . . . (4.9) is validl on1-y for z snaLl- and, so is not appl-icabJ-e near is therefore of use within tbe boundary layer on1y. e= The above t = 0. It thie sinple example an indicatiou is given on a tecbaique any probl-em of the type d,iscussed. here. For it is usuall.y Fron useful in possible i-nfinity. clescribed to find the asSnnptotic vaLue of the sol-ution aE x tends to Equation (f.4) i-rnpl-ies that the solution of the syetem ty (t,t) and (1.7) inclepend,ent of x is 6iven by 49. ....(4.10) b f ' *t = -"3+ 49. " " (4'5) ....(4.10) ....(4.10) *t = -"3+ where in tbe diffusion equat5-on t is wiitten for fit ae before' Tble means o + cr,) is coastant for aLt t and so is equa3- to its inltial value. Thus by substitutlng for o in the equation given in (1'1) t 0\ 0t = -g(e,)' ) t it is possible to fincl both € and \' In equatioa (11) g(O,I) is written for tbe u-nit as x tends to infiaity of the expression g(xr0rt) introcluce'cl ln Chapter 1' [hese ideas can be of use in other geometriee in a sj'mi]'ar llatrD€r' 3or exampJ-e, ia a finite slab which ls of suf flcient thichess so tbat the bounctary layers associated with eash boundlary d'o not ov€l- lap for smalI tine. suppose the slab is of tbickness b, then the bound.ary layers epread into the region as showo in Fig' 4't' w&: in the triaugular-shaped region eolution indlepenilent of time. Eence it is onlY one carl ase the (snaLl tine) tbat ,o, Tbeotherpenainingcaeetobeconsiderediswhentheslabie thin. Ihis means that the effect of the boundary ls felt almost innecliately throughout the whole sl-ab, A perturbatLon expanaion can be ueed to obtain an approxi-nate sol-ution to the problen' Thls has to be done in such a way so a6 to avoid obtainj-ng a singuJ'ar perturbatlon serles. consider the exa.nple froru chapter 9, that J.a the coupLecl paii of equations o2e oe -a\ ,7 = 6E+'E' Al , 5t = -le' o2e oe -a\ ,7 = 6E+'E' Al , 5t = -le' ....(4.u) ... r (4.r5) o2e oe -a\ ,7 = 6E+'E' Al , 5t = -le' ....(4.u) ... r (4.r5) o2e oe -a\ ,7 = 6E+'E' ....(4.u) ... r (4.r5) Al , 5t = -le' in the region o(x(b eupJect to o = l onx = oandxEbr and O = L, tr = l- when t = O. Recognition of tbe fact that the epeciuen is smal-L is gC.vea ln writins O = f +Eu and g*" fot x, where € is srnall- and u is a function of xrt satisfyiag: * * + =.*t*+ -1 C 0x *i = -I(r + €u) ' + =.*t*+, ....(4.r4) -1 C 0x *i = -I(r + €u) ' .,.. (4.r5) + =.*t*+, ....(4.r4) -1 C 0x *i = -I(r + €u) ' .,.. (4.r5) + =.*t*+, -1 C 0x *i = -I(r + €u) ' with honogeneous boundary anct initial conditions. Now in (14) r if the expansione with honogeneous boundary anct initial conditions. Now in (14) r if the expansione ....(4.15) . . , . (4,17) u = uo + E5 + tzurt ...t = \o + Et, + E2)rt ..., u = uo + E5 + tzurt ...t = \o + Et, + E2)rt ..., are substituted. and the coefficients of corresponcl.ing powers of are equated, it foJ-l.ows that, are equated, it foJ-l.ows that, 5L. 2 -. .? utoo olo o'oi doi-L oli .- = "#, ,;2- = # * "U1* for i = LtZ' ... . Thj-s 5ives i ' 2 -. .? utoo olo o'oi doi-L oli .- = "#, ,;2- = # * "U1* for i = LtZ' ... . Thj-s 5ives i rise to what is called a singular perturbation series' A cliscussioa of these is given in Van Oyfel8. To avoid' this, F is written for € in eo-uatfoa (14) onIy, and. j.s consiclered as a constant' Then oa equating coryespondLq powers of t as before it follows: rise to what is called a singular perturbation series' A cliscussioa of these is given in Van Oyfel8. To avoid' this, F is written for € in eo-uatfoa (14) onIy, and. j.s consiclered as a constant' Then oa equating coryespondLq powers of t as before it follows: o2uo -= ox 6Io 5r ^?0u.a ^1 ox 0u p;f + =-\o, 0u. = BF* + 0\ o aEF' aI.L AEF' .. .. (4.r8) . . . , (4.19) . . . , (4.eo) . . . . (4.2L) 0\ at = -(I, * ti-Ilo * ..... + uo\r-r) . o2uo ox 0u p;f + 0\ o aEF' o2uo ox 0u p;f + 0\ o aEF' .. .. (4.r8) o2uo -= ox 6Io 5r ^?0u.a ^1 ox 0u p;f + =-\o, 0u. = BF* + 0\ o aEF' aI.L AEF' .. .. (4.r8) . . . , (4.19) . . . , (4.eo) . . . . (4.2L) 0\ at = -(I, * ti-Ilo * ..... + uo\r-r) . bas homogeneoue -1r \1 =O Equati-ons (a0) ana (21) apply for i E 1r2r ... . oi bouadary and initial conditions (i=O includ.ed) and Io when t = 0, bas homogeneoue -1r \1 =O Equati-ons (a0) ana (21) apply for i E 1r2r ... . oi bouadary and initial conditions (i=O includ.ed) and Io when t = 0, oa solving (J.9) subject to the above initial cond'ition it is found that lo = e-t. Then substituting in (r8), taking Laplace transforns it fo].lows that 2 2 ctuo -+ -a clx where i o Ppio, subject to ilo = O on x = 0 ancl x = bi - r)e-t- ...o(4,22) a = p+1 = 7 "-Ptro(x,t)dt. to 1 '2 uo(xrt) ctuo -+ -a clx where i Ppio, subject to ilo = O on x = 0 ancl x = bi a = p+1 = 7 " Pt ( t)dt to ctuo -a clx Ppio, subject to ilo = O on x = 0 ancl x = bi a = p+1 - r)e-t- ...o(4,22) ( ) 1 '2 uo(xrt) - r)e-t- ...o(4,22) 1 '2 ? "-t*'frt/tt' uirr( zn.r)nx/\ -"E (zn*I{en*r)2n' - g^r] 4b2a 52. This er4rression, in (ee) r represents an approximation to the soLution oftbeprobJ.enasposedpreviousIy.ItistheIi@approxi- mation to the solution. Mathematically, it is the solutLon of the diffusion equation subject to heat generation which depeads on the surface tenperature and the surfac0 reactant concentration' EOw goocl this approximation is, depends on the thickness of the specinen' A better approximation can be obtalned by solving for the second-order terns \r5i given by the coupled eo.uations (20) and (2L) with i r I. This is done by taking laplace transforns of the equations and, solving, exactly as before. In (af) r use is made of the sol-ution uo given Ln (ZZ), or rather the Laplace transforn of uor to forn a first order linear ordinary differential- equation for \r. As one proceeds for higher order approximations, the prob}ens of computation increase, and so it is aot j.ntended to state the bigher terme, except to note that it is possible to solve for these ia principLe. Further, if the uo, ulr ... so obtained wben p I O are expanded. in powers of F, the term independ.ent of B should be tbat obtained by puttins I = 0 in (f8 - ef). For exampl-e, ? y p ) ? u l^ = *ax(t -r)e-t ancl tbis is the solution of 0 uo o-p=o F = " # = -"\o, subject to honogeneous bounclary conditions on x = 0 and x = b. to honogeneous bounclary conditions on x = 0 and x = b. To use these perturbation series a6 approximations to the sol-u- tion, it is necessary to put F = € = 1. ft is interesting to note that uolp=t t" the eame crpFa66iou aa that denotedl by v in Chalter ] (see pp. 4t - 4a). ,5. Thus tbe fiuile s].ab caa be rbolvedltt Ji'n tbe fo].lowtug ttays: (i) if it ls thln, tben the Sierturbation eef:Les obtaLned I'n thle section can, be ueedl (ii,) tg it i,s t-h5-ck, tben ne,err the edgee the bouudary layer soli1gl'oa ls a useful approXi.m*tlonrand in the eentne the eolutlon appro4!'- nat,es an i4fiuJ-ta prohJ-em forr sufficientl.y ana1l tlne' tr'ol] xerge tine tbe specinEA wi.1I bebave tike a thi-a epecinen, Ln tbat tbe e,ffect of the bounttary Ls f,elt tb.rouShoutl thougb it nay be that oihrtrng to tle equatio:r being nonLiaear, the pertu:sbation eerLes wi]-t not be va].idt tbere' fheee icleae cau.. ba e4eralized to anSr' type o'f rreactl'oo in aut particular geometry, Srovlded that it ie- poe,sl.ble to obta;i.n L:laear equatio;4e' ,f,o:r the tenns :bf, the pentgibatLon ser:Los' 54, TEE SPACE - AVERAGING PRoCESS A$D cBIErgAI,=qfATEs As it was mentioned in Chapter 1, many of the autbors who have stuclied. this particular system have mad.e use of what wilL be calIed a space - averaging process. It j-s intended to extrlJ-ain this in detail and connent on its vaLiclity. Tbese authors have been interested nain- 1y J-n deternioi.ng the critical conditions, So before any discussj-on of the space - averaging process can be givea, a definition of what ie meant by cri-tical conditions nust be made. 5.1. The Critica]. State. There is some difficuLty in d.efining the critical stater aadl just this problen has occupied the attention of nany authors in the last few years. A state vriLl- be said to subcrj.tical if the corresponcling soLution is stable n and. supercritical if the corresponding solution is unstabLe. The extreme case of stability shall be callecl critical stab- il.ity. However, the question is left as to how to d.ecide whether or not a given solution is stable ot unstable. This shal-L be defined here as in BeJ-lman19. That is, a soJ.utlon, o, of a partial d.ifferent- ia3- equation is said. to be stable, if any solutionrO, of the equation whose boundary values are sufficiently |tcl-oserr to Ors boundary values, remains |tcJ"oserf to O for al-l vaLues of the independent variables. The term rrcJ-oserr wj-J.J- be used in the fol-lowing 6en6e: el and. 0, are suff- iciently close in a set B if the rnaximum of lOf- Oal is sufficiently srnalL. lt shouJ.d be noted that stabiLity is not necessarily the same as being bounded. )d Stoher'" set up a criterion for the stabil-ity of an equil-ibriun set up a criterion for the stabil-ity of an equil-ibriun 55. state of a nechenical- gysten through the application snra].]- osci.ll-ation6. This has been extended to cover erned by systens of partial differentiaL equations. the crj.terion is justified for a class of parabolic sider tbe equatioa for a zero ord.er reactionr which can be rtritten of the nethod of situations gov- 2'l In McNabb--, equatione. Con- fron Cbapter J. ....(5.1) ....(5.1) ae + f(x,o) = 3E . ae + f(x,o) = 3E . Then the stabil-ity of the steady state solutions of fron considerations concerning the solutions of the ....(5.1) (L) can be decidecl equation In particular, each menber of a one parameter fanii-y of stead.y state solutions is stable in any closed reg:ion not containing points at which tbe nember touches the envelope of the f"nJlX. As an example, McNabb discussed the equation . . . . $,7) o2o + 6eo = ax2 006T' o2o + 6eo = ax2 006T' . . . . $,7) . . . . $,7) in a srab of thiol@ess 2b. This is the equatioa derived fron the Amheaius equation (1.2), by naking a binomial approxinnation. Tbe steady state solutj.oa of (7) was glveo in Kamenetskiil ae o = oo - 2logcosirf(u"?tt*"] , . . . . 5.1. The Critica]. State. (5,4) where o6 satisfied "S = corh2( o.Oyil*a . Then 6crit is the max- j-mum 6 for which a solution of this last equation is real i.e. require y = coshz{aazylz1* to have a real sor-ution. By defining tr= (oazyp1*, it fol-lows that coshrr/y = (Z/bbZ)+. Hence to find, the naxim'm 6, all that is necessary is to find the ninirnum of eoshc/q for alr pos_ itive [, This is found. by successive iterations to be ]..'5rr and (5,4) 56, hence 6crit = O'88/U2. Or if the value of 6 j-s fixect, it follows that the criticaL size of the sl-ab is g:iven by b = O' 94/6+, That is if 6b2> o'88 tfre solutions of (3) are uastable and in fact, ao ls sbown in l{cNabb, are unboqnclecl as t tends to infinity. If 6b2<O.BB the solution is stable and tends to the steady state given in (4), as t tends to infinity. Tbe critical state is given, of courset a by 6b' = o'88. This is illustrated in tbe figure below. ri,g:14. In this case it is obvious that if the system is stable, it means that the soLution is in fact boundecl, and if it is unstabl-e, that the solution is unbound.ed., though tbis is not general,ly the case. Thus there is no problern in cLassifying any zero order reaction, but in the general case of a nth ord.er reaction the picture is far from clear. The following connents wil-I apply to the most general type of system, that is as dessribed in chapter 1. when the comes- ponding temperature-time curves are d.rar,rn, it is obvious tbat the behavj-our is d.ifferent fron that illustrated ln Fi-g. .5.J-. The tenp- erature fj,rst ri-ses as though it wa.s a ze?o order reactiont then owing to the reactant coneumption becoming significant, this effect gracluaLly doninates. fhus the temperature reacbes a maximum ancl then tends asymptotically to zero. This is shown in Fig. 5.2. i FiE. 5.? Thus any disturbance ad.ded to any particuJ-ar state will eventually shrink to zero as t increasesr and it is never posslble for auy sucb d.isturbance to grow exponentially witb t. Using tbe definl- tion of sta'uility g:iven on p.t4, one rnust conclude that auy reaction of nonzero ord,er is necessaril-y stabl-e. However iu the paper by Thomas and Bowe"8 u.o attenpt i-s made to set up a cr5.terj.on for criti- ca1 stability. It is intended. to discues this further. For conditions, which are d.efiaed by Thonas and Bgwes to be stable, curves of Oo (the centre temperature) againet t increase to a maximum(and then fall) in such a way that Ago/At decreasee as t increases, i.e. azeo/otz1o. However, if the condition' are unstable (again in the sense of Thomas and Bowes) Oo ftotted against t passes through a point of inflection and thea risee much more rapidJ-y to a maxi.mum which is much higher than in the stable case. 58, This type of tenperature behaviour is conventionall-y considered as cbaracteristic of ignition, and the lowest value of any parameter (such as 6 before) for which a curve of 0o against t includes such an inflection while the temperature is rising is then defined'' again conventionally, as the critical state. with the type of reactlon that is cousidered here, this is a reasonable approximation' For therateatwbichheatisevo1vedis@w.ithtenperature. However, if there wa-s a more marked variation of heat prod'uctlon vnith temperature, then the criterion would become anbiguoUe. This criterion cau be thought of in a different way, althougb the same conclusions, as to which states are stable and which are unstabler are reached., For the stable caset any snal-L dlsturbance aclded to the system will- tend, to decrease with time. one could say that the d,isturbed state grow6 back on to the undf.sturbed' state. In the unstable caser atrY such disturbance will first grow away fron the uadisturbed, state anil then gradualJ.y decrease to zero for J-arge time. Such behaviour can be calLetl tfvariation increasingtt 4a coB- pared. to ftvariation decreasingtt in the former cerS€. Thonas et.81' wish to distinguish between these two cases. It is suggested here that what tbey are reall,y saying is that the systen shall be stable or unstabl-e according to whether it is asynptotically stable or unstabl-e. FiE. 5.? The worE traslmptoticalS.ytf is used here in the sense that a zeto order reaction can be regarded as the linit of a sequence of nth order reactions. This r*:ill- be d.j.scussed, below where a pa.rticuLar example will be dj-scussed which illustrates asynptotic instabil-ity. Consider the coupled systen, Consider the coupled systen, + + '\oe 0x 0\ 0t 00att 59' ....(5,r) . . . . (5.6) = -gArg fi i f hi k 2b L h 59' + + '\oe 0x 0\ 0t 00att ....(5,r) . . . . (5.6) = -gArg f 2b ....(5,r) rrrbere s is a parameter i in a finite sl-ab of thickness 2b, wLth €=1, \=lwhent =Oe-nd.O =L onx= b. Now, ingeneralt under the criterion of Be1lnan, this eysten is said to be stable for any s I O. If a = Or then the reaction is effectively of zero ordert ancl so the probLem j-s sjmilar to that proposed at the beginning of this section. tr'or this casel a2o 0x- 0e = f,fr . . . . (5.?) subject to I = tbe stab5-Iity o frcm I on x = b and O = 1 when t = 0. Then as ia McNabbt f the steady state soLutions of Q) can be ddcidlect frcm 2g-9+o -1 cx 0. . . . . (5.8) A family of sol-utioas satisfying the boundary condition 6O/6x = O at x = O and strrnmetrical about x = O is O(xrA) = Aco€x. The stead'y state solution O(xrA) touches the envelope at x =t*/2, e = O (in fact the envel-ope is just these two points in this case) ancl is therefore stable in aay region lxl < b if b 1 n/2. The correapondiag solutionsatisfyingO = 1 otlx = b is g = cos x/cosb, Bhus it is said that the critical size of the region is b = n/2. For this value of b, the solutioa of (7) is unbounded ae t tends to infinity. Now, returni-ng to the case o / O and tak{ng a region which exceeds the critical size, i.e. is unstable, when c = Or then it may 50, he systeu is as3mptoticalLy unstable as a tende to zero. be said Tben the that the curves systeu is as3mptoticalLy unstable as a tende to zero. f,or varioua c wil]. be shown in Fig. 5.3. o(= € Fig. FiE. 5.? 5.J Thus it can be seen how the criterion set up by Tbonas hinges on the corresponding zero order reactj-on. Suppose that the ti-me scaLe of reactant consunption is Large compared to that of tbe ti-ne taken to reach a quasi-stead.y state (tAat is the temperature curve flattena off). Note that this corresponds to a smalL in the above example. Then for sna.J.J. tine the temperature behaves as though the reaction was of zero order, in that if a snal.L ctisturbance is acldeclr tbis tends to grow exponential-Ly, or disappearsr witb ti-ne dependiug whether or not the reaction is stable. 5.2. The SDace-averaAing Process The authors with which this thesis w:iL1 be nainly concerned. are Thomas and Bowe"3'8'9 and AilLer aad Enig4. The prlmary aim of theee authors is to convert the partj.al. differentiaL equations into orclin- ary differential equations in the independ.ent variabLe t, thereby 61. meking the equations easier to solve' This is doue by an inte- gration over space and replaciug the temperature and reactant concentration by the averages of these quaatities over the whole region. consider the equations for a heated body, which can be written, using suitabl-e dimensionless variables as a6 = *?+"f!, 0\E= -.\nr(e) . ....(5,9) . . . .(5.1o) a6 = *?+"f!, 0\E= -.\nr(e) . ....(5,9) . . . .(5.1o) a6 = *?+"f!, 0\E= -.\nr(e) . ....(5,9) 0\E= -.\nr(e) . . . . .(5.1o) In (9) tbe Laplacian is in the appropi:[ate d,imension. For a s;runmetricalJ-y beated body, A€ = Oze/axt. * 00/0x where J = Orlr2 for a slab, cyliader, and sphere respectively. The region oceupied by the material- wiLl be denoted by V and its boundary by S. The function f(O) is in general a nonlinear function of temperature e.go as the Arrhenius equatiotr f = A1-B/Q. These equations are to be soLvecl subject to a prescribed value of I on S and, prescribed vai.tres of 0 ancl \ initi""Lly. Now this is done in the authors meationed previ-ousJ-yt but lt is felt that at no stage j-s it c1ear Just what assunptions are madet and horv the systen. arrived at resenbles that l+itb vrhich they startecl. 8o by integrating throughout tbe whole region V and using Gaussl theorem in the appropriate d.i:eensions it foJ-l-ows tbat -. rA jd d6 . _di raou.*} = aT + taT. . . . .(5.11) 1 F --. rA jd d6 . _di 7$ graou.*} = aT + taT. In (11) V is used. for the volu.ne of the region average temperature and reactant concentration. (10) it foll-ows that . . . .(5.11) *na 5, i are tbe Likew'ise, by usLng . . . .(5.11) d;E = -3{fr(e)av ' = -3{fr(e)av ' . . . .(5.12) . . . .(5.12) 62. The introd.uction of the quantiti"u 6 *na T explains the use of tbe term ttspace-averaging processtr. At this point two assumptionE are made. These are outlined below. (i) The second term in ecluation (fe) can be written -a'O'Ff (6), where A(t) : {rft(e)aVnlnf(6). 5.2. The SDace-averaAing Process If tbe region ie large enougb, then for sufficient3-y snal-l- tine before the boundary has had a eignificant effect on O and \, l(t)pI. Indeed this is the fLrst approximation made. Eowever, it can be noted. that this is equiva- lent to using a perturbation expansioa in which tbe first tern repre6ents the sol-ution independent of x, while tbe higher order terms represent the effect of tbe boundary J-ayer as it moves into the region. For if x/el\ is written for x in (9) ancl (fO) t and corresponding power series expansions are written for € and ) it fol-l-ows tbat l(t) = 1+€Ar(t)+€2lr(t)+... ....(5.11) l(t) = 1+€Ar(t)+€2lr(t)+... ....(5.11) Eence, the approximation made is to neglect aLL terms but the first in (rF), and so, first in (rF), and so, 4rn = -atr"f(€). dT dt 4rn = -atr"f(€). ....(5.14) dT dt ....(5.14) (ii) The other approximation made compensates for the negJ-ect of the boundary aad the conditione i:aposed there. Since the tempera- ture is now the same over the whol-e region, it follows tbat the temperature just insi-cle the boundary is d.l-ffereat fron the tenperature of the environnent (or tbe temperature inposed on the boundary). So the first term in (ff), which is nroportiona.l to the heat fJ-ux oa the surface S, is replaced by a term which suggests a heat J-oss to the environnent given by l,Iewtonts law of 6t. ,qoolj-ng. That is, $ { Sraae.q5 e -Fg , where S is the surface area of the region V. (Tbe surroundiugs are assumed to be at zero temperature). The quantity p is calJ-ed the effective heat-transfer coefficient. It is called this becauee the coeffj-cient represents the surface cobling coefficieat (see p.6), when the tenperature variation across the region i-s replaced by an averaged temperature. Tbe approxination wbich is made can be regarded a6 a truncatecl per- turbatiou e:tpansion also. For the expressions above to be equalt B woulcl nornalS-y be a function of tine. In fact P( ) / d (5 t5) ,qoolj-ng. That is, $ { Sraae.q5 e -Fg , where S is the surface area of the region V. (Tbe surroundiugs are assumed to be at zero temperature). The quantity p is calJ-ed the effective heat-transfer coefficient. It is called this becauee the coeffj-cient represents the surface cobling coefficieat (see p.6), when the tenperature variation across the region i-s replaced by an averaged temperature. 5.2. The SDace-averaAing Process Tbe approxination wbich is made can be regarded a6 a truncatecl per- turbatiou e:tpansion also. For the expressions above to be equalt B woulcl nornalS-y be a function of tine. In fact P(t) = { grad.o.fl! /sd . . . . (5.t5) Since interest is focussed. on tines near those at which O reacbes its maximum, and coadlltions are quasi-steady, F(t) is approximately constant. Thj.s is the approxinatLon made hele. (t'tote that when t=O r p raust be infinite). Ilence the equation can be written as P(t) = { g /sd Since interest is focussed. on tines near those at which O reacbes its maximum, and coadlltions are quasi-steady, F(t) is approximately constant. Thj.s is the approxinatLon made hele. (t'tote that when t=O r p raust be infinite). Ilence the equation can be written as dE . ged dt v' dl = _aET .. dE . ged dt v' dl = _aET .. .... (5.16) Thue the systen haE been reduced to the pair of ordinary differ- ential. equations (14) and (15) j'n t. The spatial- variation of O antl ) had been eliniaated, eee Fig.!.4. Fig.5.4. Fig.5.4. 64. The problen wbich arises is to ctetenniae tbie value of F for the different geonetries discuseecl. Tbonas et. al. were concerned w.ith cleternining the conclitions for rrcriticalitytt and so interest has beea confined with times prior to the temperature reaching its maxj^Bum' For zero order reactions (n= O) , Kamenetskiil obtained values fron the heat baLance (see equation (15)) i'n the steady state' To facili- tate discussion of this, the quaatities 6 anA 1 will- be si'rapLy writtea as O ancl \ , where it is now unilerstood that the forner are meant. Hence, In the steadY state ....(5.r?) r?) ....(5.r?) I(anenetekii, using a binonial approrination to tbe Arrheaius lawt wrote f(O) = 6 eg . Then, the steady state sol-ution for the tenpera- ture ls given by so1-ving the resul-tiag transcendentaL equation' Iu the cases mentioned before S/V = 1+i. This has two roots provicledl the para:neter (f+i)p is sufficientl-y large (see Fig,5,5). ('; )o o A ,f=,5e" j/- FLg,.5.5- ('; )o o FLg,.5.5- 65, The smallest value of F for whj-cb the equation hao reaL roots rep- reseats the criticaL state (i.e. if F were sma].]-er, then it is not possible to fincl a steady state solution, in fact the system is un- stable). TEE CUI-OFF PROBLS,I In this chapter, a d.etailed discuEsion of what will be called tbe cut-off problem will- be given. It is f,eLt that tbere is a class of probLens which are of conslderable mathematical- lnterest ernd are in nany ways like the cl-aseical free boundary problems 5-n fluid clynami-ce. 5.2. The SDace-averaAing Process Eence, by finding the gradi-ent of the tangent to f = 6eO which passes through the origin it follows that p = Oe,/(l+J)' Thonas9 uses tbese estimates in deternining the conditions for a critical state (in tUe sense that he forrnulatedl) for a reaction other than one of zero order. The vaLiclity of this depends on the extent to which the transient tenperature tlistribution is of the same form aE the critical eteady state' Probably the latest paper of Acll-er aad Enig4 gio"u the nost iLLustrative clescription of the systen, that is equations (f4) and (f6). Here the equatioas are combined as *i = - i(r-le/rnt(c)), .. (5.18) obtainecl. by dividing tbe equatioae, wbere A = (l+i)F antt the initial conclitioae tr = 1, O = O. t is regarded. as a paraneter for the inte- gral curves obtained by integrating equation (f8). These are iLluet- rated below j.n Fig.J.6. (See over page). .. (5.18) From equation (L2) t the significant parameters ate a and A. The rest of the paper is devoted to discussing the relationshlp between them when the conditions are critical j-.e. to finding the first curve which passes through a point of inflection, and the cor- responding naxinum temperature rise. This is calLed by the ignition teraperature by the chemists. The above anaLysis givee Or"* = 1*o*. To the chenist this represeats the highest tenperature possibl-e before ahother reaction takes over and ignites the nateriaL. The 66, 'Ilreve&tiea of 6:uah unc-oatrdLted conhustLon 5.€ of the inter.aet of, tb,Ls sy-eten arong cbeniet,s. reoponslble for m-trcb It is f,eJ.t that, Lf ttllvt-ebed to u6,e the crf,terl-on gtven by ThouaE to cJ.as if,y reactioas as Etab1e ,or U,laetabtre, :Lt uonldl be nrcb rotrs us'G,f,ltl to consicle.r the aeynptotic etabiltty as cliecueged ea.rIle:r. EbE introd,uction of the of the effectJ.ve beat-transfer coefficient i.s reasona'bx'es Ln that jr.t w:ilt glve a faLr].y accu:rate angweri provicledt the tr:r,auF':[eat teaperature dLstribrutj-on up to aad noer Lte aarJ-uun is uearlSr the saae as :i.a the cri.tical eteadltrr state. EowEver, f,t i.c qot a teebnique that natunal-lSr loadle i.teelf to hJ.gher onderr approx- *?atiorne or that rri.Il alJ.ow the arlor nad,e La Lhe aBproxinatlon to be ca3.cuJ-ated, ea"eily. 67. 6,L, The Exlgtenoe of Cut-off . It is intended. to show that tbere is a which the reaetant Ls exhausteil in a finite to exhaust the reactant w:ill-, of courser be and, so there wil-} appear a boundary, as yet no further heat is geaerated'. ft was shown 2,4) that there dlid exist a uuique solution sider the couplecl pair of equations class of problene for time. Tbe tine takea a fuaction of position unknown, beyontl which in Chapter 2 (section of this problen. Con- ae + alnf(x,e) = 8i ' .... (6.f) . . . . (6.4) 0l6T= - trnf(xro) , ae + alnf(x,e) = 8i ' .... (6.f) . . . . (6.4) 0l6T= - trnf(xro) , ae + alnf(x,e) = 8i ' . . . . (6.4) It will be assu.nect that f is a bounded positive function. (es it was mentioned in Chapt er 3 t f is nondecreasing rrritb O aLso. ) These requirements were alL satLsfied in the physical- situations fron which this analysis &roso. Equation (2) can be written gg)= r(x,o), nttl' 0t\n-1/ 58. fi (ros r/r) = f(xr0), n=L. ' ' ' ' (6'4) In the fj.rst case, \ / l, if *-o is plottecl against ti-ne for a fixed, x, the slope is 6iven by (n-1) f (xro). Assune that the react- ant is consuned' in a finite ti-ne, i'e' \= O for t = to' Then as t tends to to, \t-o tends to zero or iafinity according as a ie less than or greater than unity. This is il-lustrated below' fi fi Now, if a function of time is unbounded as t tends to tor then it Now, if a function of time is unbounded as t tends to tor then it follows that its derlvai;ive (assuning that it exists) is also unboundeil as t tends to to. This is provecl' in the following Lernrna. !s,: Assume that is unbounded t+ t . o T(t), Tr(t) erist ast4t, Then o for O-< t <t and that T(t) o T t (t) is aLso unbounded as !s,: Assume that is unbounded t+ t . o T(t), Tr(t) erist ast4t, Then o for O-< t <t and that T(t) o T t (t) is aLso unbounded as Assume to the contrary that ft(t) is bounded for aJ.J- t, and in particul-ar as t+to. Then there exlst Bl-r Ba such that Bz4 T'(t) Proof: i.e. Bat 69, which inpl.ies tnat T(t) is bor:nded as t*to i tbue contradicting the ProPosition' g Eence, if n ) 1, f(xro) tends to infinity as t tencls to to. This contradicts the requirement that f be bound'ed. Further it follows that, ia tbe case of n) l-, all the reactant cannot be consured i':r a finite time. In the case of n 1 ! Lt can be shown that the reactant will be consumed in a finite ti-me' Since f is boundecl and positive positive lq> f(xro(xrt)) 7. n for Thus fron (l) it follows tbat al-l xrt. Thus fron (l) it follows tbat * ,tt-") < (n-r)n , (n<1) i. €. .\1-o ) r-o (t-n)nt r o (6,5) * ,tt-") < (n-r)n , (n<1) i. €. .\1-o ) r-o (t-n)nt r o (6,5) (n<1) * ,tt-") < (n-r)n , (n<1) i. €. .\1-o ) r-o (t-n)nt r o where lo is tbe initial concentration of I as a function of poeitS-on. However, silce it is an obvj-ous physical requirement that \ renain positive, lt follows that,\ becones zero in a finlte timet say to. Then for t 7 to there is no further heat generation aad equatioa (l-) becones sinpJ-y de Ag = tr OE ch is the heat conduction equatlon w:ith no beat source. the heat conduction equatlon w:ith no beat source. For the case tl = lr plot l-og (VI) against time and note that Log (V\) tends to infinity as X tendls to zero. 70. 70 tshe eloBe wI11 be gd-ven by ,f (xra). Eemce the Eaae ArgF![eat wt'Il 70. tshe eloBe wI11 be gd-ven by ,f (xra). Eemce the Eaae ArgF![eat wt'Il apply aE Lt dtitl in the oase n ? 1' trhue' it, naY be eonclu.dled'l n ( I the reaef,ant r*il'x be coueuned l'n a fid.te tl.nir nV L. the reaa,tanrt cannot be consus.Ed i.n a finite tlue aait wLlL tendl assrnptotical,ly te aqrs as t becoue,s large. Thls phenonetou (tn the former case) bas pronptecl the name ttsutr-'ff problenrr. Proof: !ftea ther,e is, no outrEff (n " 1) the Broblem o'f, actual\r so1ving the equatious (1) and (2) wiJ-l be more straight-forwardlr :Lu that, aJ.thougb the equatioas are uonlLueEr, tbere Le ao dlvl-aLon of, tbe rogion in t'be (.xnt) Etrte,ee ia wb;icb tkey.apply' Inteye.st W!I.J- rror bre conf,l.nedt to n ( Ir where tbere Ls a qut-off, tise to wh1oh willl in geueral' be a firnction of !(r EEI c{r). Ehe problen may be stated as aa + a\nr(xro') = *9, r<o( *9, t) c(x) .. t.(6r?l .r,.(6.8) A9 A9 7L. (5.9) alfr = trtrf ( ) (The last of tbese is subject to tbe condition A = O when t = C(x) ) ' These equations are to be solved with gj-ven initial (€ ana I ) and boundary (O onl-y) conditions. If n is zero, the interpretation that f = 0 wb.en t z C(x) must be added'' Thie wil-I meaa a sharp cut- off in the heat source and rate of reactant consunption. There are also some coatinuity conditions on 1 = C(x). It was shown in Chap- ter 2 that tbe temperature and. its first derivatives l.|-ith respect to xi are continuous on the cut-off surface. FhysicalJ-y tbls i's nerely asserting that both the tenperature and heat fhur are continuous. Note that it is assumed here that there is no change in the ther-naI properties of the naterial brought about by tbis burning process' Otherwise these conditions (and the equations) woul-dt have to be noclified to al1ow for this. fr In the case of a nonuero order reaction it has been ebow:c tbat AO/A| is continuous on t = C(x). Further it vras notecl that in a reaction of zero orcler it is aot possible, in general, to attrlbute the discontinuity on 1 = C(x) whoIly to either AOrlAt or to the eecond d.erivatives of g witb respect to the x1. Though in the opecj-al- caoe of C(x) constant it is onJ.y Ae/Af, which can be dliscontLn- llolrs r To ill-uetrate the posi-tion further, consider the sent-infinite problem x > O in which tbe tenperature is prescribecl on x = O (and r,rhen t = O). Then the cut-off curve (as it is in this case) divictes tbe positive quad.rant of the (xrt) plane as it is shown i-n Fig.5.]. 72. Proof: eq rctron (?) ei*-t o.'" ({) eq rctron (?) ei*-t o.'" ({) As it is drawn with Ct(x))Or the cut-off curve replegenls a Irave advancing into the region Leaving bebind it a bgrnt out residue in which the ordinary heat conduction equation aptrr}ies. Ja the eol- ution of these problens one of tbe prinary u:xknownE is, of coutrset the cut-off curve itsel-f, as wel1. as the depeudent variablee g and ) She probLenr j-s etilL well-posed however, because of the iufornatLou about I on t = C(x). 6.2. Examples of Cut-off Problens. Consider the zero order reaction in wbich f ls merely a functioa of O. fhen the cut-off probl-em whea tbe equations are ind.ependent of x, is given by This For att da r\ Ei = af(o) =-"t? , t(C. ... is a conparatively si-nple system to solvet at least in example, when f(e) = a t B0 the sol-utLon subject to O . (5.10) princlple. = Or l= t The tine =oris e = ftte"Ft-r) , \ = r -${ ""Ft- 1) , da r\ Ei = af(o) =-"t? , t(C. ... l i . (5.10) i l l da r\ Ei = af(o) =-"t? , t(C. ... l i . (5.10) i l l This For att is a conparatively si-nple system to solvet at least in example, when f(e) = a t B0 the sol-utLon subject to O princlple. = Or l= t The tine =oris e = ftte"Ft-r) , \ = r -${ ""Ft- 1) , This is a conparatively si-nple system to solvet at least in princlple. 7t. of, cut-off ie grvea by \,= 'o i'e,. t =* los ( r * $) . Eence dA/:61 = O for t)C, which Lnpl5.ea that 6 = o1 aDi[ l= O for t>C. fhese a're i,Llustratecl below. Eln. 5.4 Eln. 5.4 lfhe eolutlon above would b,e tbe ae5rnBtotic ve.lue as x teode to tafS-atty of the senl-infLnl.te problen nC.th O = 0 e[ r = O. '**" 5rrobl.eu :Ls more difflsuLt to so1ve, llhat 5.e, i't Lo requlrect to s:glrile oeg as,Z 00 0tt + a(e + F0) t ( s(r) '...(5.11) 0I = | E subjectto6=Oon on t = C(x). For I having a horizontal t ) c(x) cr + pe) , t ( c(x) x = O and rvhen t = Oi I = ) O the cut-off curve wi]-L asynptotet=Casabove. ?4, ,... (6.ra) . . . . (6,11) t=0, )=0 as shown belowt a2o -ox 0e0tt J. rshen appear 0I = | E t ) c(x) cr + pe) , t ( c(x) ,... (6.ra) . . . . (6,11) a2o -ox 0e0tt subjectto6=Oon on t = C(x). For I having a horizontal x = O and rvhen t = Oi I = ) O the cut-off curve wi]-L asynptotet=Casabove. t=0, )=0 as shown belowt J. rshen appear so the solution so obtaiaed was not in fact taking into account what it shouLd have been, i'e' that tbe reactant is exhausted at some points earlier than it is at others' Thj'e tech- nique would bave worked for the solution iaclepend'ent of x l'e' 4s iD equation (tO). Eere O and, \ coulct have been expanded in power eeries in 9i the solution obtained being exactly as given, except that it wouldbeexpandedinpowersofB.Thisls,ofcourse,becausethe cut-off curve, for any F, occurs at the sane time for any point' l d The problem discussed above has not been solved completeLyt however, it is hoped that it se1rres to illustrate some of tbe proper- ties of the systen. It ls now inteatled to discuss in d'etall- another probLen of interest, whieh can be solved. using a similarity technique' Ehe solution of (ff) is easily obtainecl for t < C = nin C(x) t by taking Laplace transformsi, as e = cs I u9*"rttvr"*l ill Eowever, it does not appear possible to o", "1, such technique for poiats for which t ) C. For exarnpJ-e, at the point B (%rtB), the solution must in sone way take into account the fact that the reactant has been burnt out alread.y at sone points. At this stage of tbe problem, it was considered, that one way of obtaining the solution would be to u6e a perturbation expansion l-u F. Fod when I = O, the cut-off curve is t = constant (passing through A 1n Fig. 6.5), Deaote this by t = Co. Substitute 0 = Oo * 961 . ., t \ = \o + B), + .. . Aad. further it was thou6ht tbat the continuity condition€i on t = C(x) could be transferred to t = Co by using suitable 75. Taylor expansions , However, when this was done lt r'+as noticed' that the expausions were singuJ-ar. on cl-oser inspection of tbe problen it:i.s found. that this should have been expected', for, the zero order problen has as cut-off curve a cbaracteristic of the parabolic dif- ferential equation. so the solution so obtaiaed was not in fact taking into account what it shouLd have been, i'e' that tbe reactant is exhausted at some points earlier than it is at others' Thj'e tech- nique would bave worked for the solution iaclepend'ent of x l'e' 4s iD equation (tO). Eere O and, \ coulct have been expanded in power eeries in 9i the solution obtained being exactly as given, except that it wouldbeexpandedinpowersofB.Thisls,ofcourse,becausethe cut-off curve, for any F, occurs at the sane time for any point' The problem discussed above has not been solved completeLyt however, it is hoped that it se1rres to illustrate some of tbe proper- ties of the systen. It ls now inteatled to discuss in d'etall- another probLen of interest, whieh can be solved. using a similarity technique' Taylor expansions , However, when this was done lt r'+as noticed' that the expausions were singuJ-ar. on cl-oser inspection of tbe problen it:i.s found. that this should have been expected', for, the zero order problen has as cut-off curve a cbaracteristic of the parabolic dif- ferential equation. 5.4. A Sinilarity Solutiou. This problem, is again a zero order reaction, whicb as sbown in section J-, will exhaust the reactant in a finlte tine. Eeat will be generated according to f = cr + 9g/x2. Eence the problem can be stated a2e Be ae - + " *5 = ;;, t<C(x) . . . . (6.14) 6xt x # : **, t>c(x) " " (5'15) 26, *,,""1 ,"- :::"':': ,1.,.1 . .. . (6.16) There wirl be solvecl subject to the initial cond'itLons e = ax2r \ = Yx2 and boundary condition'Q = bt s1 3 = O' It is possib3'e to el-ininate one of the constants. By a suitable s{aling of tbe cle- pendent variabLes, it follows that & can be taJren ae uuity' This can be done by writing o€ and g) for I and \ "e"pectivel-y, and reclefining a and b as qa and ob, the equations and boundary con- diti.ons are then as abovel with c = 1. In effect all this is d.oing, is choosi-ng a particular member of a cl-ass of probLemet which are defined for variou6 &' The particular member is that obtained. when o = I. 0n first glaace, it appears that this problen possesses al-I the difficulties of the one in the previous section. Ilowever, this problem possesses a similarity solution wbich enabl-ee the partial differential" equations to be written as ordinary ctif- ferential equations in a sj.ngle variable. For if the transforma- tions *,,""1 ,"- :::"':': ,1.,.1 . .. . (6.16) . .. . (6.16) tions a r-> FX 2 t +r-t o -+r2o I * r2\, a r-> FX 2 t +r-t o -+r2o I * r2\, conditlons these terns of the are appl,ied to both the equations and the bou.ndary are !gg[34g1],. Eence the probJ.em can be written in 1 single variabLe v = x/t?. Define o=r2l\\ r g\z/, I ^l \ = xth.(z), ) . . . . (6.12) o=r2l\\ r g\z/, I ^l \ = xth.(z), ) . . . . (6.12) o=r2l\\ r g\z/, I ^l \ = xth.(z), ) . . . . (6.12) 77, Then equation (16) 'l . in'(z) = reduces to L2+ z- i fi i ' Then equation (16) 'l . in'(z) = reduces to L2+ z- Then equation (16) reduces to Then equation (16) reduces to subJect to tbe conditi.on that h tends to il as z teuds to infiaity' The cut-off curve in tlre xrt pJ-ane is defined, by \(xrt) = 01 i'€' b(z) = 0, which lnplies z = K. Eeace the curve'g = C(x) wil'I be the 1 parabo1ar x = i(tU . Equati,on (1-B) is appl-lcable for only z) K andl is subJect to the definl-ng conclition for K, h(K) = O' Likernrise eguations (L4) ana (r5) reduce to Likernrise eguations (L4) ana (r5) reduce to 2 z 2 z g,,(z) + (42+u7/ilgt(z) +(Fe ile,k) +1 = Orz)K,..(5.19) gtt(z) +(42+23/2)g,'(il + zg,Q) E Of z<K "'(6'20) 2 z 2 z g,,(z) + (42+u7/ilgt(z) +(Fe ile,k) +1 = Orz)K,..(5.19) gtt(z) +(42+23/2)g,'(il + zg,Q) E Of z<K "'(6'20) The a.nd matchiag condition6 on the cut-off curve z = K become slnply g dg/d"z continuous on 2 = I(. The matchiag condition6 on the cut-off curve z = K become slnply g t T<K e1**,ir lzo) T>( eounC rin [rt) .{,*,t*; tt't) ztE I o q,lr \ = Trsr EE:6.9. EE:6.9. The bounclary antl inj.tial conditions on O and I can now be appJ-J.ed f,o g ancl h respectively Hencet The bounclary antl inj.tial conditions on O and I can now be appJ-J.ed f,o g ancl h respectively. Hencet g--ra) I as z+cD u+UJ g, t\t A/uZ as z*O, i.e. zzg(d-tb as z{o. g--ra) I as z+cD u+UJ g, t\t A/uZ as z*O, i.e. zzg(d-tb as z{o. 78, the situati.on On is looking at the z Plane for as g-A/zZ BrBt continuous g+8 r z from zero to infinitYn the situati.on On is looking at the z Plane for as g-A/zZ BrBt continuous g+8 r z from zero to infinitYn BrBt continuous Since the function g depeuds on z a1one, lt follows that the isotherms (lines joining points of the same function value) are parabolas in the x,t pLane also. Equation (eO) can be sol-ved by latti4g g(z| = w( il/22 and so wr.t +zw'/z-w -0, w(o)=b. (5.21) One solution of this differential equation other can be found by substituting w = (f substitutingn and solving for w, aud hence is t + z2/z and so + zz/z)v?) in (2L). g it foll-ovrs that the On eG) = r(j[+ t/22) + n7"-"2/47, + n*(t*t/zz)etb/il), eG) = r(j[+ t/22) + n7"-"2/47, + n*(t*t/zz)etb/il), ) wbere R is an arbitrary constant. Tbe latter wil:. o) ie finite. Then it woul-d. be expected that g(o) is o" r.""u: "rt:.::.t by using the matching conditions at z = K. In general-r R w'il-L be a function of K, Howevern equatS-on (fg) presents sone clifficulty. By writing J = 7-/z it folLows that y2g" (ey+L/zfllr +(B+z)e + 1 -o. .... (6,2t) Ilence it fol-lows that X = 0 (i,e. z = (D ) is an iregular singular point and so there w:lll not exist a solution in serj-es. Further the sol-ution of (19) wtren I = O (c.f, Qz)) is lows that X = 0 (i,e. z = (D ) is an iregular singular point and so there w:lll not exist a solution in serj-es. Further the l ti of (19) t I O (c f Qz)) is so = 1 t P(* + t/z?1 + q!-l"2/4/z +,r*C+ + r/22)ect(z/il), That is Bo(o) ie finite. Then it woul-d. be expected that g(o) is so = 1 t P(* + t/z?1 + q!-l"2/4/z +,r*C+ + r/22)ect(z/il), That is Bo(o) ie finite. Then it woul-d. be expected that g(o) is ?9, finlte aIso, d,iff erential- ting since p / 0 does not al-ter greatly the character of.the .found equation. The conplementary function of (19) is/Uy let- finlte aIso, d,iff erential- ting since p / 0 does not al-ter greatly the character of.the .found equation. The conplementary function of (19) is/Uy let- ting e(z) where u = u2 = soe(s), ....(6.a4) and 6 i.s a constant. Hence G(s) satj-sfies e(z) where u = u2 = soe(s), ....(6.a4) and 6 i.s a constant. Hence G(s) satj-sfies e(z) where u = u2 = soe(s), ....(6.a4) and 6 i.s a constant. Hence G(s) satj-sfies 4sGf I provicled,462 +66 + B+2 =0. The differenti.alequation can be solved by taking a contour integral . (6.2j) in (25) +(Bo+Lo+6)Gr+oG = or ,.. provicled,462 +66 + B+2 =0. The differenti.alequation can be solved by taking a contour integral in (25) provicled,462 +66 + B+2 =0. The differenti.alequation can be solved by taking a contour integral in (25) e(E) = f "rsr(r),b, where c is an appropriate contour in the conpJ.ex plane. This gLves ( ), , where c is an appropriate contour in the conpJ.ex plane. This gLves G(s) = / ers r6-1(r+t/4)6'1/2 ar, l c -r^-r | ' ' (5'?5) where c is such th.at I-""u J!+t/4>u'r/'7 o = o.J L since 6 must satisfy only the quad.ratic equatloa above, tbeu it can be chosen as the positive root, whlch imlJ-ies -3/4 -{ Real(6) <+f provided 9V O, G(s) = / ers r6-1(r+t/4)6'1/2 ar, l c -r^-r | ' ' (5'?5) where c is such th.at I-""u J!+t/4>u'r/'7 o = o.J L since 6 t satisfy l th d ti tl b b it where c is such th.at I-""u J!+t/4>u r/ 7 o = o.J L since 6 must satisfy only the quad.ratic equatloa above, tbeu it can be chosen as the positive root, whlch imlJ-ies -3/4 -{ Real(6) <+f provided 9V O, Two possJ-bJ'e choices for C are: Two possJ-bJ'e choices for C are: Two possJ bJ e choices for C are: Thus the two ind.ependent solutione for G(e) ate -To Oot l+ e-rs{r/a - ,16+3/2 e-ru(t/4 - 116+3/2 - 0/4)o'll2]ro-ra" , "6-1dr , -To Oot l+ e-rs{r/a - ,16+3/2 e-ru(t/4 - 116+3/2 - 0/4)o'll2]ro-ra" , "6-1dr , Bo. where u = u2 and 6 greater real part. is the root ot 402 + 56 + 9+2=0whichhasthe fter a litt]-e manipulation these caa be written as rrb 'B) After a litt]-e + t n\ I to I.r\ZrPl = lrfr-=J r 6za't2 o manipulation these "-ultuz/4 - s 16+3/2 caa be written as + (o + i/z)Qz/+ - ')bq"6d", ....(6.27) . . .. (6.e8) = ja*z fr;" {u214 - ")u'3/2 s6-1ds ' rrb 'B) After a litt]-e + t n\ I to I.r\ZrPl = lrfr-=J r 6za't2 o manipulation these "-ultuz/4 - s 16+3/2 caa be written as + (o + i/z)Qz/+ - ')bq"6d", ....(6.27) . . .. (6.e8) = ja*z fr;" {u214 - ")u'3/2 s6-1ds ' Thus the solution of (f9) can be written as e(z) = ,'u\rrr(zrg)+orr(z,g)J #t ....(6.29) ( O) i e(z) where C sanne as P is set ( compare = ,'u\rrr(zrg)+orr(z,g)J #t ....(6.29) and D are arbi.trary constants. Since equatioo (eO) is tUe that satisfied by the complementary function of (L9) when equal- to zero, then tbe general sol-utj-on of the forner is (29) ) : e(z) where C sanne as P is set ( compare = ,'u\rrr(zrg)+orr(z,g)J #t ....(6.29) and D are arbi.trary constants. Since equatioo (eO) is tUe that satisfied by the complementary function of (L9) when equal- to zero, then tbe general sol-utj-on of the forner is (29) ) : e(z) .f..(6.70) = rtuoto rr(zro) + Bt2?ro) ], where 6o = 6 when F =O i.e. 6o is the larger root of 4p2+ 5p + 2 = Ot which means that 69 = 4 . It is a simple exercise to verify that both I1(zrO) ancl lr(zrO) are 1-inear combinations of the J-inearl-y ln- dependent solutions given in (ZZ). In fact, it can be shown that they both behave iri-lke-lF/z as z tends to zero. Then in order to satisfy the recluirement for g( z) as z o, it fol-Lows tbat A+B = -b/r#. llow as z4(D, g(z)-ra, and so the asymptotic behaviour of the integrals in (27) and (28) nust be cal"culated. It can be showu that fot z Iarge, 6 3/2 ' (6':r) (6'tz) g , u'u -r(u, p) + Ctl 226 rz(u rg) '+ 6+3/2 I' (r+o ),/o o I as z.+(D . Henee, C = t" * g|r#' (zo) can be "r;r".: Thus the soLutj.on of the equations (19) and gL. e(il = -r-{u rrG,il/n*+_s(K)(rr(z,o) - rz(z,o)) J, z1K . . (6.17) rr^ = "utl . u*t,#ri"rrrz,F) + u(r) rr(z,u)i- # , z> K 'UUrU'i''n' vrhere BrD are functions of K given by the cond.itionsi ,; are continuous on z = K. AlL the way through this conputation the known expression for zlK (see equation Qe)) was of assista.nce in checking tb.e soLutions obtained. The quautity I(, as nentioned prevS-ously, is given by h(K) = O. By integration of equation (f8) , using (f9), it foLlows that K sat- isfies the following equation # + ge(K){t *::olt<z) + eFe'(K) - ag - lf(rz + p1 = o. (6.t5) Hence, using (55) and the equations obtainea rron (]3) ana (94) for g and d,g/dz continuous on z=Kr it is possi-b1e to find tbe conpJ-ete answer to the problem. Ilowever, the resuLting equation for K is extremery diffi-cult to use in practice. The author has actually found tbis equation, but it vriIl- not be written here. rn order to demonstrate the type of equatj-ons obtained^, the asymptotic vaLue of K wi].l be obtaineil for K Iarge. ThLs can easiry be done using the as}rnptotic behaviour of the integral-s glven in ( j1) ana 32). Thus using the expressions Ln (33) and (74) at z = I(r for z large it follow that, B * (za-ilt# for K large. Then this nay be used tn the equation in using asynptotic expansions of I, the respective orders of nragnitude. . . (6,36) for K, i.e, (15). Bowever, in and I^ r care must be taken hr:tth 1' For, us5-ng (j6) , (6,36) g(K) = a(t+z/zz) -..., -l g, (K) = -4a/zt -... , J for large z ' 6.t?i (6.r8) If juet the first order terms are ta.ken it fo1lows that L2F+ i. €. K= ('f rfrlEiu )* aF(l+lord)-aF -f(ra"p) = o, f,or K J-arge. ....$r3g) This expression repreaents the first approxination to tbe behavLour of K for enaLL f. By taking higher ord.er terms a better approximat- l-oa could be obtained,. f,ikewise the behaviour of K r"Jtth F can be obtained by findiug as)reptotLc expansions of I, and I, for J.arge p. It wouLd be expectecl that B J.arge would inply that E is large aLeo. Eence it ean be seen that it is possibLe to use the equations to obtal-n estjrnates for K. of course, it is possible in the generaI caser but tbis vrould invoLve an enormous anount of calcul-ation. This expression repreaents the first approxination to tbe behavLour of K for enaLL f. By taking higher ord.er terms a better approximat- l-oa could be obtained,. f,ikewise the behaviour of K r"Jtth F can be obtained by findiug as)reptotLc expansions of I, and I, for J.arge p. It wouLd be expectecl that B J.arge would inply that E is large aLeo. Eence it ean be seen that it is possibLe to use the equations to obtal-n estjrnates for K. of course, it is possible in the generaI caser but tbis vrould invoLve an enormous anount of calcul-ation. 8r, e.!@,. c0Ncl,usIoNS It is felt that the most interesting features of tbe systen described. in this thesls have been discussed'. This is botb fron the point of view of previous investigations into tbe probl-ent and of tbat of the natbenatician. Ijflth reference to tbe aspects dis- cussed in Chapters 2 and J, it is boped tbat as our knowleclge anfl scope of the nachinery Decessary to establ-ish the eXistencet unique- rressr etc. of solutions expands, so vrlLl the general-ity of the results obtained. Tbere is, of course, a ]ot of interest in these qualitative properties at present ancl ao doubt sone si6:rificant advances wiLl be made in the near future. It is felt tbat there is a more general- comparison theorem possibl-e, which wou1d. have embodietl the resuJ-ts given in ChaPtet 3. From the technical poiut of view there are, of courser a 1ot of itifficult problems to solve with this systen. Many of the pro- blems for which it is possib3-e to obtain solutions aaalytlcall-y are given in the book by Carslaw and J""g""22. Eowever, there is a much greater nunber of probl-ems for whicb there is no such eol-ution. For these, oae is forced to rnake varioua approxinations and find pertur- bation expaireions j-n different reg;ions of the ind.ependlent varia.b1e6. By doing this, one often gains an insight i-nto the difficult features of such problems, This ie usually of assistance when tbe problen is to be computed on an electronic computer. With the tremendous devel-opments in computing, it is now possible to solve many of the 84, extren€l-y nonlJlnqar probJ.6m6 whiob were too forl{id.e.b16 in the past, 'i:his approach ie the oae which wi-Ll.r'usuaa3.y be tbe nost fruitful for the plractieing ee.iantLst. &oweve:r, this ba.s not beea attempt,ed here. llhene ere nary aspects of this probJ.em that bave not been coa- s1dered' AE it has been meqtLo,nerd. in the conte,:rt of this thesd.e, there a?e llafly sf-npl5.f,y1.ng assuslrtio.ne trhLeh hato bee.n inade to the physieal rao'd,e;L fton whi-ch iibie analysia arore. Probably the most obvious of theee is. the one that suppooed the t,berual propertj.ee o.f the ne*itur are unchanged.by the conbustioa. Sone of the requiredl uae.hiner,yr for provlng, the exi.stence of a eol-utLon to sueh a eyeitea barg been eover6d. in C-hapter 2, and this woul-d foro ari i:rterest&ag generalization of the refiltrts. c0Ncl,usIoNS extren€l-y nonlJlnqar probJ.6m6 whiob were too forl{id.e.b16 in the past, 'i:his approach ie the oae which wi-Ll.r'usuaa3.y be tbe nost fruitful for the plractieing ee.iantLst. &oweve:r, this ba.s not beea attempt,ed here. 85. APPE}IDIX A.1. Existence of Sol-utions to ],(o)= lL(x.t.O). 2 lnd can, L satisfiee the conditions I Assume S* bel-ongs to C- t and II .(as in Chapter 2), anci\(xrtrO) is ll6lcler continuous (exponent a) in 5', for each fixed value of O. Further asaume that t[ satisfies a J,ipschitz cond.ition iu e, with Lipschitz constant M| and that there exists two functions O and 6 satisfylng the inequalitiee given in (2.15). Finally assune tbat there exists a functior y of class C2*o iu d,n whicb I coincides w'ith the given boundary conditions Oo(xrt) on Sn and. on B at t = O. Then there exists a soLution of the system 1,(O) = r|,(xrtrO), e = eo(xrt) on S" and on E at t = Or such that 0 is of class Cl*F in D* for any o < F <l- and of class "2+t for sone {> o. of Proof: Couslder the eet functions defined by t'{o1 Moo = \(x,tr6) - Md = $xrtrOo_1) MOo_L for a= 2111. , , ; = Ootxrt) on S* and on B at t = Or nV1 . r(01) li(on) oo Now if Or._1 is Holcier continuoua with expoaent c, J.a Dgr then so j j-s the functioa /o_rtxrt) = ilxrtrOo_r(xrt)). Then by Theorem 2 j-s the functioa /o_rtxrt) = ilxrtrOo_r(xrt)). Then by Theorem 2 o exists and is E6'lder continuous w"ith expouent e 5-n F*. Furthernore, from Theorem J, for any 0{6(1 leolr+o < P( ll"-tlo + lrll2 + I"Ilon-rlo). rt is shown in r.rcl{abb1o th"t 95. ( 95. g ( on \< on_I that is (Oo) is a monotone decreasing eeqrience which j-s boundecl beLow. Thls inp].ies that frn(*rt) is a].so bounded in iln and so leolf+O is bouncled by a constant Pt ind.epenclent of n. Hence the Ii-uit of this sequence exists anct ctefines a functlon O in Dr. Furtber O is HoLder contlauous of erponent 6. r}om Theoren 2, this neans that the systen L(ot) Mer = tlxrtro) U9, Or = Oo on the bound,- ariesi has a soLution Or of cLass Cl+p in D, for any 04p<l- and of cLass ,2+( for some f,>0. A.1. Existence of Sol-utions to ],(o)= lL(x.t.O). Now Now Now L(o' - oo) - M(er - oo) = f,(x,t) = = |(x,tie) - ?(*rt,o1-1) - M(e - on-I) and Ot- On = 0 on the boundaries of D, while L(o' - oo) - M(er - oo) = f,(x,t) = = |(x,tie) - ?(*rt,o1-1) - M(e - on-I) and Ot- On = 0 on the boundaries of D, while l0(x,t) | l0(x,t) | Sluce, by Theoren J, le'-€rrl < Kr lfrkrt) | anct the rJ-ghthand side tends to zero as n tends to infinity, or coincid.es vf,ltb o in ilr. Hence this o is the sol-ution of the system as requLred, and is of class cltg io Dn for any o <B<L aad of cr-ase c2+6 for some T> o. Moreover, siace each oo is bound.ed, it foLlows fron tbe L+6 estinate above that lolfrO ( prn where pr is depend.ent on only 6rLrlrand t . 6rLrlrand t . 6rLrlrand t . 8?. A,2. Existence of Solution to the Complete Systen. Assune s, be3-ongs to c2 and c2*n, r, satisfiee the condtitions r ancl rr, as in chapter 2, t and g satisfy the l,ipschitz conditions givea in (2.12) and (2.t3), and g is negative for \) O and zero whea tr= o' It is further assumed that there exLsts a functionlof claee ^2+a . = u--_- in D? which coincides with the boundary conditioas oo of o on s* and E'at t = o. elso the initial cond,ition \o(xro) is assuned. to be of cl-ass cl on B, and it is assumed. that there exists two fuactiona p and 5 continuous in iln and having continuous bounded clerivatlves ln D*r ,sucb that for O -( \ (Io, r(g) - f(x,t,9,)) > o > \(6) _ f(x,t,6rl), g, (eo h i r(g) - f(x,t,9,)) > o > \(6) _ f(x,t,6rl), g (eo Thea there exists a solutlon of the systen Thea there exists a solutlon of the systen L(9) = f(xrtrer\), 0) of = g(xrtrorlr), e = Oo(xrt)onSrandonFatt=0, I = Io(xr0)on Batt=0r such that € i-s of class cl-+p in D, for any 0( p<t ancl of class "2+T in D* for some [) O, an6 I is of class Cl in dr. Proof: Consider the set of functions defined by 9Ir at = g(xrt 16, If ) , L(On) = f(xrtrOn, \n), n=1r2r... gb ot = g(x, t ron_1 , Io) , n7. Z 88. in Dr, where in Dr, where in Dr, where en = 0o (n)rl) oa S, ancl on E at t = or en = 0o (n)rl) oa S, ancl on E at t = or \o = \o |.nVD onFatt=O, \o = \o |.nVD onFatt=O, Now if I' i" Hiild.er conti.nuous w:i-th exponeat a in so is tbe function fo(xrtrg) = f(xrtrOr\(xrt)). By in Appendi-x 1, Oo exists and. is certainly of claes C1 tloreover leolf*O ( Pr, D-*, th"o the result in D-. I' where Pr depeads on 6, Lrprand, fo. This neaas that Pr depends on Oorlu r blrt as it vr:iIl- be shown that tbe6e are unifornly bound.ed. it follows that there wiJ-l exist a unLform bounct for the seguence (l0nl1+6) . fn fact it can be shorflo tbat Now if I' i" Hiild.er conti.nuous w:i-th exponeat a in so is tbe function fo(xrtrg) = f(xrtrOr\(xrt)). By in Appendi-x 1, Oo exists and. A,2. Existence of Solution to the Complete Systen. is certainly of claes C1 tloreover leolf*O ( Pr, D-*, th"o the result in D-. I' 9, ( en { 5 for each nrin iln. 9, ( en { 5 for each nrin iln. Theoren 4 inpJ.ies that )o(xrt) exists and., provid.ecl Oo_, is of class Cl tn Dry then so too is \o, From the requiremente on g it folLows that each \o satisfies O(\n ( \o in D-r. Next it is shown that o ( on ( 6 J-n Dr. Thi.s forLows fron the comparisoa theorems proved by McNabblo. For L(en) - f(xrtrenrln) = o ( Lgo) - f(xrtrerlo), r,(On) - f(xrtren,U) = o ) L(d) - f(xrtr6, o). And since I { urr( 6 on the bound.aries of D* , these inequalitJ-es aLso hoLd. in the interior. Also siuce eacb co is EiiLder coutln- uous of exponent c (at Least) , then obvJ.ously each oo is equi- continuous on D-r. Dy the Ascoli Lenma, there exists a subsequ- ence tgnl , d'efined for k= Lrlr,., of (on), converging 'nifornly to a L{mit functioa g ctefinecr on dr. rt is sinple to show from lolr*o { Pt that I is of cr.ass co in 51 , and, further that o ie 89. of class cl in x, of class cl in x, of class cl in x, cLa66 cl io D*, Each \o is of cl-aes cI La Dr, wbere T'ikew"ise the same procedure foLlows from Oi\n(lo which inplies the existeace of a Lirn{t \. Thie linit is shomr to be of 9lt', of g(xrtron_1rln), Denote hrr(t) _= \o(xrt) - \o{*r 't). Therefore, 9lt', of g(xrtron_1rln), Denote hrr(t) _= \o(xrt) - \o{*r 't). Therefore, !!l. ot = B(xrtrorr-1(xrt) r\o(xrt) )- s(t'rtroa-l(xrrt) rln(rfrt)) r t'"'i8t1 € uflu"l tloo-r(x r)-on-r(x'rt)l] + Klx-x'1 Denote hrr(t) _= \o(xrt) - \o{*r 't). Therefore, !!l. ot = B(xrtrorr-1(xrt) r\o(xrt) )- s(t'rtroa-l(xrrt) rln(rfrt)) r t'"'i8t1 € uflu"l tloo-r(x,r)-on-r(x'rt)l] + Klx-x'1, !!l. ot = B(xrtrorr-1(xrt) r\o(xrt) )- s(t'rtroa-l(xrrt) rln(rfrt)) r t'"'i8t1 € uflu"l tloo-r(x,r)-on-r(x'rt)l] + Klx-x'1, !!l. ot = B(xrtrorr-1(xrt) r\o(xrt) )- s(t'rtroa-l(xrrt) rln(rfrt)) r t'"'i8t1 € uflu"l tloo-r(x,r)-on-r(x'rt)l] + Klx-x'1, us5-ng the Lipschitz cond.ition on g. Note Maad. K are ind.ependent of n. Then since each oo-, ie of crass clin x, by consid.erin8 UZ O separateJ.y, it foJ-lows that us5-ng the Lipschitz cond.ition on g. Note Maad. K are ind.ependent of n. A,2. Existence of Solution to the Complete Systen. Then since each oo-, ie of crass clin x, by consid.erin8 UZ O separateJ.y, it foJ-lows that lho(t) | = l\(xrt) (xf rt) | -( gr lx-x'l ror a13- xrxr in B. Likeraise by r,riting Io(x,t) = Io(x,o) + t rr*rz,oo-1(x,z),\(x, z)) dz r it follows that Io(x,t) = Io(x,o) + t rr*rz,oo-1(x,z),\(x, z)) dz r it follows that llo(xrt) - Io(xi!') | since (0o) and (lo) are unifornry bound.ed for arl n, it folLows that ll(xrt) - I(x,t') I So it can be coucLudecl that \ ts of class cl in dr. ll(xrt) - I(x,t') I So it can be coucLudecl that \ ts of class cl in dr. Thusr under the assumptions of the theorem o and I are certainLy HoJ-der conti-nuous with expoaent a, in Dr. This is sufficlent to ens[re tbat the systen \ ts of class cl in dr. Thusr under the assumptions of the theorem o and I are certainLy HoJ-der conti-nuous with expoaent a, in Dr. This is sufficlent to ens[re tbat the systen 90. L(er) = f(xrtrortr), # = g(xrtrel\) r or = go onSnandonBatt=0r )r = Io oagatt=Or has a solution Or, It ulth gt of class Cl+P for any O <9<L in D, and a].so of class ,2+t for some T >o in D-T. Now Now Now L(et-en) = p (xrt) s f(xrtrorl) - l(xrtronr\n), aodl Ot-eo = O on the boundaries of D, whiLe, from lbLeclnante estimates (Theoren J) lo'- onl ( P' l/(x,t) | . lfi|:l-fit l of crass gr*F in D, anct of cLass t2+T in Dr. and so aE n teads to l-afiaity through the val.ues nk the righthanil side tend.s to zero. f,ence Or coLncicleE w:ith O in il, and so O ie A si-ciJ.ar argunent using the equatlon A si-ciJ.ar argunent using the equatlon 0It 51- = g(xrtrerl) shows that )t ancl \ coLncide in 01,. Thus 0/0t exl-sts and, more- over is of class cl i.:r Dn., since g, O, and \ are aLso. I 9L. BIBI,IOGRAPHT 1. D.A,Frank-Kamenet6kii, trDiffUSion and Eeat Exchaage in chenical Kineticsrt, Prinoetou (1955). 2, P.L.Chanbre, cr.Chem.Phys, Vol 2o (L952)i 1795, 7. P.E.Tbonas, Trans.Faraday Soc, VoJ- 55r Part 6 (1960) i 81r' 4. J.Ailler and J.W.Enig, Combustion and Flane, VoJ. 8 (fg5kl , 97. 5, I.K.Walker and W.J.Earrison, N,Z,tI.Agr.Res, VoI , (J96O)i 861. 6, r.K.warkerI N.Z..J.sci, vor 4 (l-96L) i tog, ?. G.c.wake ancl LK.wal-ker1 N.Z.J.Scin VoI ? (1964) i 227, 8. P,B.Thomas and P.C.Bowes, J.Appliect Phys, VoL l-0 (1961) i 222, g. P.B.Thomas, Proc.Roy.Soc, Vo1 262 (195L) i 192. Lo. A,McNabb, J.l,lath.AnaJ., VoI 3 Q)6L)i Ltt, 1l-. P,K.Zetagijar Soobsc.Acad.Nauk.Gruzin SSR, Vol 26 (1961) i 257. 12. H.Batenan, rPartiaL Differential Equatioas of l'latbenatical- 1. D.A,Frank-Kamenet6kii, trDiffUSion and Eeat Exchaage in chenical Kineticsrt, Prinoetou (1955). 2, P.L.Chanbre, cr.Chem.Phys, Vol 2o (L952)i 1795, Fhyslcstr, Dover, New York (1944) i l-35. LJ. A.Frieclnau, J.Math.and, Mech, VoJ- 9 11p6O) i 539, 14. E.A.Coddington and N.Levinsoa, ttTheory of Ordinary Differentlal LJ. A.Frieclnau, J.Math.and, Mech, VoJ- 9 11p6O) i 539, 14. E.A.Coddington and N.Levinsoa, ttTheory of Ordinary Differentlal Equationstf , McGraw-Sil1 (Jg|j) i 7. L5, tf-J.Stoker, ttNonLinear Vibrationstt, Intersci-ence (1950) i 18. L6. J.R.PhiLip, Aust.J.Phys, VoI l? \L964) i 423. If. A.Erctelyi, ttTables of Integral fransformsrr, Vol 1 (1954) i 245. 18. M.Van Dyke, ttPerturbation Metbods in tr'Iuid l,Iechanicstr, Acadernic Press (1954) i 45. J-9. R.Bellnaa, Trans.An.t+ath.soc, Vo]. 54 (L948)i Z]-. 20. J.J.Stoker, Com'n.Pure and. App].ied Maths, Vo]. 8 (f_955) a L33, 21. A.rurcNabb, J.Iulath.rr,na1, Vo1 4 (lg6ili Lgl, 22' H',s.carslaw and. J.c.'Jaeger, ftconduction of Eeat J-a solid,srt, $94? J-9. R.Bellnaa, Trans.An.t+ath.soc, Vo]. 54 (L948)i Z]-. 20. J.J.Stoker, Com'n.Pure and. App].ied Maths, Vo]. 8 (f_955) a L33, 21. A.rurcNabb, J.Iulath.rr,na1, Vo1 4 (lg6ili Lgl, 21. A.rurcNabb, J.Iulath.rr,na1, Vo1 4 (lg6ili Lgl, 22' H',s.carslaw and. J.c.'Jaeger, ftconduction of Eeat J-a solid,srt, $94?)';
https://openalex.org/W4292003007
https://zenodo.org/records/6995508/files/jucs_article_27466.pdf
English
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Spline-Fourier Approximations of Discontinuous Waves
Zenodo (CERN European Organization for Nuclear Research)
1,998
cc-by
2,466
Journal of Universal Computer Science, vol. 4, no. 2 (1998), 110-113 submitted: 25/9/97, accepted: 1/11/97, appeared: 28/2/98  Springer Pub. Co. Journal of Universal Computer Science, vol. 4, no. 2 (1998), 110-113 submitted: 25/9/97, accepted: 1/11/97, appeared: 28/2/98  Springer Pub. Co. Spline-F ourier Appro ximations of Discon tin uous W a v es Roumen Anguelo v (Vista Univ ersit y anglv-r@marlin .vi sta.ac.za) Abstract: In the F ourier series appro ximation of real functions discon tin ui ti es of the functions or their deriv ativ es cause problems lik e Gibbs phenomenon or slo w uniform con v ergence. In the case of a nite n um b er of isolated discon tin ui ti es the problems can b e to a large extend recti ed b y using p erio dic splines in the series. This mo d- i ed F ourier series (Spline-F ouri er series) is applied to the n umerical solution of the w a v e equation (in p erio dic form) where discon tin ui ti es in the data functions or their deriv ativ es app ear quite often. The solution is sough t in the form of a Spline-F ouri er series ab out the space v ariable and close b ounds are obtained using a certain iterativ e pro cedure of Newton t yp e. Key W ords: V alidated n umerics, F ourier h yp er functoid, W a v e equation  In tro duction This w ork is inspired b y the ideas for v alidated appro ximations of function space problems [] and essen tially it is a further application of the F ourier Hyp er F unc- toid describ ed in [] where the co ecien t v ector c = (: : : ; c  ; c  ; c 0 ; c  ; c  ; : : :) of the F ourier series f (x)  X k = c k e ik  x f (x)  X k = c k e ik  x of a function f : [; ] ! R is appro ximated b y a v ector of the form c(k ) =  > > > > > < > > > > > : c k : 0  k  N c k : N  k   () k p P j = a j  k j : jk j > N () () Let fs j : j = ; ; : : : g b e a set of p erio dic splines de ned as p olynomials on (; ) suc h that s  (x) = x; x  (; ), s 0 j = s j  , s j  C j  (; ), j = ; ; : : : . These splines are closely related to the monosplines, whic h are w ell- kno wn in the spline theory , as w ell as the Bernoulli p olynomials. In fact n!s n and the nth monospline di er b y a constan t and s n (x) =  n n! B n ( x+  ) where B n is the nth Bernoulli p olynomial.  Spline-F ourier Series Let M b e the set of all p erio dical function with p erio d  whic h are p-times di eren tiable at ev ery p oin t of (; ] except for a nite n um b er of p oin ts  ;  ; : : : ; M and let d p f dx p  L  [; ]. There is a unique represen tation of ev ery function f  M in the form f (x) = a 0 + M X m= p X j = a mj s j (x +  m ) +  X k = k =0 b k e ik  x () () w her e  X k = k =0 b k e ik  x  C p (; ) The co ecien ts are obtained as follo ws a j =    d j  f dx j  ( 0) d j  f dx j  ( + 0)  =   Z   d j f (x) dx j dx ; j = ; : : : ; p a 0 =   Z   f (x)dx () b k =  (ik  ) p Z   d p f (x) dx p e ik  dx ; k = ; ; : : : The rounding S N : M ! S N (M) is de ned b y () b k =  (ik  ) p Z   d p f (x) dx p e ik  dx ; k = ; ; : : : The rounding S N : M ! S N (M) is de ned b y The rounding S N : M ! S N (M) is de ned b y S N (f ; x) = a 0 + M X m= p X j = a mj s j (x +  m ) + N X k =N k =0 b k e ik  x i.e. b y truncating the series at k = N . The error is estimated as follo ws i.e. b y truncating the series at k = N .  In tro duction F rom the F ourier series expansions s j (x) =  (i ) j  X k = k =0 () k k j e ik  x ; j   111 Anguelov R.: Spline-Fourier Approximations of Discontinuous Waves w e can see that the appro ximation of the F ourier co ecien ts of the form ()is equiv alen t to appro ximating the function f b y linear com binations of fs j : j = ; : : : ; pg [ fe ik  x : k = 0; ; : : : ; N g F or practical reasons that can b e seen b elo w w e w ould prefer to use the spline notations rather then the ab o v e ansatz () for the co ecien ts.  Numerical Metho d for the W a v e Equation W e consider a w a v e equation of the form u(x; t) tt u(x; t) xx = (t)u(x; t) + (x; t)) u(x; 0) = g  (x) ; u t (x; 0) = g  (x) with p erio dic b oundary conditions at x =  and x = , assuming that , g  , g  or some of their deriv ativ es ma y b e discon tin uous but they can b e represen ted as a spline-F ourier series (). p ( ) An in terv al enclosure of the solution is calculated in the form U (x; t) = A 0 (t) + M X m= p X j =  X "= A mj " (t)s j (x + "t +  m ) + N X k =N k =0 B k (t)e ik  x using an iterativ e pro cedure U (k +) = ( )U (k ) +   g +   R R G(x;t) U (k )  U (k +) = ( )U (k ) +   g +   R R G(x;t) U (k )  where G(x; t) is a triangle with v ertices (x; t); (x t; 0); (x + t; 0) and g (x; t) =   g  (x + t) + g  (x t) + Z x+t xt g  ( )d + Z Z G(x;t) (y ;  )dy d !   where G(x; t) is a triangle with v ertices (x; t); (x t; 0); (x + t; 0) and g (x; t) =   g  (x + t) + g  (x t) + Z x+t xt g  ( )d + Z Z G(x;t) (y ;  )dy d ! Essen tial part of eac h iteration is the ev aluation of the in tegral. The follo wing form ulas are used: Z Z G(x;t)  q q ! s j (y )dy d = s j +q + (x + t) + ( ) q s j +q + (x t)  q X l=0 lev en t q l (q l )! s j +l+ (x) Z Z G(x;t)  q q !  Spline-F ourier Series The error is estimated as follo ws jf (x) S N (f ; x)j  " N = jf (x) S N (f ; x)j  " N =   Z    d p f (x) dx p   dx M X m= a  mp  N X k = (k  ) p jb k j  !     (p ) p N p    Therefore w e ha v e the inclusion Therefore w e ha v e the inclusion f (x)  a 0 + M X m= p X j = a mj s j (x +  m ) +  X k = k =0 b k e ik  x + [" N ; " N ] 112 Anguelov R.: Spline-Fourier Approximations of Discontinuous Waves  Numerical Examples  Numerical Examples Example . u tt u xx =  (t + )  u +   (t + )  sin( x) + (t + )(x; t) where (x; t) is a piecewise constan t function in [0; ]  [0; ) as de ned on the sk etc h u(0; t) = 0 u(; t) = 0 ; t  0 u(x; 0) = sin ( x) +  (x) u t (x; 0) =  sin ( x) +  (x); 0  x   where (x) =  x 0  x  0:  x 0:  x   - x q  q   t 0  q q @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ -  - 0 0 0 0 0 0 Exact Solution where (x; t) is a piecewise constan t function in [0; ]  [0; ) as de ned on the sk etc h u(x; 0) = sin ( x) +  (x) u t (x; 0) =  sin ( x) +  (x); 0  x   where (x) =  x 0  x  0:  x 0:  x   Exact Solution u(x; t) = (t + )  (sin( x) + s  (x + t + 0:) + s  (x t + 0:) s  (x + t 0:) s  (x t 0:)) E xampl e : u tt u xx =    +  (t + )   u u(x; 0) = (s  (x + ) s  (x)) u t (x; 0) = 0 In b oth examples it is demonstrated that using relativ ely small v alues of p and N ,e.g.p = ; N =  close b ounds for the solutions are obtained. In example  since the exact solution is in S N (M) the computed b ounds are particularly close giving up to 0 correct digits of the solution. In example  using similar v alues of p and N - correct digits of the solution are obtained. All programs are written in P ASCAL-XSC.  Numerical Metho d for the W a v e Equation s j (y )dy d = s j +q + (x + t) + ( ) q s j +q + (x t)  q X l=0 lev en t q l (q l )! s j +l+ (x) Z Z G(x;t)  q q ! s j (y +  )dy d = q + X l=0     l t q +l (q +  l )! s j +l+ (x + t)     q + s j +q + (x t) Z Z G(x;t)  q q ! s j (y  )dy d = q + X l=0     l t q +l (q +  l )! s j +l+ (x + t) +     q + s j +q + (x t) Z Z G(x;t)  q q ! e ik  y dy d =   ik   q + e ik  (x+t) +   ik   q + e ik  (xt)  q X l=0 lev en t q l (q l )! e ik  x =   X l=0 lev en (ik  ) l t q +l+ (q + l + )! e ik  x =   X l=0 lev en (ik  ) l t q +l+ (q + l + )! e ik  x l=0 lev en The in tegration o v er G(x; t) pro duces splines with larger indexes. The splines s j that are sucien tly smo oth (e.g. j > p) are rounded as follo ws s j (x)   (i ) j N X k =N k =0 () k k j e ik  x +  (j ) j N j  [; ] 113 Anguelov R.: Spline-Fourier Approximations of Discontinuous Waves . Anguelo v R., Guaran teed b ounds for the Solution of the W a v e Equation, Questiones Mathematicae, V ol  (-),  . . Kauc her,E.,Baumhof,C., A V eri ed Computation of F ourier-Represen tatio n of Solu- tions for F unctional Equation, Computing, Suppl. , 0-( ), Springer-V erlag, Vienna,  . . Kauc her,E.,Mirank er,W., Self-v alidati ng Numerics for F unction Space Problems, Academic Press,  . . Anguelo v R., Guaran teed b ounds for the Solution of the W a v e Equation, Questiones Mathematicae, V ol  (-),  . . Kauc her,E.,Baumhof,C., A V eri ed Computation of F ourier-Represen tatio n of Solu- tions for F unctional Equation, Computing, Suppl. , 0-( ), Springer-V erlag, Vienna,  . . Kauc her,E.,Mirank er,W., Self-v alidati ng Numerics for F unction Space Problems, Academic Press,  .
https://openalex.org/W2058157145
https://link.springer.com/content/pdf/10.1007/s00455-014-9580-3.pdf
English
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Influence of the Chin-Down and Chin-Tuck Maneuver on the Swallowing Kinematics of Healthy Adults
Dysphagia
2,014
cc-by
6,228
Influence of the Chin-Down and Chin-Tuck Maneuver on the Swallowing Kinematics of Healthy Adults Ja-Ho Leigh • Byung-Mo Oh • Han Gil Seo • Goo Joo Lee • Yusun Min • Keewon Kim • Jung Chan Lee • Tai Ryoon Han Received: 10 March 2014 / Accepted: 23 September 2014 / Published online: 31 October 2014  The Author(s) 2014. This article is published with open access at Springerlink.com Abstract The purpose of the study was to investigate the influence of the chin-tuck maneuver on the movements of swallowing-related structures in healthy subjects and formulate standard instructions for the maneuver. A total of 40 healthy volunteers(20menand20women)swallowed10 mLofdiluted barium solution in a ‘‘normal and comfortable’’ position (NEUT), a comfortable chin-down position (DOWN), and a strict chin-tuck position (TUCK). Resting state anatomy and kinematic changes were analyzed and compared between pos- tures. Although angles of anterior cervical flexion were com- parable between DOWN (46.65 ± 9.69 degrees) and TUCK (43.27 ± 12.20), the chin-to-spine distance was significantly shorter in TUCK than in other positions. Only TUCK showed a significantly shorter anteroposterior diameter of the laryngeal inlet (TUCK vs. NEUT, 14.0 ± 4.3 vs. 16.3 ± 5.0 mm) and the oropharynx (18.8 ± 3.1 vs. 20.5 ± 2.8 mm) at rest. The maximal horizontal displacement of the hyoid bone was sig- nificantly less in TUCK (9.6 ± 3.0 mm) than in NEUT (12.6 ± 2.6 mm; p\0.01) or DOWN (12.1 ± 3.0 mm; p\0.01). TUCK facilitated movement of the epiglottic base upward (TUCK vs. NEUT, 15.8 ± 4.7 vs. 13.3 ± 4.5 mm; p\0.01). In contrast, DOWN increased the horizontal excur- sion of the epiglottic base and reduced movement of the vocal cords. These results quantitatively elucidated the biomechanical influences of the chin-tuck maneuver including reduced hori- zontal movement of the hyoid bone, facilitation of vertical movement of the epiglottic base, and narrowing of the airway entrance. Comparing DOWN and TUCK, only TUCK induced significant changes in the airway entrance, hyoid movement, and epiglottic base retraction. Keywords Deglutition  Deglutition disorders  Rehabilitation  Biomechanics Keywords Deglutition  Deglutition disorders  Rehabilitation  Biomechanics Keywords Deglutition  Deglutition disorders  Rehabilitation  Biomechanics Dysphagia (2015) 30:89–98 DOI 10.1007/s00455-014-9580-3 Dysphagia (2015) 30:89–98 DOI 10.1007/s00455-014-9580-3 ORIGINAL ARTICLE J. C. Lee Department of Biomedical Engineering, Seoul National University College of Medicine, 101, Daehang-ro, Jongno-gu, Seoul 110-744, Republic of Korea J.-H. Leigh  B.-M. Oh (&)  H. G. Seo  G. J. Lee  Y. Min  K. Kim  T. R. Han Department of Rehabilitation Medicine, Seoul National University College of Medicine, 101, Daehang-ro, Jongno-gu, Seoul 110-744, Republic of Korea e-mail: keepwiz@gmail.com Introduction Various neurological disorders and mechanical injuries can cause swallowing difficulties, namely dysphagia [1]. To alleviate dysphagia, restorative or compensatory approaches have been adopted [2]. Compensatory approaches include food modification, postural changes, and compensatory maneuvers. Postural changes and maneuvers are simple and effective ways of improving the safety and efficacy of swallowing in many cases. The chin-down, head rotation, head tilting, supraglottic swallowing, and Mendelsohn’s maneuvers are examples of commonly used strategies [2, 3]. Among these, the ‘‘chin-down’’ or ‘‘chin-tuck’’ posture has been recommended to various patients [4] with the expectation that it can reduce the risk of laryngeal pene- tration [5] or aspiration [6]. With the chin tucked, the anterior pharyngeal wall is pushed backward, thus nar- rowing the airway entrance. The vallecular space is also widened [1], which mitigates the risk of subglottic aspi- ration from premature food spillage. Welch et al. [7] reported that airway protection improves with the chin tucked by narrowing the laryngeal entrance. Bu¨low et al. [8] revealed that the chin-tuck posture decreased resting state distances from the hyoid bone to the larynx and J.-H. Leigh  B.-M. Oh (&)  H. G. Seo  G. J. Lee  Y. Min  K. Kim  T. R. Han Department of Rehabilitation Medicine, Seoul National University College of Medicine, 101, Daehang-ro, Jongno-gu, Seoul 110-744, Republic of Korea e-mail: keepwiz@gmail.com J.-H. Leigh  B.-M. Oh (&)  H. G. Seo  G. J. Lee  Y. Min  K. Kim  T. R. Han Department of Rehabilitation Medicine, Seoul National University College of Medicine, 101, Daehang-ro, Jongno-gu, Seoul 110-744, Republic of Korea e-mail: keepwiz@gmail.com J. C. Lee Department of Biomedical Engineering, Seoul National University College of Medicine, 101, Daehang-ro, Jongno-gu, Seoul 110-744, Republic of Korea J. C. Lee 12 3 J.-H. Leigh et al.: Swallowing Kinematics of Various Chin Positions 90 posture was ‘‘move your chin down’’ and for the ‘‘strict chin-tuck’’ (TUCK) posture, ‘‘tuck your chin as close to your sternum as possible’’ or ‘‘intentionally bring or touch your chin to your chest’’, which was adapted from the instruction by Logemann [1]. The instructions were pro- vided with brief illustrations (Fig. 1). No further explana- tion was given about the proposed mechanisms of the three different neck postures. Each subject was instructed in the positions and allowed to practice the swallow in those positions prior to imaging. Videofluoroscopic Study (VFS) of Swallowing Videofluoroscopic Study (VFS) of Swallowing The entire process of analysis is shown in Fig. 2. Images were acquired on a mobile fluoroscopy system (Medix 3000, Hitachi, Japan), and two-dimensional (2D) digitiza- tion of the swallowing motion was performed with the same system, as described previously [10]. Subjects were seated upright in a chair for the duration of the study and ingested 10 mL of 35 % w/v diluted barium solution (Solutop Suspension, Tae Joon Pharm Corp., Ltd., Seoul, Korea) using a spoon. A coin 24 mm in diameter was taped under the subject’s chin at the midline to serve as a ref- erence ruler for radiographic magnifications. All video clips were cropped from when the head of liquid reached the lower mandibular margin through the end of the liquid’s passage through the upper esophageal sphincter (UES). This study aimed to investigate the influence of different chin-down postures on swallowing kinematics and to pro- vide a more concrete rationale for the use of these maneuvers. Subjects The study group consisted of 40 healthy volunteers (20 men and 20 women) ranging in age from 26 to 79 years (mean ± SD, 52.9 ± 17.9 years). We stratified partici- pants into three recruitment groups (10 subjects for those of 20–39 years old, 10 for those of 40–59 years old, and 20 for those older than 60 years old). Also, the same number of men and women were recruited in each group (i.e., 5 men and 5 women each). They had no symptoms or signs of swallowing problems and reported no history of neu- rologic disease such as cerebral infarction, syncope, or transient ischemic attack, or history of pulmonary disease. Each participant additionally filled out questionnaires about their previous medical history. The number of sub- jects needed ranged from 25 to 37 for all outcome measures to achieve a statistical power of C0.80 with an alpha level of p B 0.05 based on power analyses. The study protocol was approved by the Institutional Review Board of our hospital, and all participants were informed of the potential experimental risks and signed an informed consent docu- ment before the study. A rater with 2 years’ research experience in swallowing motion analysis, who was blinded to the study design and purpose, analyzed the video clips. The following points of interest in each video frame were analyzed using motion analysis software (Ariel Performance Analysis System; Ariel Dynamics, Inc., Trabuco Canyon, CA, USA). The anterior–superior margin of the hyoid bone; base-to-tip of the epiglottis; head of the barium fluid; anterior–posterior margin of the mandible; anterior–posterior ends of the upper margin of the subglottic airway column, which represents the vocal cords; and the tip of the arytenoids were digitally coordinated in each frame, as was the mental protuberance in the resting position frame. To calculate the coordinates for each point, we operationally defined the y-axis as a straight line connecting the anterior–inferior border of the fourth cervical vertebra (the origin) to the anterior–inferior border of the second cervical vertebra; the x-axis was a straight line perpendicular to the y-axis crossing the origin (Fig. 3), as described previously [11, 12]. All the numbers, i.e., distances and excursions, were calculated and presented as the actual distances in milli- meter. All digitized data were then filtered using a quintic Introduction The video fluoroscopic record- ing was performed in the order of NEUT, DOWN, and then TUCK. Only a single swallow in each position was recorded and analyzed from each subject. mandible. Despite the wealth of studies, changes in the movement of swallowing structures in different neck pos- tures have not been evaluated extensively. Considering that a specific neck posture can affect not only the anatomy at rest, but also dynamic physiology during swallowing, the current understanding of the chin-down effect does not provide a complete picture. There have also been discrepancies in the terminology and practical instructions regarding the maneuver. Because there is no consensus on what an ‘‘effective’’ chin-down posture is, variations of its effects in the literature are inevitable [9]. Although patients are usually taught to ‘‘position the chin toward the chest and look down toward the knees,’’ instructions vary from clinic to clinic [1]. Understanding the exact biomechanical consequences of a specific posture can provide valuable clues for the devel- opment of standard instructions for the position. J.-H. Leigh et al.: Swallowing Kinematics of Various Chin Positions J.-H. Leigh et al.: Swallowing Kinematics of Various Chin Positions J.-H. Leigh et al.: Swallowing Kinematics of Various Chin Positions 91 Fig. 1 The instructions for the study postures (upper pane). The radiographic difference with each posture (lower pane) spline algorithm. All binary data were exported for sub- sequent analysis. A script was written using MATLAB (R2007a, The MathWorks, Inc., Natick, MA, USA) for the adjustment of potential errors and calculations. Measurement The following variables were measured: (a) the chin-cervi- cal spine, epiglottic base-cervical spine, and epiglottic base- Fig. 2 The process and potential errors of the kinematic swallowing analysis. An asterisk represents the potential error in each step, and a dotted line represents a correction of the error Fig. 2 The process and potential errors of the kinematic swallowing analysis. An asterisk represents the potential error in each step, and a dotted line represents a correction of the error ential errors of the kinematic swallowing analysis. An asterisk represents the potential error in each step, and a dotted of the error spline algorithm. All binary data were exported for sub- sequent analysis. A script was written using MATLAB (R2007a, The MathWorks, Inc., Natick, MA, USA) for the adjustment of potential errors and calculations. spline algorithm. All binary data were exported for sub- sequent analysis. A script was written using MATLAB (R2007a, The MathWorks, Inc., Natick, MA, USA) for the adjustment of potential errors and calculations. Positions Subjects were first instructed to drink the liquid in a ‘‘neutral’’ (NEUT) or ‘‘normal and comfortable’’ position. The instruction for the ‘‘comfortable chin-down’’ (DOWN) 12 123 Measurement The following variables were measured: (a) the chin-cervi- cal spine, epiglottic base-cervical spine, and epiglottic base- 12 3 92 J.-H. Leigh et al.: Swallowing Kinematics of Various Chin Positions Fig. 3 The coordination of the kinematic analysis and the selected anatomical points Fig. 3 The coordination of the kinematic analysis and the selected anatomical points for anatomical differences among the subjects [13], a spatial normalization was implemented. The vertical length from C4 to C2 was normalized to 40 mm, and the horizontal distance from the chin to the cervical spine was normalized to 100 mm. After the temporal–spatial normalization, the pooled average trajectory and distributed range of motion were presented, respectively, as a series of mean values and band ranges obtained from elliptical distributions within 95 % confidence intervals (mean ± 2SEM) in both the x-axis and y-axis directions at each time step. arytenoid distances (mm) at rest just before swallowing; (b) the maximal vertical and horizontal excursions (mm) of the hyoid, epiglottic base, and vocal cords (upper margin of the subglottic airway column), defined as the maximal dis- tance from the starting point of each structure to the point of maximal excursion along each direction; the maximal 2D excursion of the hyoid, epiglottic base, and vocal cords, defined as the maximal distance from the starting point of each structure to the point of maximal excursion; and the maximal flip angle (degrees, ) of the epiglottis during swallowing; (c) the maximal vertical and horizontal 2D velocities (mm/s) of the hyoid, epiglottic base, and vocal cords, defined as the points with maximal velocity along each direction; and the velocity of the bolus head. Evaluation of Reliability To evaluate the intra-rater reliability of the swallowing kinematics analysis, 10 VFS cases were utilized. Dis- placement, angle, and velocity for the hyoid, epiglottis, and vocal cords were calculated and compared. To determine intra-rater reliability, each rater analyzed the same cases twice at an interval of 1 month. Although there was an interval of just 1 month, the brightness characteristics of the images were slightly modified and the case names were changed prior to the second analysis. The distance from the ‘‘zero’’ point to the anterior– inferior border of the second cervical vertebra was calcu- lated to serve as a covariate of the vertical spatial relation, and the distance from the mental protuberance perpendic- ular to the y-axis in the resting position as that of the horizontal spatial relation in the pharynx. Pooled Averages for Trajectories of the Hyoid Bone and Epiglottic Base Fig. 3 The coordination of the kinematic analysis and the selected anatomical points Statistical Analysis Pooled averages were calculated for the purpose of trajec- tory figure generation for the hyoid bone and epiglottic base. Because each subject spent different amounts of time per- forming the swallowing test, a temporal normalization was required. The digitized data from each subject were inter- polated for a total of 100 time steps. In addition, to correct A repeated-measure analysis of variance (RM-ANOVA) was used to compare variables among the posture sub- groups using the within-subject effect. Variability in the size of body structures might have influenced the results, but did not reject the sphericity assumption of all variables. 123 123 J.-H. Leigh et al.: Swallowing Kinematics of Various Chin Positions 93 Table 1 Spatial variables for neutral, comfortable chin-down, and chin-tuck postures Neutral Comfortable chin-down Strict chin-tuck Distance at rest (mm) Epiglottic base-posterior wall* 19.70 (3.28)e 19.38 (4.25)f 17.89 (3.26)e,f Epiglottic base-arytenoid* 16.36 (5.00)e 15.72 (4.90) 14.03 (4.27)e Hyoid bone-vocal cord* 36.92 (6.19)d,e 34.14 (6.73)d,f 30.78 (7.22)e,f Chin-posterior wall* 80.66 (7.00)e 80.19 (8.13)f 74.67 (8.22)e,f Hyoid bone, maximal excursion (mm) Vertical 11.43 (4.80) 10.63 (5.21) 12.02 (4.89) Horizontal* 12.55 (2.59)e 12.14 (2.99)f 9.64 (3.03)e,f 2D 14.59 (3.43) 14.29 (3.89) 13.93 (3.92) Epiglottic base, maximal excursion (mm) Vertical* 13.33 (4.48)e 13.43 (4.68)f 15.79 (4.67)e,f Horizontal* 10.73 (3.97)d 12.66 (4.52)d 11.61 (3.72) 2D (Posterior-upward)* 13.26 (4.71)e 14.16 (4.91)f 16.25 (4.38)e,f Epiglottic flip angle, degrees () 111.37 (21.65) 118.55 (22.32) 119.68 (31.31) Vocal cords, maximal excursion (mm) Vertical* 21.13 (5.56)a 18.86 (5.82)a 19.01 (6.18) Horizontal* 6.64 (2.13)b,d 4.69 (1.55)d 5.32 (2.61)b 2D 21.84 (6.28)a,b 19.50 (5.65)a 19.24 (6.83)b 2D two-dimensional * p \ 0.01 using a repeated-measure ANOVA a,b,c Significantly different at p \ 0.05 by Bonferroni-corrected pairwise comparisons d,e,f Significantly different at p \ 0.01 by Bonferroni-corrected pairwise comparisons Table 1 Spatial variables for neutral, comfortable chin-down, and chin-tuck postures the hyoid bone was significantly less in TUCK than in NEUT (p \ 0.001) or DOWN (p \ 0.001) postures. How- ever, the maximal vertical displacement of the hyoid bone was not significantly different among the three postures. The maximal 2D excursion distance of the hyoid bone exhibited borderline significance between NEUT and TUCK (p = 0.059) positions. Figure 4 shows an example of the change in trajectory of the hyoid bone in each posture. When there were significant differences among the groups, a Bonferroni-corrected pairwise comparison was used for the post hoc analysis according to the homogeneity of the variables. Results Distances from the chin to the posterior pharyngeal wall were measured to confirm the differences among the three postures, and were significantly different between TUCK and NEUT (p \ 0.001) and between TUCK and DOWN (p \ 0.001) postures. The Epiglottis and the Laryngeal Entrance The Epiglottis and the Laryngeal Entrance The distance between the epiglottic base and the arytenoids in the lateral fluoroscopic projection, which represents the anteroposterior diameter of the laryngeal entrance, was narrower in TUCK than in NEUT (p = 0.001) or DOWN (p = 0.001) postures. The minimum distance from the epiglottic base to the posterior pharyngeal wall was nar- rower in TUCK than in NEUT (p \ 0.001) or DOWN (p \ 0.001) postures. TUCK showed greater vertical and 2D excursions of the epiglottic base compared to NEUT (p = 0.004 and p \ 0.001) and DOWN (p = 0.006 and p \ 0.001) postures. In contrast, DOWN, not TUCK, showed a greater horizontal excursion of the epiglottic base than NEUT (p = 0.025). The maximal angle of epiglottic rotation also increased from NEUT to DOWN and TUCK, but the trend was not significant. Statistical Analysis Values of p B 0.05 were considered statistically significant for all comparisons. To evaluate intra-rater reliability, an intraclass correlation coefficient (ICC) was used. Statistical analyses were performed with SPSS 17.0 for Windows (SPSS Inc., Chicago, IL, USA). Values are given as the mean (standard deviation). The Hyoid Bone Mean displacement magnitudes and standard deviations of the hyoid bone and larynx in NEUT were similar to data reported in previous kinematic analysis studies [14]. Table 1 shows the changes in spatial variables for each posture. The distance of maximal horizontal excursion of 12 3 J.-H. Leigh et al.: Swallowing Kinematics of Various Chin Positions 94 The Vocal Cords The displacement of the upper margin of the subglottic airway column, which represents the vocal cords’ motion during swallowing, was reduced in both DOWN and TUCK postures. As compared to NEUT posture, DOWN resulted in a reduction of the vertical (p = 0.026) and horizontal (p \ 0.001) displacements of the vocal cords. TUCK showed reduced horizontal displacement of the vocal cords (p = 0.049). Fig. 4 The pooled average trajectories of the hyoid bone and epiglottic base left column. Arrow heads in each trajectory indicate the starting point. The hyoid movement trajectories during swallowing in a neutral (a), a comfortable chin-down (b), and a strict chin-tuck (c) posture are shown. The chin- tuck posture shows a marked reduction in hyoid excursion in the horizontal direction and a slight increase in the vertical direction (c). Right column. The trajectories of the epiglottic base during swallowing in a neutral (d), a comfortable chin-down (e), and a strict chin-tuck (f) posture are shown. The backward retraction and elevation of the epiglottic base is distinctively enhanced (f) 94 J.-H. Leigh et al.: Swallowing Kinematics of Various Chin Positions Fig. 4 The pooled average trajectories of the hyoid bone and epiglottic base left column. Arrow heads in each trajectory indicate the starting point. The hyoid movement trajectories during swallowing in a neutral (a), a comfortable chin-down (b), and a strict chin-tuck (c) posture are shown. The chin- tuck posture shows a marked reduction in hyoid excursion in the horizontal direction and a slight increase in the vertical direction (c). Right column. The trajectories of the epiglottic base during swallowing in a neutral (d), a comfortable chin-down (e), and a strict chin-tuck (f) posture are shown. The backward retraction and elevation of the epiglottic base is distinctively enhanced (f) TUCK postures. As compared to NEUT posture, DOWN resulted in a reduction of the vertical (p = 0.026) and horizontal (p \ 0.001) displacements of the vocal cords. TUCK showed reduced horizontal displacement of the vocal cords (p = 0.049). The Vocal Cords Intra- and Inter-Rater Reliability In the determinations of the two raters, all measurements, except for the maximal horizontal velocity of the hyoid bone (0.717, 0.792), displayed almost perfect intra-rater reliability coefficients that ranged from 0.894 to 0.997. Inter-rater tests of the measurements also showed compa- rable reliabilities from 0.748 to 0.995. The Vocal Cords The displacement of the upper margin of the subglottic airway column, which represents the vocal cords’ motion during swallowing, was reduced in both DOWN and 123 123 J.-H. Leigh et al.: Swallowing Kinematics of Various Chin Positions 95 Table 2 Maximal velocities of the swallowing structures Neutral Comfortable chin-down Strict chin-tuck Hyoid, maximal velocity (mm/s) Horizontal* 55.77 (30.45)b 50.36 (29.90)c 36.71 (20.37)b,c Vertical 53.26 (22.88) 56.11 (25.47) 60.83 (35.74) 2D 125.16 (51.12) 113.53 (43.18) 104.51 (57.06) Epiglottic base, maximal velocity (mm/s) Horizontal 83.88 (49.31) 83.44 (45.44) 77.73 (35.07) Vertical 72.82 (32.28)a 86.86 (41.47) 98.68 (49.02)a 2D 127.76 (54.28)a 148.95 (72.51) 159.16 (73.63)a Vocal cords, maximal velocity (mm/s) Horizontal 28.67 (16.48) 32.72 (13.53) 35.29 (25.27) Vertical 103.76 (44.80) 90.91 (53.38) 91.14 (49.16) 2D 109.10 (42.57) 101.19 (52.08) 105.45 (60.56) 2D two-dimensional * p \ 0.01 using a repeated-measure ANOVA a Significantly different at p \ 0.05 by Bonferroni-corrected pairwise comparisons b,c Significantly different at p \ 0.01 by Bonferroni-corrected pair- wise comparisons major structures. The in-depth analysis in this study revealed that the forward flexion of head and neck in the TUCK posture reduced the anteroposterior distance of the oropharynx as well as the laryngeal inlet at rest; whereas the DOWN posture had no effect on these distances. In terms of dynamic motions during swallowing, TUCK restricts the maximal horizontal excursion of the hyoid bone, epiglottic base, and larynx. The peak velocity of the horizontal excursion of the hyoid bone was also reduced in the TUCK posture. The maximal vertical and 2D displacements of the epiglottic base were significantly increased in the TUCK posture. On the other hand, DOWN restricted the vertical and horizontal excursion of the larynx compared to the NEUT posture. In addition, the horizontal excursion of the epiglottic base was increased in the DOWN posture. The most unique feature of the present study was that our method presented the locations of the major anatomical structures at each time point during swallowing in different head and neck postures. In this way, our study demon- strated the trajectories of the structures as well as vertical and horizontal components of the movements, which have not been previously reported with regard to these postures. 2D two-dimensional The epiglottic movements during swallowing, including the tilt angle and base movement, were also novel findings. Movement Velocities of Anatomical Structures During Swallowing Traditionally, the ‘‘chin-down’’ or ‘‘chin-tuck’’ posture has been known to reduce the risk of aspiration by nar- rowing the airway entrance [7]. The distance from the epiglottic base to the arytenoid, which represents the lar- yngeal inlet, is one of the most important markers for airway protection [15, 16]. The present study showed that only TUCK, not DOWN, reduced the laryngeal inlet at rest. In addition, our results suggest that TUCK may ease swallowing in patients with weak tongue-base retraction by reducing the width of the oropharynx. Table 2 shows the velocities of the swallowing structures. TUCK resulted in a reduced maximal horizontal velocity of the hyoid bone compared to NEUT (p = 0.001) and DOWN (p = 0.025) postures. During swallowing, the epiglottic base moves upward and backward, to the naso- pharynx and posterior pharyngeal wall, respectively. Swallowing in TUCK generated a faster upward velocity of the epiglottic base (p = 0.016). The TUCK posture inhibited horizontal hyoid bone movement. This can be explained by the tongue and sub- mental muscles being compressed by the mandible in the TUCK posture. Another explanation is that a reduced excursion distance can result from decreased resting mus- cle length. Muscle operates with greatest contractile force when close to its resting length in an anatomical position [17]. In TUCK, the submental muscle length is shorter than in NEUT. The reason why the maximal horizontal velocity of the hyoid bone was reduced significantly may be understood in this way. Therefore, it should be noted that TUCK may deteriorate the UES opening because hori- zontal hyoid motion plays an important role in the opening of the UES [18]. On the other hand, DOWN had no sig- nificant effect on hyoid bone movement, which suggested that this comfortable posture did not compress or shorten these muscles. Discussion In the present study, we demonstrated that the movements of the pharyngeal and laryngeal structures during swal- lowing are differentially influenced by head and neck pos- tures. We evaluated not only the maximal excursion distances and velocities, but also the trajectories of the We suppose that the epiglottic base can, at least in part, play a role as a surrogate marker for tongue base 12 3 J.-H. Leigh et al.: Swallowing Kinematics of Various Chin Positions 96 movement, because it is located at the lower end of the tongue base. In NEUT, the epiglottic base initially moved upward and backward, then descended toward the anterior (Fig. 4d). The initial upward and backward movement may represent tongue base retraction in the pharyngeal phase of swallowing. Our results suggest that TUCK enhances tongue base retraction, while DOWN does not, because the 2D excursion distance of TUCK was greater than in NEUT or DOWN, and the 2D velocity of TUCK was greater than that of NEUT (Table 1, Figs. 4e, f). According to a pre- vious study concerning the mechanism of epiglottic tilt, a superior movement of the thyroid cartilage compresses the pre-epiglottic fat pad, which limits the downward move- ment of the epiglottic base during swallowing [19]. TUCK may enhance the dynamic compression of the fat pad, which leads to increases in the vertical and 2D movement of the epiglottic base. As the tongue base is known as a major pressure generator in swallowing [20], great and rapid tongue base retraction in TUCK can exert a higher pressure on the descending bolus. age group showed more definite differences on the maxi- mal excursion of three anatomical structures. The differ- ence of hyoid horizontal excursion of three postures was similar with whole group analysis. Additionally, vertical displacement was markedly increased in TUCK than DOWN. The vertical and 2D excursion distance of TUCK of young age groups was also greater than in NEUT or DOWN, which is comparable with the results of whole group analysis. The laryngeal motions were smaller in DOWN as compared to NEUT, but not in TUCK. Older group showed similar trends with results on Table 1 and 2, but statistical significance was compromised on all vari- ables about the epiglottis and vertical displacement of the vocal cords. Our results, which showed kinematic differences between DOWN and TUCK postures, suggest that we should empha- size patient education on correct postures. Discussion Okada et al. [9] revealed that clinicians have various understandings of the sameposture and thata single-term represented morethantwo postures. Furthermore, they questioned the effect of different headandneckpositionswithambiguousterms,whichresulted in unstandardized effects of swallowing. The results of the effectiveness of the chin-down and chin-tuck postures are controversial between studies. A recent study of 176 healthy volunteers that calculated the average angle of the chin-down posture reported that simple instructions could achieve an angle in the range reported by Welch et al. [7] as yielding a clinicalbenefit inradiographic studies [26].Onthe other hand, subtlechangesinneckflexionandheadflexionproducedquite different changes in the kinematics of the pharyngeal struc- tures [9]. Comparisons among head flexion only, neck flexion only, and head and neck flexion could provide more infor- mation about the effects of various postures. Although we initiallytrieda headflexiononlyposture,there wasnoobvious chin position point, and individual variations in neck cir- cumference and head flexibility also influenced the posture. A previous study that measured the craniovertebral angle formed by the MacGregor plane and the odontoid plane [27] (occiput-C2) reported no changes in the anteroposterior or vertical hyoid bone position from changes in head posture. Because this result demonstrated that head flexion only was not enough to create an effect, we hypothesized that the combination of both head and neck flexion could meaning- fully affect the positions and movements of the laryngeal and pharyngeal structures. As a result, DOWN and TUCK pos- tures combined both head and neck flexion. The laryngeal motions were decreased in both TUCK and DOWN compared to NEUT, although it was more remarkable in DOWN. Reduced hyolaryngeal elevation is usually considered a negative finding that can cause impaired airway protection [21, 22]. However, Bu¨low et al. [8] reported that the chin-tuck posture effectively decreases the distance of the anatomical structures, which causes shortening of the route necessary for laryngeal elevation. Because the laryngeal inlet was shortened in TUCK, reduced laryngeal motion might be sufficient to protect the airway from aspiration. Therefore, TUCK may be helpful for dysphagic patients with decreased laryngeal motion. In terms of pressure, previous studies that measured the pressure of pharyngeal constriction and pharyngoesopha- geal space by manometry give us clues to pressure changes in accordance with neck posture changes [8, 23–25]. Bu¨low et al. [19] reported that chin-tuck posture can increase inferior pharyngeal sphincter pressure in healthy subjects, but not in patients with pharyngeal dysfunction. Discussion Recently, McCulloch [23] and Balou [25] examined manometric studies on both chin-down and chin-tuck postures, which revealed that a more tucked posture increased the duration of relaxation and decreased UES pressure. When we refer to their findings in light of the present study, TUCK resulted in no changes in total excursion, but the influence of horizontal (anterior) movement waned, which resulted in decreased UES pressure. A previous study emphasized the role of horizontal (anterior) hyoid movement on the opening of the UES [18]. References 1. Logemann JA. Evaluation and treatment of swallowing disorders. 2nd ed. Austin: Pro-Ed; 1998. 2. Clark HM. Neuromuscular treatments for speech and swallowing: a tutorial. Am J Speech Lang Pathol. 2003;12:400–15. 3. Logemann JA. Behavioral management for oropharyngeal dys- phagia. Folia Phoniatr Logop. 1999;51:199–212. The anatomical reference for the coordinate system also could be a limitation. The y-axis was defined as the line connecting the anterior–inferior corners of the C2 through C4 vertebrae. When the volunteer flexes their neck, the axis can change through bending of the vertebral alignment, which could lead to changes in vertical and horizontal coordinate values. Using C2-C4 as the y-axis is not a gold standard, but an optimal standard, because this segment is less influenced than the maxilla or the mandible by chin movement and is a more inert segment than other spinal segments including C4-C5 and C5-C6, where the greatest amount of neck flexion occurs [28, 29]. 4. Ertekin C, Keskin A, Kiylioglu N, Kirazli Y, On AY, Tarlaci S, Aydogdu I. The effect of head and neck positions on oropha- ryngeal swallowing: a clinical and electrophysiologic study. Arch Phys Med Rehabil. 2001;82:1255–60. 5. Bulow M, Olsson R, Ekberg O. Supraglottic swallow, effortful swallow, and chin tuck did not alter hypopharyngeal intrabolus pressure in patients with pharyngeal dysfunction. Dysphagia. 2002;17:197–201. 6. Shanahan TK, Logemann JA, Rademaker AW, Pauloski BR, Kahrilas PJ. Chin-down posture effect on aspiration in dysphagic patients. Arch Phys Med Rehabil. 1993;74:736–9. 7. Welch MV, Logemann JA, Rademaker AW, Kahrilas PJ. Chan- ges in pharyngeal dimensions effected by chin tuck. Arch Phys Med Rehabil. 1993;74:178–81. 8. Bulow M, Olsson R, Ekberg O. Videomanometric analysis of supraglottic swallow, effortful swallow, and chin tuck in healthy volunteers. Dysphagia. 1999;14:67–72. To measure the possible bias following flexion, each axis rotation angle (ARA) of the C2-C4 axis was analyzed against the truevertical axisineachposture.ThemeanARA inNEUT had a significantly smaller angle (11.6 ± 6.5 degrees) than in TUCK (43.3 ± 12.2 degrees, p\ 0.002) or DOWN (45.7 ± 9.7 degrees, p \0.001) postures, but a significant difference was not observed between TUCK and DOWN. 9. Okada S, Saitoh E, Palmer JB, Matsuo K, Yokoyama M, Shigeta R, Baba M. What is the chin-down posture? A questionnaire survey of speech language pathologists in Japan and the United States. Dysphagia. 2007;22:204–9. 10. Lee SH, Oh BM, Chun SM, Lee JC, Min Y, Bang SH, Kim HC, Han TR. References The accuracy of the swallowing kinematic analysis at various movement velocities of the hyoid and epiglottis. Ann Rehabil Med. 2013;37:320–7. 11. Paik NJ, Kim SJ, Lee HJ, Jeon JY, Lim JY, Han TR. Movement of the hyoid bone and the epiglottis during swallowing in patients with dysphagia from different etiologies. J Electromyogr Kine- siol. 2008;18:329–35. Limitations Considering that our study has broad age spectrum of subjects and swallowing physiology might differ along the age, we performed subgroup analysis in two age groups (patients aged \60 years, and aged C60 years). Younger A limited number of swallowing trials and individual variability remain methodological concerns. The present study analyzed only one swallow per posture. To reduce 123 123 J.-H. Leigh et al.: Swallowing Kinematics of Various Chin Positions 97 variability, volunteers in this study practiced a dry swallow three times in each posture and swallowed a barium bolus without radiation before capturing one swallow for image analysis. The mean values of each kinematic parameter may be influenced by one swallow per posture and the individual variability in a single volunteer. Conclusion This study substantiates the alleged effects of the chin-tuck maneuver through quantitative kinematic data such as maximal displacements, velocities, and tilt angles, which verify the difference between the chin-tuck and similar chin- down postures. The chin-down posture has no remarkable effect, except on horizontal epiglottic movement. In contrast, the exact chin-tuck posture represents distinct kinematics from the neutral and chin-down postures, and facilitates airway protection and enhances tongue base retraction, but has the possibility of reducing the UES opening. 12. Logemann JA, Pauloski BR, Rademaker AW, Colangelo LA, Kahrilas PJ, Smith CH. Temporal and biomechanical character- istics of oropharyngeal swallow in younger and older men. J Speech Lang Hear Res. 2000;43:1264–74. 13. Kang BS, Oh BM, Kim IS, Chung SG, Kim SJ, Han TR. Influ- ence of aging on movement of the hyoid bone and epiglottis during normal swallowing: a motion analysis. Gerontology. 2010;56:474–82. 14. Molfenter SM, Steele CM. Physiological variability in the deglutition literature: hyoid and laryngeal kinematics. Dysphagia. 2011;26:67–74. 15. Shaker R, Dodds WJ, Dantas RO, Hogan WJ, Arndorfer RC. Coordination of deglutitive glottic closure with oropharyngeal swallowing. Gastroenterology. 1990;98:1478–84. Therefore, in accordance with the patient’s pathologic severity, only the chin-down posture may be effective to adequately widen the vallecular space, and it is important to instruct patients in the exact chin-tuck posture, which can provide essential airway protection. 16. Ohmae Y, Logemann JA, Kaiser P, Hanson DG, Kahrilas PJ. Timing of glottic closure during normal swallow. Head Neck. 1995;17:394–402. 17. Gordon AM, Huxley AF, Julian FJ. The variation in isometric tension with sarcomere length in vertebrate muscle fibres. J Physiol. 1966;184:170–92. Acknowledgments This study was supported by a Grant from the Korean Geriatrics Society (2008). 18. Shaw DW, Cook IJ, Gabb M, Holloway RH, Simula ME, Pana- gopoulos V, Dent J. Influence of normal aging on oral-pharyngeal and upper esophageal sphincter function during swallowing. Am J Physiol. 1995;268:G389–96. Conflict of interest The authors have no conflicts to declare. Conflict of interest The authors have no conflicts to declare. Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, dis- tribution, and reproduction in any medium, provided the original author(s) and the source are credited. 19. Vandaele DJ, Perlman AL, Cassell MD. Intrinsic fibre architec- ture and attachments of the human epiglottis and their contribu- tions to the mechanism of deglutition. J Anat. 1995;186(Pt 1): 1–15. 12 3 J.-H. Conclusion Leigh et al.: Swallowing Kinematics of Various Chin Positions 98 on the hyoid bone position and sternocleidomastoid electro- myographic activity in young adults? Cranio. 2005;23:204–11. on the hyoid bone position and sternocleidomastoid electro- myographic activity in young adults? Cranio. 2005;23:204–11. 20. McConnel FM. Analysis of pressure generation and bolus transit during pharyngeal swallowing. Laryngoscope. 1988;98:71–8. 21. Logemann JA, Kahrilas PJ, Cheng J, Pauloski BR, Gibbons PJ, Rademaker AW, Lin S. Closure mechanisms of laryngeal vesti- bule during swallow. Am J Physiol. 1992;262:G338–44. 28. Fielding JW. Cineroentgenography of the normal cervical spine. J Bone Joint Surg Am. 1957;39(A):1280–8. 29. Lind B, Sihlbom H, Nordwall A, Malchau H. Normal range of motion of the cervical spine. Arch Phys Med Rehabil. 1989;70:692–5. 22. Matsuo K, Palmer JB (2008) Anatomy and physiology of feeding and swallowing: normal and abnormal. Phys Med Rehabil Clin N Am 19: 691–707, vii. 23. McCulloch TM, Hoffman MR, Ciucci MR. High-resolution manometry of pharyngeal swallow pressure events associated with head turn and chin tuck. Ann Otol Rhinol Laryngol. 2010;119:369–76. Ja-Ho Leigh MD Byung-Mo Oh MD, PhD Han Gil Seo MD Goo Joo Lee MD Yusun Min MD Keewon Kim MD Jung Chan Lee PhD Tai Ryoon Han MD, PhD Ja-Ho Leigh MD Byung-Mo Oh MD, PhD Han Gil Seo MD Goo Joo Lee MD Yusun Min MD Keewon Kim MD Jung Chan Lee PhD Tai Ryoon Han MD, PhD 24. Bulow M, Olsson R, Ekberg O. Videomanometric analysis of supraglottic swallow, effortful swallow, and chin tuck in patients with pharyngeal dysfunction. Dysphagia. 2001;16:190–5. 25. Balou M, McCullough GH, Aduli F, Brown D, Stack BC Jr, Snoddy P, Guidry T. Manometric measures of head rotation and chin tuck in healthy participants. Dysphagia. 2013;29(1):25–32. y p p y p g 26. Steele CM, Hung D, Sejdic E, Chau T, Fraser S. Variability in execution of the chin-down maneuver by healthy adults. Folia Phoniatr Logop. 2010;63:36–42. 27. Valenzuela S, Miralles R, Ravera MJ, Zuniga C, Santander H, Ferrer M, Nakouzi J. Does head posture have a significant effect 123 123
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Probing Oral Microbial Functionality – Expression of spxB in Plaque Samples
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Probing Oral Microbial Functionality – Expression of spxB in Plaque Samples Lin Zhu1, Yifan Xu2,3, Joseph J. Ferretti2, Jens Kreth2,4* 1 Department of Periodontics, College of Dentistry, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America, 2 Department of Microbiology and Immunology, College of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America, 3 Department of Surgical Oncology, The First Hospital of China Medical University, Shenyang, People’s Republic of China, 4 Department of Oral Biology, College of Dentistry, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America Abstract The Human Oral Microbiome Database (HOMD) provides an extensive collection of genome sequences from oral bacteria. The sequence information is a static snapshot of the microbial potential of the so far sequenced species. A major challenge is to connect the microbial potential encoded in the metagenome to an actual function in the in vivo oral biofilm. In the present study we took a reductionist approach and identified a considerably conserved metabolic gene, spxB to be encoded by a majority of oral streptococci using the HOMD metagenome information. spxB encodes the pyruvate oxidase responsible for the production of growth inhibiting amounts of hydrogen peroxide (H2O2) and has previously been shown as important in the interspecies competition in the oral biofilm. Here we demonstrate a strong correlation of H2O2 production and the presence of the spxB gene in dental plaque. Using Real-Time RT PCR we show that spxB is expressed in freshly isolated human plaque samples from several donors and that the expression is relative constant when followed over time in one individual. This is the first demonstration of an oral community encoded gene expressed in vivo suggesting a functional role of spxB in oral biofilm physiology. This also demonstrates a possible strategy to connect the microbial potential of the metagenome to its functionality in future studies by identifying similar highly conserved genes in the oral microbial community. Citation: Zhu L, Xu Y, Ferretti JJ, Kreth J (2014) Probing Oral Microbial Functionality – Expression of spxB in Plaque Samples. PLoS ONE 9(1): e86685. doi:10.1371/ journal.pone.0086685 Editor: Ping Xu, Virginia Commonwealth University, United States of America Received October 28, 2013; Accepted December 17, 2013; Published January 29, 2014 Copyright: ß 2014 Zhu 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 National Institutes of Health - National Institute of Dental and Craniofacial Research grant (DE022601) to J.K. 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: JKreth@ouhsc.edu lead to large amounts of diverse sequences reads requiring specific software to adequately manage the data [5–7]. Alternatively, the metagenomic information could be used to identify an ecological relevant oral community encoded gene. The expression of this gene in human plaque samples could be determined directly thus probing its functionality in vivo. Ideally several members of the oral microbial biofilm share this gene. We previously identified a high homology of the pyruvate oxidase gene spxB between the oral commensal Streptococcus sanguinis and Streptococcus gordonii [8]. SpxB is an oxido-reductase, catalyzing the conversion of pyruvate to acetyl phosphate, CO2 and H2O2 under aerobic conditions [9,10]. The gene provides several advantages for the encoding species. Initially, we identified H2O2 as inhibiting substance in the dual species competition with cariogenic Streptococcus mutans [11,12]. Deletion of the spxB gene in both S. sanguinis and S. gordonii rendered them non-competitive against S. mutans demonstrating that SpxB is responsible for competitive H2O2 production [12]. Besides the inhibitory H2O2 action, two additional effects increasing commensals competitiveness are obvious: i) ATP production from acetyl phosphate for energy generation and ii) H2O2 induced release of extracellular DNA (eDNA) [12]. eDNA is a major component of the extracellular polymeric substance (EPS) of biofilms, promoting cell-cell and cell-tooth contact [13,14]. H2O2 induced release of eDNA can also serve in horizontal gene transfer promoting genetic Introduction The Human Oral Microbiome Database (HOMD) contains DNA sequence information from over 1300 genomes [1]. This number is most likely to grow in the future. The wealth of information provided allows the interested researcher to screen the available sequence data for common features and investigate a biological relevant relation to the community structure. For example is a specific gene or a set of genes associated with a healthy community or a predictor of a pathogenic oral biofilm? One caveat of this approach is that the metagenome only gives a static snapshot of the oral bacterial community potential at the time of sampling [2]. It will not allow determining a dynamic relationship or the functionality of the bacterial community at any given time. While the potential is determined by the metagenome, the functionality of the bacterial community is driven by its metatranscriptome and metaproteome, thus at the RNA and protein level [2,3]. However, investigating the metatranscriptome and the metaproteome comes with certain challenges, including the amount of RNA or protein required to successfully cover the entire metatranscriptome or metaproteome of any given plaque sample. For example detection and quantification of low abundance transcripts by RNA Seq can be challenging [4] since the starting material for RNA isolation, dental plaque, is limited. Furthermore, the amount of species open reading frames would PLOS ONE | www.plosone.org 1 January 2014 | Volume 9 | Issue 1 | e86685 In Vivo spxB Expression diversity as we demonstrated recently [15]. In addition, the ability to produce inhibitory amounts of H2O2 seems to be limited to oral streptococci [16]. In this pilot study, we demonstrate that spxB is a suitable candidate gene encoded by several important commensal streptococci, identified by homologous sequence search using the HOMD database. Oral streptococcal production of H2O2 as measured with specific indicator plates seems to correlate with the presence of the spxB gene as determined with PCR and spxB specific oligonucleotides. Furthermore, we isolated RNA from freshly isolated human plaque samples and demonstrate that spxB is expressed with Real-Time RT-PCR, suggesting a functional role in the oral biofilm. procedure lasted not longer than five minutes. Resuspended plaque samples in TRIzol were immediately frozen at 280uC until further processing the next day. Plaque samples for plating were removed from the teeth using a cotton swap and directly plated onto the H2O2 indicator plates. RNA Isolation and cDNA Synthesis To isolate RNA, cells were disrupted three times for 30 seconds each using 0.1 mm zirconia/silica beads (BioSpec, Inc) in a FastPrep FP210 Homogenizer (Thermo Scientific) using the highest speed setting. Total RNA isolation from TRIzol was carried out according to the manufacturer’s instructions (Isolation of total RNA using TRIzol, lifetechnologies). RNA samples were treated with Turbo DNase to remove traces of chromosomal DNA after manufacturers recommendations (Ambion). RNeasy MiniElute cleanup kit (Qiagen) was used to purify RNA samples after DNase treatment. 2 ml RNA was immediately run on a 1% agarose gel to check for integrity. cDNA was synthesized using qScriptTM cDNA synthesis kit (Quanta Biosciences) according to the manufacturer’s protocol. Materials and Methods Ethics Statement The Institutional Review Board of University of Oklahoma HSC approved the study protocol for human subjects. IRB protocol # 1934. Participants signed a written consent form following an approved procedure by the IRB. RNA Integrity Bacterial Strains and Growth Conditions RNA integrity was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.). The yield of the extracted RNA and intactness of rRNA was assayed from 1 ml RNA after DNase treatment and RNeasy MiniElute cleanup in a 2100 BioAnalyzer using the protocol of the RNA 6000 Nano Lab Chip Kit (Agilent Technologies, Inc.). The chip allows for analyzing 12 samples simultaneously. RNA was therefore collected first and stored at 280uC until all 8 samples were available for the RNA 6000 Nano Lab Chip run. Bacterial strains are listed in Tab. 1. Bacteria were routinely grown aerobically in 5% CO2 at 37uC overnight in BHI medium (Brain Heart Infusion; Difco, Sparks, MD) or on BHI plates, or as otherwise indicated. Subjects and Plaque Sampling The present study sole intention was to determine the feasibility of measuring spxB gene expression in dental plaque samples. Therefore, no subject related data were collected from the 8 volunteers asked to donate plaque samples. From each individual, supragingival dental plaque samples were recovered between 16 and 18 hours after tooth brushing, from interproximal, vestibular and lingual surfaces of all teeth using a dental probe. The collected plaque samples were immediately removed from the dental probe after a visible amount has built up using a sterile tip and resuspended in 1 ml TRIzol (lifetechnologies). The sampling RT-PCR and Real-Time PCR RT-PCR was performed as described earlier [17]. Real-time RT PCR was performed to determine specific cDNA copies with the comparative threshold cycle (CT) method using a MyiQ single-color real-time PCR detection system (Bio-Rad) and PerfeCtaTM SYBRH Green SuperMix for iQTM (Quanta Biosciences). Relative changes in cDNA copies representing differential gene expression were calculated using the DCT method described previously [18]. The 16S rRNA gene was used as the housekeeping reference gene. Oligonucleotides were synthesized by IDTDNA. Oligonucleotide sequences are: 16S rRNA F - 59AAGCAACGCGAAGAACCTTA-39; 16S rRNA R - 59GTCTCGCTAGAGTGCCCAAC-39; universal spxB F - 59CATCATGGGTGACGGTGCAT-39; universal spxB R - 59GCGTTAGGGAAGTCACAACC-39. Table 1. Bacterial strains. Strain Reference S. mutans UA140 [31] S. mutans UA159 [32] S. mitis 12261 [33] S. gordonii DL1 [34] S. gordonii V288 [35] S. oralis MC3-1 [36] S. oralis J22 [36] S. sanguinis SK36 [37] S. sanguinis 133–79 [38] S. salivarius Clinical isolates PCR Colony PCR was performed by scraping a small amount of cells from the respective agar plate using a sterile pipet-tip and resuspending the cells in a pre-aliquoted PCR reaction mix. Alternatively, cells were inoculated in 2 ml BHI overnight; 1 ml was transferred into tubes containing lysing matrix B (MP Biomedicals, Solon, OH) and cells were homogenized in a FastPrep FP210 homogenizer (Thermo Scientific) (speed setting of 6.5). After centrifugation for 10 min at 13.200 rpm in a tabletop centrifuge, 2 ml supernatant containing chromosomal DNA was used as PCR template. PCR was performed with a G-Storm GS1 thermocycler (Gene Technologies) according to the manufacturer’s protocol. GoTaq-DNA polymerase was obtained from Promega, and oligonucleotides specific for spxB and 16S rRNA were the same as listed above. S. infantis S. parasanguinis S. mutans serotype F S. mutans serotype K S. gordonii doi:10.1371/journal.pone.0086685.t001 PLOS ONE | www.plosone.org 2 January 2014 | Volume 9 | Issue 1 | e86685 In Vivo spxB Expression Detection of H2O2 Production Table 2. Distribution of spxB among oral streptococci. Indicator plates for H2O2 production were prepared and used as described [19]. This indicator plates allow for the detection of bacterial H2O2 production resulting in a blue pigment (Prussian blue, ferric ferrocyanide) that forms in the presence of H2O2. Plates were aerobically cultured in 5% CO2 at 37uC overnight. Strain Results Prevalence of the spxB Gene in the Oral Biofilm Community A BLAST search was performed using the S. gordonii CH1 nucleotide sequence as query against all available oral microbial genomes on the HOMD server. In addition to S. gordonii, the following oral Streptococci encode spxB homologs: Streptococcus sanguinis, Streptococcus mitis, Streptococcus infantis, Streptococcus oralis, Streptococcus oligofermentans and Streptococcus cristatus (Tab. 2). Furthermore, Streptococcus pneumoniae encodes also spxB as reported in the literature [20] and found during the BLAST search, but was omitted since it is usually not found in the dental associated oral biofilm. The homology of the spxB genes encoded by the oral streptococcal community is high ranging from 93% to 97% on the nucleotide level (against S. gordonii CH1) with a core sequence of about 1700 bp when compared to the 2308 bp of S. gordonii CH1. This suggests that the spxB gene is highly conserved among oral Streptococci. Interestingly, no homolog was found in cariogenic Streptococcus mutans confirming our previous observation that S. mutans does not produce competitive amounts of H2O2 [11]. Similarity Streptococcus gordonii CH1 2308/2308 (100%) Streptococcus sanguinis SK49 2000/2073 (96%) Streptococcus mitis SK321 1978/2061 (95%) Streptococcus mitis NCTC 12261 1975/2061 (95%) Streptococcus sanguinis SK36 2061/2180 (94%) Streptococcus mitis SK579 1969/2061 (95%) Streptococcus mitis SK1080 1966/2061 (95%) Streptococcus sanguinis SK72 2153/2309 (93%) Streptococcus sanguinis SK115 1973/2071 (95%) Streptococcus infantis X 1951/2061 (94%) Streptococcus oralis SK255 1772/1824 (97%) Streptococcus sanguinis SK340 1960/2073 (94%) Streptococcus sanguinis ATCC 29667 1960/2073 (94%) Streptococcus sanguinis ATCC 49296 1769/1824 (96%) Streptococcus oralis SK1074 1768/1823 (96%) Streptococcus mitis bv. 2 str. F0392 1768/1823 (96%) Streptococcus mitis bv. 2 str. SK95 1763/1817 (97%) Streptococcus sanguinis SK150 1953/2071 (94%) Streptococcus sanguinis VMC66 2033/2180 (93%) Streptococcus oralis SK304 1758/1817 (96%) Streptococcus sanguinis SK160 1951/2073 (94%) Detection of spxB with spxB Universal Oligonucleotides Streptococcus oralis Uo5 1751/1809 (96%) The available sequence data for spxB from oral Streptococci was used to design an spxB specific set of oligonucleotides. Initially, the oligonucleotides were tested for their ability to amplify spxB from several Streptococci present in our laboratory culture collection. The H2O2 production potential of the here used streptococci was assessed first by inoculating 10 ml of an overnight culture on specific H2O2 indicator plates. The plates were incubated overnight to allow for growth and H2O2 production. As shown in Fig. 1 A, with the exception of S. mutans and S. salivarius, all other Streptococci produced H2O2 evident from the formation of a blue pigment. Subsequently, cells were removed and chromosomal DNA isolated to perform PCR with 16S rRNA and spxB specific oligonucleotides. The 16S rRNA control showed amplification for all strains tested. A clear correlation was evident between positive spxB amplification in S. mitis, S. gordonii, S. oralis, S. sanguinis, S. infantis and S. parasanguinis and the formation of the blue pigment (Fig. 1B). No significant amplification was observed for all S. mutans strains and S. salivarius, but we did recognize weak bands after longer integration during picture documentation (Fig. 1B). To determine if the observed faint bands would interfere with our intention to measure spxB expression by giving false positive amplification products, RNA was isolated from S. mutans, S. gordonii and S. salivarius and cDNA synthesized. RT-PCR with spxB oligonucleotides showed only amplification for S. gordonii in the RT-PCR reaction, but no amplification for the no RT control (Fig. 2). 16S rRNA amplification was positive in all three RT-PCR reactions (data not presented). This suggests that the newly designed oligonucleotides are able to amplify spxB from H2O2 positive oral Streptococci and are suitable to be used in gene expression analysis. Streptococcus mitis ATCC 6249 1742/1798 (96%) Streptococcus sanguinis SK353 1948/2071 (94%) Streptococcus mitis SK575 1754/1816 (96%) Streptococcus oralis SK100 1740/1798 (96%) PLOS ONE | www.plosone.org Streptococcus mitis SK1073 1738/1798 (96%) Streptococcus oralis ATCC 35037 1753/1823 (96%) Streptococcus mitis SK564 1751/1822 (96%) Streptococcus infantis SK1302 1742/1813 (96%) Streptococcus cristatus ATCC 51100 1745/1817 (96%) Streptococcus mitis B6 1728/1796 (96%) Streptococcus oligofermentans AS 1.3089 1748/1824 (95%) Streptococcus infantis ATCC 700779 1723/1797 (95%) Streptococcus oralis SK610 1743/1824 (95%) Streptococcus mitis SK616 1723/1798 (95%) doi:10.1371/journal.pone.0086685.t002 PCR Amplification of spxB from H2O2 Positive Plaque Colonies To further evaluate the spxB amplification potential of the newly designed spxB specific oligonucleotides, dental plaque was collected from five subjects. The plaque samples were inoculated on H2O2 indicator plates to separate single colonies. Fig. 3A represents plaque samples from two subjects and illustrates that colonies with and without blue pigment can be distinguished. Ten blue colonies from each subject were picked and used for colony PCR with the spxB specific oligonucleotides (Fig. 3B). PCR amplification was successful for all colonies in the five subjects. Obvious, however, was the difference in PCR efficiency. While subject 1 had strong bands for all ten colonies, subject 4 showed some heterogeneity in the band intensity (Fig. 3B). In addition, 45 3 January 2014 | Volume 9 | Issue 1 | e86685 In Vivo spxB Expression Figure 1. Correlation of H2O2 production and genomic spxB presence. A) H2O2 production assay of common laboratory strains. Cells were spotted on H2O2 indicator plates (Prussian Blue pigment development indicates H2O2 production) and incubated over night under aerobic conditions. Blue color development indicates H2O2 production. B) PCR using universal spxB oligonucleotides. Cells were scraped from the H2O2 indicator plates, grown overnight and chromosomal DNA isolated and used for PCR with universal spxB oligonucleotides. 16S rRNA amplification was performed as control for successful bacterial lysis during colony PCR. doi:10.1371/journal.pone.0086685.g001 refrain from tooth brushing in the morning to collect a sufficient amount of plaque in the afternoon. The plaque samples were immediately processed for RNA isolation. Isolated RNA was analyzed and RIN (RNA Integrity Number) determined. Fig. 4A and 4B showing agarose gel-electrophoresis and Bioanalyzer gel visualization. The respective RIN numbers and RNA concentrations are presented in Fig. 4C. Sample 1 and 2 showed very low RIN and were not further used. The remaining samples ranged from RIN 4.8 to RIN 7.8. The RNA concentration range was from 50 ng/ml to 418 ng/ml. white colonies were subject to PCR amplification with the spxB oligonucleotides. No amplification was observed (Fig. 3C). In summary, the spxB specific oligonucleotides are able to amplify spxB from freshly isolated H2O2 positive plaque bacteria suggesting that the oligonucleotides could be used in general to evaluate the presence of this gene in human plaque samples. RNA Isolation from Plaque Samples The goal of this study is to detect the expression of spxB from freshly isolated dental plaque samples to evaluate the in vivo expression of this gene. The major challenge in the detection of gene expression from host-derived biofilms is the isolation of sufficient amounts of high quality RNA [21]. To test feasibility of RNA isolation, plaque was sampled from 11 subjects asked to Detection of spxB Expression from Plaque Samples Initially the expression of spxB in 9 individual plaque samples was compared using 16S rRNA as housekeeping reference. The expression was normalized to the subject with the highest spxB expression relative to the others. The result presented in Fig. 5 demonstrates that there is a high degree of interpersonal variation in spxB expression. The observed fold difference reached about 88 fold when the highest and the lowest expression were compared (subject 8 vs. subject 3). Contamination by chromosomal DNA was excluded by running a no RT control along with a RT-PCR (data not shown). In addition, the expression of spxB from a single subject on five different time-points was determined to learn how stable the expression is on a day-to-day basis. As shown in Fig. 6, time point 1 and 2 as well as 3 and 4 were taken on two subsequent days, one hour apart while time point 5 was taken several days later. Although slight variations are visible, the expression of spxB in one subject seems to be constant over time. Furthermore spxB expression can be detected repeatable from RNA isolated from human plaque samples. Figure 2. RT-PCR of cDNA from selected oral streptococci. RNA was isolated from the H2O2 producer S. gordonii DL1 and expression of spxB compared to H2O2-non-producer S. mutans UA140 and S. salivarius. RT-PCR was performed for 28 cycles. doi:10.1371/journal.pone.0086685.g002 PLOS ONE | www.plosone.org 4 January 2014 | Volume 9 | Issue 1 | e86685 In Vivo spxB Expression Figure 3. H2O2 production and spxB presence in plaque samples. A) Plaque samples for spreading on the H2O2 indicator plate were collected by swapping over several tooth with a sterile cotton swap. After inoculation, the indicator plates were incubated over night aerobically. Shown are representative plates from two subjects. B) Respective blue colonies indicating H2O2 production were picked and used for genomic spxB amplification. 16S rRNA was used as positive control, with bands for all tested colonies (not shown). C) 45 white colonies were also tested for spxB amplification. L = ladder,+ = chromosomal DNA S. gordonii DL1. doi:10.1371/journal.pone.0086685.g003 Figure 4. RNA integrity and concentration. A) Gel-eletrophoretic separation of isolated total RNA after DNase digest and clean-up on 1% agarose. B) Gel images of RNA samples generated by the Agilent Bioanalyzer using RNA 6000 Nano Lab Chip. C) RNA concentration and RIN as determined by the Agilent Bioanalyzer. RIN = RNA Integrity Number; L = RNA Ladder. Green line in Figure 4B: Bioanalyzer internal marker. doi:10.1371/journal.pone.0086685.g004 PLOS ONE | www.plosone.org 5 January 2014 | Volume 9 | Issue 1 | e86685 In Vivo spxB Expression Figure 5. Expression of spxB in freshly isolated human oral plaque samples. One time expression measurements of spxB from 8 different subjects. Expression was normalized to 16S rRNA expression and subject 8 arbitrary set as 1. Error bars represent standard deviations of technical repeats (n = 3). doi:10.1371/journal.pone.0086685.g005 which goes beyond the determination of what is present in the oral cavity or what kind of potential metabolic pathways are encoded [3,22]. The next logical step is to move away from the static information of sequence data to determine if the microbial potential of the chromosomal sequences is converted to a Discussion The HOMD contains DNA sequence information from over 1300 genomes (www.homd.org). An eminent question is what kind of information can be extracted from the deposited sequence data Figure 6. Expression of spxB from one subject over three different days. Time point 1, 2 and 3, 4 were taken on two subsequent days, one hour apart; time point 5 was taken several days later. Expression was normalized to 16S rRNA expression and time point 1 arbitrary set as 1. Error bars represent standard deviations of technical repeats (n = 3). doi:10.1371/journal.pone.0086685.g006 PLOS ONE | www.plosone.org 6 January 2014 | Volume 9 | Issue 1 | e86685 In Vivo spxB Expression functional response in vivo. The here presented study uses the sequence data provided by HOMD to identify an oral microbial community shared gene, spxB and demonstrates its functional expression in freshly isolated human plaque samples. Several oral commensal Streptococci encode the spxB gene as we identified in this study. Up to 80% of the detected initial colonizers belong to the genus Streptococcus. Some species are even discussed as constant members constituting a core group for initial biofilm formation [23,24]. The spxB gene is therefore highly abundant during initial biofilm formation and might be considered as core gene involved in oral biofilm formation. The production of an antimicrobial substance like H2O2 could therefore be regarded as an important protection mechanism of the initial colonizers of the resident biofilm community against invading and competing species like the extremely H2O2 sensitive S. mutans [11]. More importantly, it might also be a mechanism to shape the colonization process towards a specific species composition. Only species co-evolved with oral streptococci and therefore adapted to withstand H2O2 can integrate or colonize in close proximity to the initial colonizers and extend the developing biofilm community [8]. The gene is expressed in the different subjects suggesting a function in the biofilm mode of growth of oral streptococci. The next step is to design a large-scale study to relate the expression of spxB with an assessment of the oral health status. A positive association of spxB expression with oral health would allow for a better caries risk assessment in the future. Expression studies on infection relevant genes in vivo in humans and animal models have been done before as summarized in [25]. Three in vivo studies are relevant in the context of our study: i) An investigation of in vivo gene expression in the human host was carried out with Streptococcus pyogenes (Group A Streptococcus, GAS) [26]. The author’s demonstrated GAS host-pathogen interactions by analyzing the expression of 17 GAS genes in throat swab specimens sampled from 18 pediatric patients with pharyngitis. Several known and putative virulence genes and regulatory genes were highly expressed during infection [26]; and ii) In a study with the periodontal pathogen Porphyromonas gingivalis, Shelbourne et al. demonstrated a clear correlation between periodontal disease status and elevated expression of dnaK and htpG encoding general stress response proteins [27]; iii) while another study established the expression of an uncharacterized gene in P. gingivalis (G1334) as more frequent in diseased sites compared to healthy sites [28]. The importance of the G1334 gene in virulence was confirmed in a mouse abscess model of infection [28]. The here presented expression of a community-encoded core gene of oral biofilm formation and the single-species focus by other groups detecting virulence and stress related gene expression in vivo demonstrate the feasibility to specifically determine the expression of genes of interest in their ecological context. Limitations of the study became apparent when the RIN where determined. Although all samples where processed after the same protocol, RNA degradation was a problem for some samples. Two RNA samples were severely degraded and not further considered, while others showed varying degrees of degradation. In general, an RIN of 10 would indicate no degradation. RIN greater than five indicates good total RNA quality for reverse transcription [29]. Some of the samples were close to 5 and therefore would be considered not ideal for spxB expression quantification. Nonetheless, we used the total RNA of subject 3 and 4 for cDNA synthesis to determine spxB expression. The respective relative expression of spxB from subject 3 and 4 were among the lower spectrum, however, subject 5 and 10 showed a similar relative expression level albeit higher RIN. Another caveat of this study lies in the fact that the oral microbial diversity among subjects varies [30] and the spxB sequences will have sequence dissimilarities. This will result in nucleotide mis-pairing of the spxB oligonucleotides used for spxB expression. As a consequence, non-ideal PCR amplification conditions will occur due to different primer efficiencies when plaque samples with undefined species composition are used. This might be prevented in the future if more spxB sequences become available to optimize oligonucleotides for amplification. In addition, the main advantage we take in our approach is the wide distribution of a highly conserved gene among the most prominent genus in the oral biofilm. If other similar important genes can be identified needs to be determined. In summary, we showed in vivo expression of an oral biofilm community encoded gene in its ecological context, thus suggesting an active role of spxB in oral biofilm physiology. This study demonstrates how the HOMD database can be used to determine a dynamic relationship or the functionality of the potential encoded in the metagenome of the oral biofilm. Author Contributions Conceived and designed the experiments: LZ JJF JK. Performed the experiments: LZ YX. Analyzed the data: LZ JK. Wrote the paper: LZ JK. References 10. Carlsson J, Edlund MB, Lundmark SK (1987) Characteristics of a hydrogen peroxide-forming pyruvate oxidase from Streptococcus sanguis. Oral Microbiol Immunol 2: 15–20. 11. Kreth J, Merritt J, Shi W, Qi F (2005) Competition and coexistence between Streptococcus mutans and Streptococcus sanguinis in the dental biofilm. J Bacteriol 187: 7193–7203. 12. Kreth J, Zhang Y, Herzberg MC (2008) Streptococcal antagonism in oral biofilms: Streptococcus sanguinis and Streptococcus gordonii interference with Streptococcus mutans. J Bacteriol 190: 4632–4640. 13. Kreth J, Vu H, Zhang Y, Herzberg MC (2009) Characterization of hydrogen peroxide-induced DNA release by Streptococcus sanguinis and Streptococcus gordonii. J Bacteriol 191: 6281–6291. 14. Das T, Sharma PK, Krom BP, van der Mei HC, Busscher HJ (2011) Role of eDNA on the adhesion forces between Streptococcus mutans and substratum surfaces: influence of ionic strength and substratum hydrophobicity. Langmuir 27: 10113–10118. 15. Itzek A, Zheng L, Chen Z, Merritt J, Kreth J (2011) Hydrogen peroxidedependent DNA release and transfer of antibiotic resistance genes in Streptococcus gordonii. J Bacteriol 193: 6912–6922. 16. Ryan CS, Kleinberg I (1995) Bacteria in human mouths involved in the production and utilization of hydrogen peroxide. Arch Oral Biol 40: 753–763. 17. Zhang Y, Whiteley M, Kreth J, Lei Y, Khammanivong A, et al. (2009) The twocomponent system BfrAB regulates expression of ABC transporters in Streptococcus gordonii and Streptococcus sanguinis. Microbiology 155: 165–173. 1. Chen T, Yu WH, Izard J, Baranova OV, Lakshmanan A, et al. (2010) The Human Oral Microbiome Database: a web accessible resource for investigating oral microbe taxonomic and genomic information. Database (Oxford) 2010: baq013. 2. Maron PA, Ranjard L, Mougel C, Lemanceau P (2007) Metaproteomics: a new approach for studying functional microbial ecology. Microb Ecol 53: 486–493. 3. Zaura E (2012) Next-generation sequencing approaches to understanding the oral microbiome. Adv Dent Res 24: 81–85. 4. Haas BJ, Chin M, Nusbaum C, Birren BW, Livny J (2012) How deep is deep enough for RNA-Seq profiling of bacterial transcriptomes? BMC Genomics 13: 734. 5. Fang Z, Cui X (2011) Design and validation issues in RNA-seq experiments. Brief Bioinform 12: 280–287. 6. Kvam VM, Liu P, Si Y (2012) A comparison of statistical methods for detecting differentially expressed genes from RNA-seq data. Am J Bot 99: 248–256. 7. Oshlack A, Robinson MD, Young MD (2010) From RNA-seq reads to differential expression results. Genome Biol 11: 220. 8. Zhu L, Kreth J (2012) The role of hydrogen peroxide in environmental adaptation of oral microbial communities. Oxid Med Cell Longev 2012: 717843. 9. Carlsson J, Edlund MB (1987) Pyruvate oxidase in Streptococcus sanguis under various growth conditions. Oral Microbiol Immunol 2: 10–14. PLOS ONE | www.plosone.org 7 January 2014 | Volume 9 | Issue 1 | e86685 In Vivo spxB Expression 28. Walters S, Rodrigues P, Belanger M, Whitlock J, Progulske-Fox A (2009) Analysis of a band 7/MEC-2 family gene of Porphyromonas gingivalis. J Dent Res 88: 34–38. 29. Fleige S, Pfaffl MW (2006) RNA integrity and the effect on the real-time qRTPCR performance. Mol Aspects Med 27: 126–139. 30. Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE (2005) Defining the normal bacterial flora of the oral cavity. J Clin Microbiol 43: 5721–5732. 31. Qi F, Chen P, Caufield PW (2001) The group I strain of Streptococcus mutans, UA140, produces both the lantibiotic mutacin I and a nonlantibiotic bacteriocin, mutacin IV. Appl Environ Microbiol 67: 15–21. 32. Ajdic D, McShan WM, McLaughlin RE, Savic G, Chang J, et al. (2002) Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc Natl Acad Sci U S A 99: 14434–14439. 33. Kawamura Y, Hou XG, Sultana F, Miura H, Ezaki T (1995) Determination of 16S rRNA sequences of Streptococcus mitis and Streptococcus gordonii and phylogenetic relationships among members of the genus Streptococcus. Int J Syst Bacteriol 45: 406–408. 34. Pakula R, Walczak W (1963) On the nature of competence of transformable streptococci. J Gen Microbiol 31: 125–133. 35. Kili AO, Herzberg MC, Meyer MW, Zhao X, Tao L (1999) Streptococcal reporter gene-fusion vector for identification of in vivo expressed genes. Plasmid 42: 67–72. 36. Yoshida Y, Ganguly S, Bush CA, Cisar JO (2005) Carbohydrate engineering of the recognition motifs in streptococcal co-aggregation receptor polysaccharides. Mol Microbiol 58: 244–256. 37. Xu P, Alves JM, Kitten T, Brown A, Chen Z, et al. (2007) Genome of the opportunistic pathogen Streptococcus sanguinis. J Bacteriol 189: 3166–3175. 38. Herzberg MC, Gong K, MacFarlane GD, Erickson PR, Soberay AH, et al. (1990) Phenotypic characterization of Streptococcus sanguis virulence factors associated with bacterial endocarditis. Infect Immun 58: 515–522. 18. Zheng L, Chen Z, Itzek A, Ashby M, Kreth J (2011) Catabolite control protein A controls hydrogen peroxide production and cell death in Streptococcus sanguinis. J Bacteriol 193: 516–526. 19. Saito M, Seki M, Iida K, Nakayama H, Yoshida S (2007) A novel agar medium to detect hydrogen peroxide-producing bacteria based on the Prussian blueforming reaction. Microbiology and Immunology 51: 889–892. 20. Spellerberg B, Cundell DR, Sandros J, Pearce BJ, Idanpaan-Heikkila I, et al. (1996) Pyruvate oxidase, as a determinant of virulence in Streptococcus pneumoniae. Mol Microbiol 19: 803–813. 21. Giannoukos G, Ciulla DM, Huang K, Haas BJ, Izard J, et al. (2012) Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes. Genome Biol 13: R23. 22. Segata N, Haake SK, Mannon P, Lemon KP, Waldron L, et al. (2012) Composition of the adult digestive tract bacterial microbiome based on seven mouth surfaces, tonsils, throat and stool samples. Genome Biol 13: R42. 23. Rosan B, Lamont RJ (2000) Dental plaque formation. Microbes Infect 2: 1599– 1607. 24. Diaz PI, Chalmers NI, Rickard AH, Kong C, Milburn CL, et al. (2006) Molecular characterization of subject-specific oral microflora during initial colonization of enamel. Appl Environ Microbiol 72: 2837–2848. 25. Shelburne SA, Musser JM (2004) Virulence gene expression in vivo. Curr Opin Microbiol 7: 283–289. 26. Virtaneva K, Graham MR, Porcella SF, Hoe NP, Su H, et al. (2003) Group A Streptococcus gene expression in humans and cynomolgus macaques with acute pharyngitis. Infect Immun 71: 2199–2207. 27. Shelburne CE, Gleason RM, Germaine GR, Wolff LF, Mullally BH, et al. (2002) Quantitative reverse transcription polymerase chain reaction analysis of Porphyromonas gingivalis gene expression in vivo. J Microbiol Methods 49: 147– 156. PLOS ONE | www.plosone.org 8 January 2014 | Volume 9 | Issue 1 | e86685
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Teacher and Student Facing Learning Analytics
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ABSTRACT Learning analytics systems are increasingly being designed for and implemented in classroom teaching and learning in K-12 and post-secondary contexts. For analytics to play a constructive role, it is important to consider how they are being used by teachers and students and how they can be designed to enhance and complement human decision making. In this chapter, we first discuss issues that teachers and students face in the sensemaking of learning analytics systems as well as in the subsequent phase of acting on the information provided by such systems. We then discuss the following aspects for teacher facing and then student facing analytics: (a) theoretical models underlying analytics use; (b) ways analytic systems have been designed and implemented; (c) evidence of impact the systems have had on teaching and learning. The chapter ends with an overarching discussion of challenges that concern both teacher and student facing analytics and introduces the possibilities for co-design of analytics systems to address some of these challenges. Keywords: Student facing analytics, teacher facing analytics, learning analytics systems, learning analytics dashboards, learning analytics use, self-regulated learning, sensemaking, learning analytics design, human-centered learning analytics, participatory design of learning analytics 1Throughout this chapter we use the term teacher generally to refer to those holding instructional roles in both K-12 and post-secondary educa- tion. Chapter 13: Teacher and Student Facing Learning Analytics Anouschka van Leeuwen, 1 Stephanie D. Teasley, 2 Alyssa Friend Wise 3 1 Faculty of Social and Behavioral Sciences, Utrecht University, Utrecht, Netherlands 2 School of Information, University of Michigan, Ann Arbor, USA 3 Learning Analytics Research Network, New York University, New York, USA DOI: 10.18608/hla22.013 1 Faculty of Social and Behavioral Sciences, Utrecht University, Utrecht, Netherlands 2 School of Information, University of Michigan, Ann Arbor, USA 3 Learning Analytics Research Network, New York University, New York, USA 1 IMPORTANT CONSIDERATIONS FOR LEARNING ANALYTICS USE Much of the work of learning analytics designers and re- searchers revolves around challenges of how to extract, process, and present data in ways that are useful to ed- ucational stakeholders. However, system design alone does not ensure successful uptake [26, 24, 32]: “analyt- ics exist as part of a sociotechnical system where human decision making and consequent actions are as much a part of any successful analytics solution as the technical components” [84, p. 4]. Thus, learning analytics designers and researchers need to attend to the human activity of working with these tools in their various contexts of use. In this chapter, we specifically address the use of learning analytics systems by teachers1 and students. We first dis- cuss issues in making sense of and acting on information provided by learning analytics systems. We then detail, first for teachers and then for students: (a) theoretical models underlying analytics use, (b) ways systems have been designed and implemented, and (c) evidence of the impact these systems have had on teaching and learning. We conclude with a focus on obstacles and opportunities to the development of effective and adoptable tools. Using learning analytics effectively involves making sense of the information presented and taking action based on it [77, 15]. While analytics are often developed for use across a range of situations, the answer to questions of meaning and action are inherently local. In the case of teachers and students, the design of learning analytics systems needs to be sensitive to the anticipated contexts of use (e.g. daily classroom routines) and potential unin- tended consequences of use (e.g. taking student metrics as a proxy for teacher competence). Wise and colleagues [96, 95, 93] have pointed to several well-documented issues in using analytics to inform educational decision-making that relate to processes of interpretation and taking ac- tion. These considerations must be taken into account by those designing learning analytics systems and those implementing them for use of the systems to be effective. g In this chapter, we specifically address the use of learning analytics systems by teachers1 and students. We first dis- cuss issues in making sense of and acting on information provided by learning analytics systems. 2.1 Theoretical Models of Teachers’ Learning Analytics Use With respect to taking action, there are two core issues. First, analytics provide a retrospective lens to evaluate past activity, but do not always indicate how to make changes to the situation in the future. For example, a social network diagram can show that a certain student is not receiving replies from their classmate without pro- viding information about what would encourage greater responsiveness. Second, even when desirable action is identified, most change does not occur instantaneously — incremental improvement and intermediate stages of progress are often required. For example in Wise, Zhao and Hausknecht [97] students took multiple cycles of goal- setting and feedback to change their learning behaviors. Action may also be deferred when teachers (or students) are not certain of their interpretation and want to wait for more data to become available [94]. These issues have con- sequences for analytics design and implementation. To support teachers and students, designers cannot assume that providing data alone is enough. Support is needed to translate information on past activity into future action (and track progress towards this goal) either as part of the analytics system or the surrounding practices with which it is implemented. It also means that when study- ing use of analytics systems, researchers may need to take a longitudinal approach to reveal changes that happen in- crementally over time rather than directly after dashboard use. It is also important to consider the larger culture of trust and transparency around analytics amidst concerns of surveillance. Teachers may fear that their data can be used by administrators to assess performance or compli- ance with mandated standards [35, 35]. Students are often unaware of how they are being monitored, why, and who can view this data [79]. If data processing prior to analytic presentation is “blackboxed,” teachers and students may perceive that the collection of these data is primarily de- signed for the benefit of the institution and be less likely to trust and use the information provided [78]. One way analytics can support teachers is to inform learn- ing design. Learning designs document teachers’ peda- gogical intentions, providing a conceptual frame for ask- ing questions about learning activities and supporting sense-making of the information provided by the analyt- ics [18]. Data can help teachers understand the effects of a specific instructional approach on student activity and learning [20], which in turn provides feedback to improve the design [65, 60]. 2 TEACHER-FACING LEARNING ANALYTICS are aware of the fact that actions in digital spaces leave trace data. The conceptual leap is understanding how the high level representations of learning activity shown in analytic systems are produced from these data. In addi- tion, there is the question of what reference point the data should be compared to (e.g. a pre-determined standard or relative values for peers, [95]). Even when information is understood, it may not be believed as accurate, relevant or useful [42, 96], thus questions of trust and validity present additional consideration for the interpretation of analytics [15, 52]. Another limitation is that analytic systems tend to provide the same kinds of metrics over time; however, different information may be more or less relevant to dif- ferent parts of the learning process [92]. Finally, students and teachers each have their own goals for learning; thus designers cannot rely on a one-size fits all solution to be relevant for everyone [82]. Students and teachers need to prioritize the relative value they assign to the available analytic feedback. Teachers are a natural audience for learning analytics as they are already engaged in examining student learning to inform their practice. While such teacher-inquiry tra- ditionally depended on qualitative methods (e.g. student observations, examination of learning artifacts; [16]), there is increasing interest in the use of quantitative data to in- form the process [89]. Analytics can also be a powerful tool to help teachers with other dimensions of their prac- tice, for example identifying and meeting diverse student needs [22]. While the discussion below focuses on cogni- tive and pedagogical models of use, research suggests that affect also plays a role as teachers may feel encouraged, disheartened or even upset about what the information tells them [94]. 1 IMPORTANT CONSIDERATIONS FOR LEARNING ANALYTICS USE We then detail, first for teachers and then for students: (a) theoretical models underlying analytics use, (b) ways systems have been designed and implemented, and (c) evidence of the impact these systems have had on teaching and learning. We conclude with a focus on obstacles and opportunities to the development of effective and adoptable tools. With respect to interpretation, analytics are abstracted representations of past activity, yet intended to inform concrete future activity. This makes it critical for users to have an understanding of the context, purposes and processes of the learning activity in which the analytics were generated and a means by which to connect this in- formation to possible future action [50, 27]. Most people PG 130 | HANDBOOK OF LEARNING ANALYTICS 2.1 Theoretical Models of Teachers’ Learning Analytics Use Lockyer and colleagues [50] provide a specific model for aligning learning analytics use with learning design that describes how teachers can map the learning processes intended by their design, pre-identify patterns indicating (un)successful student engagement in the processes, and use analytics to track student pro- gression towards the desired state (an absolute reference frame for interpreting the data; [94]). Setting incremental stages to target along the way or using prior activity to judge progress are other comparison strategies that can be employed. In addition to point-in-time judgements, tem- poral analytics can also be used for dynamic evaluation of learning progress [59]. Another way analytics can support teachers is by provid- ing feedback on activities inside the classroom as they occur [85]. Here the analytics are used in (relatively) real- time as a tool to monitor activity, support the diagnosis of situations needing attention, and prompt teachers to offer support according to the students’ needs. These analytics support classroom orchestration [69] in which teachers use data as continuous formative assessment to adapt learn- ing at the classroom, small group, or individual level [41]. Several authors have provided descriptive models of how teachers make sense of the information provided and se- lect a pedagogical response [54, 58, 85, 94]. In the first stage, analytics aggregate information for manageable presentation through visualizations comparing students’ current activity to prior activity or absolute standards [85, 94]. To arrive at an interpretation of students’ activi- ties, teachers triangulate and contextualize the data with other information they have, noticing differences across CHAPTER 13: TEACHER & STUDENT FACING ANALYTICS individuals or groups, to answer goal-oriented, problem- oriented or instructional modification questions [49] . In the second stage, teachers use the information to inform pedagogical responses, which could be scaffolds targeted at the whole class, subgroups of students, or individual students. There is great potential for analytics to support teachers’ classroom orchestration by enhancing their in- sight into the classroom situation, their confidence in this insight, and thus inclination to act [46]. In this way ana- lytics enable teachers to make informed decisions that are aimed at students’ needs in-the-moment [58, 85]. ers by providing information not only about students but also about their own actions. Here the analytics take on a role of stimulating self-reflection, albeit with the same goal of optimizing student learning. 2.3 Use and Impact of Teacher-Facing Learning Analytics Systems The impact of teacher-facing learning analytics has largely been studied in terms of effects on teaching: teachers’ per- ceptions of usability, their awareness of student activities, and the actions they may take as a result [86]. This is a complex process [93, 86] requiring specific competencies such as data literacy and the ability to integrate knowl- edge from the analytics with existing teaching knowledge [54]. Multiple studies have found that analytics increase the specificity of teacher diagnoses in their classroom [75, 47]. However, for teacher-facing learning analytics to have an impact on students, teachers need to act on these diag- noses by selecting appropriate response actions. A small number of studies have examined the subsequent actions teachers select based on their interpretation of the ana- lytics. Molenaar and Knoop-van Campen [58] showed that when activating pedagogical knowledge in the sense- making stage, teachers use more diverse types of feedback in the response-stage. Wise and Jung [93] also showed diversity in teachers’ responses to learning analytics, in- cluding a non-action response of adopting a “wait-and- see” posture. Xhakaj, Aleven, and McLaren [98] found that analytics use influenced teachers’ subsequent lesson plannings (e.g. what topics to cover in a class session). Knoop-van Campen, Wise & Molenaar [43] found dash- board consultation led to relatively greater amounts of process feedback and that the difference was especially large for low-ability students. Teacher dashboards are a form of extracted analytics: data traces of students’ learning activity are provided in an interface separate from the learning environment that gen- erated them. An alternative is embedded analytics, when the data traces of learning activity are shown directly in the learning environment that generated them [92]. For exam- ple, Alavi and Dillenbourg [3] created ambient displays in the form of small lamps placed in the classroom that provided information on whether students had a question for the teacher and how long they had been waiting. In more recent work, Holstein, McLaren, and Aleven [34] developed augmented reality systems that displayed in- formation visible through the teachers’ enhanced glasses showing whether students were off-task or stuck on a problem. Going beyond teacher actions, very few studies have yet to follow the prolonged causal chain to examine effects on student activities or learning. 2.2 Teaching-Facing Learning Analytics Systems The most prominent form of learning analytics for teach- ers thus far are dashboards: visual displays that provide information about students’ activities and progress on the task at hand (for recent overviews, see [75, 47]). An impor- tant distinction in teacher-facing analytics is the amount of interpretational aid they provide [48]. Some early teacher dashboards left all interpretation to the teacher. For ex- ample, Schwarz and Asterhan [73] showed teachers infor- mation about students’ argumentation in a collaborative learning setting, but did not prescribe when to intervene or what situations might need attention. Similarly, [58] reported on a dashboard that displayed information about individual student performance on mathematics exercises; teachers were free to decide how to interpret and use them for follow up interventions. Other teacher dashboards have gone a step further to provide alerts about the occur- rence of problems that specific students or groups might be facing (e.g. [13, 30], or even alerts plus advice regard- ing what kind of problem students might be facing in a particular situation [86]). Most existing dashboards have focused on supporting teacher sense-making; however many teachers also experience difficulty in determining what action to take in response [75, 86]. While few dash- boards have yet to explicitly target the action-taking phase of analytics use, there are some notable exceptions. For example, Olsen, Rummel, & Aleven [63] developed a sys- tem which advised the teacher on which students to pair up and when to switch to a different activity. In an earlier example, the Assistant program offered advice to teachers on what feedback to provide to students [14]. 2.3 Use and Impact of Teacher-Facing Learning Analytics Systems 2.1 Theoretical Models of Teachers’ Learning Analytics Use For example, Anh et al [2] used small lamps on the tables of collaborating students to display how long the teacher had visited each group, thus providing information to the teachers about their circula- tion around the classroom. The lamps provided neutral information without enforcing or encouraging teachers to divide their attention equally - that decision remained with the teacher. Despite their potential, systems that ad- vise on specific teacher behaviors are rare and hard to design since the impact of teachers’ actions can be very context-specific. 3 STUDENT-FACING LEARNING ANALYTICS Second, while other sys- tems tend to provide relatively simple static feedback, dashboards offer visual displays which are often complex and/or interactive, allowing students to filter or select specific information. In addition, prior feedback systems typically provided information to students after they had finished a problem, activity or assignment, whereas dash- board information can be available on-demand, so stu- dents have flexibility and control of when they consult this information. Third, while many feedback systems benchmark using normative standards, in dashboards stu- dent performance is often also visualized in relation to that of local peers. Sometimes, students are also provided with information specifically in the context of “students like them” [40]. Finally, in cognitive tutors and similar systems, the computer is in control, whereas dashboards offer information to students, who decide on any possible follow-up actions. These dashboards are quickly becom- ing a standard feature in applications aimed at personaliz- ing learning, such as Learning Management Systems, as well as in newer applications for personalizing learning like gameful approaches to pedagogy (e.g. Gradecraft [1]) and as part of tailored messaging systems (e.g. eCoach; [36]). While SRL has been the dominant theoretical paradigm thus far, several other theoretical frameworks could also contribute to the system design for student-facing analyt- ics. Expectancy Value Theory (EVT; e.g., [25]) posits stu- dents are motivated based on their expectancy of success, value, and cost of their options to accomplish learning goals. Investigation of dashboard use under EVT could reveal in which contexts students consider dashboards to have lower utility, and thus lower value, such as students taking a course to fulfill a requirement versus those who want to perform well (e.g [42]). Self-Determination Theory (SDT: e.g. [68]) posits that motivation is primarily based on the satisfaction of three needs: autonomy, relatedness, and competence. Based on SDT, dashboard design and evaluation could be oriented toward how effectively they contribute to students’ need satisfaction. For example, students’ motivation to engage with a dashboard may de- pend on their belief that it provides (a) a sense of control over their ability to accomplish course goals, (b) a greater sense of belonging within the course or discipline, and/or (c) information that increases their competence in meeting course requirements. 3 STUDENT-FACING LEARNING ANALYTICS to be theoretically grounded with respect to pedagogy and learning (e.g. [74, 6, 7, 37]), most system designs are still driven primarily by available data. When theory does drive system design, models of Self-Regulated Learning (SRL) are commonly employed [56]. Students are an important audience for analytics use, as their learning is the ultimate goal of educational systems and much of the data collected in learning analytics sys- tems is generated by or about them. There is a presump- tion that students will benefit from exposure to their own learning data and many argue that students have the right (and responsibility) to review their own data [64]. As such, an increasing number of analytics systems are being designed to provide information about learning directly to students. These both follow and diverge from a long history of educational technologies used to provide feed- back to students (e.g., cognitive tutors, [45]; homework practice and assessment tools such as Assistments, [57]; open educational resources such as Kahn Academy, [38]). Zimmerman [100, p. 4] described self-regulated learning as students that are “metacognitively, motivationally, and behaviorally active participants in their own learning”. This includes planning, monitoring, and evaluation of one’s own learning, and using these strategies to achieve academic goals. As a positive relationship exists between self-regulation and learning performance [10, 90], SRL is seen as a promising way for learning analytics to sup- port students by making these processes more explicit and allowing students to see and assess their own learn- ing. Drawing on SRL theory, Wise [97] proposed a spe- cific model of student learning analytics use involving goal-setting, action and reflection. These engendered four principles for pedagogical practice to support students’ analytics use, with initial empirical validation in Wise et al. [96]: Integration, Agency, Reference Frame, and Dia- logue. Later work by Klein et al. [42] validated the central importance of Agency in shaping students’ relationships to analytics and offered four additional factors to consider in their sense-making: Accuracy, Relevancy, Trust and Relationships. Learning analytics dashboards differ from prior feed- back systems in a number of ways. First, other systems typically provide feedback about correctness of answers, whereas dashboards often combine performance feedback with information on students’ learning processes (e.g., planning, tracking progress). 3 STUDENT-FACING LEARNING ANALYTICS Students who experience a higher level of control in the learning process are more likely to be intrinsically motivated and improve their perfor- mance [19] and dashboards may be an excellent avenue to provide students with a greater sense of agency and autonomy. 2.3 Use and Impact of Teacher-Facing Learning Analytics Systems In one notable excep- tion, Martinez-Maldonado, Clayphan, Yacef, and Kay [55] report a comparison of impact of two dashboards, one providing information only and one providing informa- tion plus alerts. Teacher interventions informed by the system with alerts resulted in an improvement in student learning, those informed by the system with information only did not. This study points to the importance of work- ing towards studies that document the ultimate goal to impact students’ learning. Learning analytics can also play a role in supporting teach- PG 132 | HANDBOOK OF LEARNING ANALYTICS 3.1 Theoretical Models of Students’ Learning Analytics Use Student-facing learning analytics aim to support students in conscious attention to and improvement of their own learning processes [93]. Feedback is provided in the con- text of dynamic cognitive processing whereby students se- lect, adapt and generate tactics and strategies for learning and monitoring their learning [12]. Affective considera- tions come into play as well as how students use analytics depends not only on what the information helps them know, but also how it makes them feel [92, 42]. Although there have been calls for student facing learning analytics 3.2 Student-Facing Learning Analytics Systems Fritz [28] conducted one of the first wide-scale deploy- ments of a dashboard specifically aimed at students, called Check My Activity (CMA), that allowed university stu- dents to compare their LMS activity and grades against CHAPTER 13: TEACHER & STUDENT FACING ANALYTICS | PG 13 their classmates. Student focus on grade views has been observed repeatedly, including Young’s [99] analysis of students using a commercial LMS (Blackboard). Follow- ing Blackboard’s design, Corrin and de Barba [17] cre- ated a dashboard with data on students’ formative and summative assessment scores and their LMS engagement. Students were able to articulate and interpret feedback presented through a dashboard, but there was little evi- dence of students’ ability to understand the connection be- tween feedback and their current learning strategies. Wise and colleagues [92] implemented an analytics-enhanced discussion forum called Starburst that incorporated goal- setting and reflective prompts to encourage analytics use as part of an SRL cycle. Students’ use of the analytics showed that comparison with peers played an influen- tial — though not always positive —role on changes in behaviour, and students’ mistrust in how some analyt- ics were computed may have dampened use [96]. Khan and Pardo [39] implemented Data2U, a system provid- ing students with feedback about their interactions with the online resources. They characterized different types of student dashboard use, providing insight into when different students utilize the dashboard (i.e. beginning, middle or end of a study session). However, there was no statistically significant relationship between students’ use of the dashboard and their academic performance. Taking a participatory approach to analytics with a critical lens, Knox [44] developed the Learning Analytics Report Card (LARC) to give students choices about what data to include and exclude in the reports it generated. Most recently, Kia et al. [40] implemented a student dashboard into their campus’ LMS, and found that students’ SRL behaviors and academic achievement influenced how stu- dents used the dashboards. tool was to increase the percentage of their peers’ posts that they read. This is a behavior thought to contribute to learning theoretically (through increasing the diversity of ideas to which a student is exposed), but direct evidence of learning outcomes gains was not available. 3.2 Student-Facing Learning Analytics Systems Further, only a few recent studies have investigated differences in how students use dashboard information [4, 33, 40], such as the particular tactics and strategies they take to work with the information [29], that may have important effects on subsequent outcomes. From these studies it is clear that the use and impact of student-facing analytics is a crucial topic for future research to understand how, when, and to which students we should provide these sys- tems. In the preceding sections we described teacher- and student-facing learning analytics systems independently. However, there are important issues that bridge across these categories. A central question for teacher- and student-facing learn- ing analytics is what kinds of information is most useful to distribute across which parts of the overall system at different points in time. What support should analytics offer to students directly, which information is best passed to the teacher first, and which decisions can effectively be made by the analytics system autonomously? One example is suggested by Rummel, Walker, and Aleven [70] as they describe an “utopian” vision of adaptive sup- port for collaborative learning in which the analytic sys- tem nudges a student directly to engage more with her partner during a learning task, supports her review and reflection on her engagement once the task is complete, and provides information and suggestions to the teacher for assigning her a subsequent collaborative partner and task. They also consider what analytic systems can learn from teachers and students to help them provide more useful information and/or guidance. This represents a move towards considering hybrid teacher-analytic and student-analytic systems as part of the classroom ecology. 3.3 Use and Impact of Student-Facing Learning Analytics Systems In addition, the triangular interplay between teachers, stu- dents and analytic tools is a growing area of focus and research. Two particular issues to consider are symme- try (to what extent do students and teachers have access to the same kinds of information) and transparency (to what extent do students know what teachers can see). In situations where teachers and students are able to work with data jointly to improve learning processes, analytics can be seen to act as a third “voice” in the conversation between teacher and student [92]. For example, in two re- cent studies of teacher-facing dashboards at the university level, teachers expressed the desire to have a deidentified view of the analytics so they could show their students evidence about why they were concerned about their per- formance in the class [48, 94]. Analytics can also act as a mediational object for interactions between teachers and students as seen in Tan, Koh, Jonathan, and Tay [81] who documented a 9th grade school teacher sharing visualiza- tions of her students’ online discussion comment types and interaction network with them as an object to support collective reflection about the quality of their collabora- tion. Similarly, Lonn, Aguilar, and Teasley [51] described how when a dashboard designed specifically for academic There has been limited research exploring how students interpret and act on learning analytics and the resulting ef- fects on their motivation, behavior, knowledge and skills [6, 74, 87]. This problem is not unique to learning an- alytics, however. Regarding the broad research on the effectiveness of feedback, Winstone [91, p. 227] points out “there are very few examples where researchers ex- plore the use of feedback on a behavioral level, and even fewer examples where researchers collect data to follow up and see how students’ engagement influences them later in time”. With the notable exception of collaborative learning analytics (particularly group awareness tools, e.g. [5]), existing research on student-facing learning analytics systems has concentrated more on dashboard usability and usefulness, rather than an understanding how they support educational practices in the wild [29]. When how students use analytics is studied in authentic educational settings, their interactions with technology (e.g., counting views, files accessed, time on task) are usu- ally the main marker of impact. 3.3 Use and Impact of Student-Facing Learning Analytics Systems For example, Wise [92] found the most common change that students made af- ter the introduction of the analytics-enhanced Starburst PG 134 | HANDBOOK OF LEARNING ANALYTICS shift towards human-centered learning analytics [76]. Co- design of learning analytics can involve users in decisions about the content of the analytics (what information is pro- vided) and/or the visualization of the analytics (how the information is provided). When co-design is employed, it has most often involved teachers (e.g. [2, 23, 34, 55, 83, 84, 88]). Recent efforts have started to engage students in the process of analytics design as well (e.g. [67, 66, 71]). advisors was shared in an advising session, it became a tool for advisors and students to talk about the student’s academic progress. With the introduction of this third voice, a recalibration of student and teacher classroom roles is needed. For students, analytics offer the opportunity to be explic- itly prompted and supported to monitor and reflect on their learning, allowing them to develop metacognitive skills and take responsibility for their own learning. Re- search has shown that some students arrive in the class- room better equipped to make use of analytic information than others [53]; thus there is often a need to develop data literacy and self-regulation skills in tandem with analytics use. However, there is also a risk that providing too much information, automation or guidance (whether from the learning analytics systems or by the teacher) may create dependency, robbing students of the opportunity to dis- play agency in their own learning. Educators worry about the rise of “helicopter analytics” where institutional tools and processes assume a decision-making role for students that many parents have been criticized for playing [35]. On the whole, a balance is needed to provide guidance that both helps students make better-informed choices in the short-term [62] and increases their ability to be inde- pendent learners over the long-term [9]. The potential benefits of co-design are substantial: by giving teachers and students a role in the creation of learn- ing analytics we are not only better able to design tools that fit their contexts and needs, but also allow them to surface their hopes and fears related to the use of ana- lytics. There is a long tradition of work in HCI that can inform our processes of co-design (e.g. REFERENCES [1] Stephen J. Aguilar, Caitlin Holman, and Barry J. Fishman. “Game-inspired design”. In: 13.1 (Aug. 2015), pp. 44–70. DOI: 10 . 1177 / 1555412015600305. URL: https://doi.org/ 10.1177%2F1555412015600305. 3.3 Use and Impact of Student-Facing Learning Analytics Systems [21]); however there are also challenges specific to learning analytics, par- ticularly varying levels of data literacy and asymmetric power dynamics. These issues may also intensify exist- ing tensions in co-design for learning between what users want and what others want for them. Techniques from established co-design methodologies are being adapted for learning analytics to address such challenges [34, 66, 71]. A basic tenet of learning analytics is to provide infor- mation that is actionable by its users. 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[97] Alyssa Friend Wise, Yuting Zhao, and Simone Nicole Hausknecht. “Learning analytics for online discussions: Embedded and extracted approaches.” In: Journal of Learning Analytics 1.2 (2014), pp. 48– 71. [98] Françeska Xhakaj, Vincent Aleven, and Bruce M McLaren. “Effects of a teacher dashboard for an intelligent tutoring system on teacher knowledge, lesson planning, lessons and student learning”. In: European Conference on Technology Enhanced Learn- ing. Springer. 2017, pp. 315–329. [99] JR Young. “What clicks from 70,000 courses reveal about student learning”. In: Chronicle of Higher Ed- ucation 63.3 (2016). [100] Barry J. Zimmerman. 4 CO-DESIGN OF LEARNING ANALYTICS AS A WAY FORWARD “Self-regulated learning and academic achievement: An overview”. In: Educa- tional Psychologist 25.1 (1990), pp. 3–17. PG 140 | HANDBOOK OF LEARNING ANALYTICS
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https://journals.wlb-stuttgart.de/ojs/index.php/bjb/article/download/9481/9372
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mittelalterliche deutsche Predigthandschrift aus dem Augustiner-Chorherrenstift Backnang
Backnanger Jahrbuch
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Eine mittelalterliche deutsche Predigthandschrift aus dem Augustiner-Chorherrenstift Backnang Von Carsten Kottmann Herzog Ulrich von Württemberg (1487 bis 1550) ließ während der Reformation im Jahr 1535 ein Inventar des Stifts und der Stiftskirche Backnangerstellen. Dieses Inventar ist leider ver­ loren gegangen, und mit ihm auch die Chance, Einblicke in die materielle Ausstattung des Stifts zu erhalten. Das wäre umso interessanter gewe­ sen, als bisher über die Kulturgeschichte des Stifts nur wenig bekannt ist. Damit bleiben Fra­ gen nach dem alltäglichen Leben im Stift, nach der Bildungssituation, nach Frömmigkeit und Liturgie, nach Schreibstube, Literaturrezeption und -produktion weitgehend unbeantwortet, ja, es lässt sich der gesamte Bereich „der literari­ schen und grammatischen Bildung, der nach aller Wahrscheinlichkeit im Stift ebenfalls ge­ pflegt wurde, [...] aus den schriftlichen Quellen nicht mehr fassen".1 Dabei wurde in der Reformationszeit dem Stift Backnang eine köstlich liberei, also eine gut aus­ gestattete Bibliothek attestiert.2 Doch von dieser ist so gut wie nichts erhalten geblieben. Die Wirren der Reformationszeit,3 der Dreißigjährige Krieg mit seinen Streitereien um das Backnanger Stift4 und nicht zuletzt der Franzoseneinfall 1693 in Südwestdeutschland, bei dem Backnang komplett niederbrannte,5 boten genügend Gele­ genheiten, die Backnanger Bibliothek in alle Winde zu zerstreuen bzw., was wahrscheinlicher ist, zu vernichten. Neben diplomatischem Schrifttum wie Urkun­ den sind bisher vor allem kalendarische und Memoria-Fragmente erhalten, so ein Kalender mit Jahrtagseinträgen und eine Wohltäterliste aus dem letzten Viertel des 15. Jahrhunderts,6 zudem der „Backnanger Nekrolog", der zwar erst zwi­ schen 1569 und 1598 niedergeschrieben, aber wohl schon nach 1453 und vor 1466 zusammen­ gestellt wurde.7 Zudem wurden Einbandfrag­ mente mit Resten liturgischer Handschriften, vor allem des frühen 13. Jahrhunderts gefunden, die aufgrund der verwendeten liturgischen Eigen­ heiten nachweislich in Backnang verwendet und vielleicht auch dort geschrieben wurden.8 Der nun ausführlicher vorzustellende Hand­ schriftenfund betritt damit Neuland auf dem Ge­ biet der Geschichte des Augustiner-Chorherren­ stifts Backnang, da es sich um Texte handelt, von deren Existenz in der bisherigen Forschung nicht die Rede war. Dabei ist die Handschrift und auch ihre Provenienz in Backnang seit Beginn des 20. Jahrhunderts grundsätzlich bekannt,9 hatte aber 1 Gerhard Fritz: Backnang und Umgebung im 13. Jahrhundert, Backnang 1982 (= Schriftenreihe des Heimat- und Kunstvereins Backnang 1), S. 30. Für eine knappe übersichtliche Darstellung der Geschichte des Stifts Backnang mit kulturgeschichtlicher Ausrichtung vgl. Carsten Kottmann: Liturgische Handschriftenfragmente aus dem Augustiner-Chorherrenstift Backnang. - In: ZWLG 62 (2003), S. 101 bis 11 7. - Folgende Abkürzung wird an dieser Stelle eingeführt: 2VL = Die deutsche Literatur des Mittelalters. Verfasserlexikon. Hg. von Kurt Ruh / Burghart Wachinger u.a., 14 Bde, Berlin / New York 1978 bis 2008. 2 Sabine Beate Reustle: Stift und Stadt Backnang im 16. Jahrhundert. Territorialisierung und Reformation in einer württembergischen Amtsstadt, Backnang 1996 (= Backnanger Forschungen 2), S. 160. 5 Ebd., S. 158 bis 161. 4 Vgl. Carsten Kottmann: Geschichtsschreibung als Legitimation. Die Jesuiten und das Backnanger Stift in den Jahren 1635 bis 1648. - In: Glaube -- Bildung - Gesellschaft. Leben in der Frühen Neuzeit (16. bis 18. Jh.). Beiträge der Backnanger Tagung vom 21. Juni 2003, Hg. von Carsten Kottmann und Bernhard Trefz, Backnang 2006 (= Backnanger Forschungen 7), S. 1 77 bis 208. 5 Vgl. Gerhard Fritz: Backnang und die Franzoseninvasion von 1693. - In: BJb 2, 1993/94, S. 64 bis 95. 6 Karl Otto Müller: Neue Quellen zur mittelalterlichen Geschichte Backnangs. - In: ZWLG 7 (1943), S. 181 bis 208. 7 Gerhard Fritz: Der Backnanger Nekrolog. Studien zur Geschichte des Augustiner-Chorherrenstifts Backnang. - In: ZWLG 44 (1985), S. 11 bis 64. . 8 Kottmann (wie Anm. 1). 9 Handschriftenarchiv der Berlin-Brandenburgischen Akademie der Wissenschaften: Stuttgart, WLB, cod. theol. et. philos. fol. 64, beschr. von Otto Leuze, Stuttgart 1915, 93+5 Bll. 59 ihren Weg in die Backnanger Stadtgeschichts­ schreibung dessen ungeachtet nicht gefunden. Weite Verbeitung von Heiligenprecligten Es handelt sich um eine Sammlung von deut­ schen Heiligenpredigten, die in der literaturwis­ senschaftlichen Forschung den „Schwäbischen Heiligenpredigten" zugeordnet wird.10 Heiligen­ predigten waren das gesamte Mittelalter hin­ durch beliebt und verbreitet. Quelle für die Predigten waren in aller Regel Sammlungen von Legenden, also Lebens- und Tatenbeschreibun­ gen von Heiligen, die schon früh zusammen­ gestellt wurden, so zum Beispiel in den „Dialogi de vita et miraculis patrum italicorum" Gregors des Großen (540 bis 604). Im 13. Jahrhundert versuchte der Dominikaner Jacobus de Voragine (um 1230 bis 1298) die ihm verfügbaren Legen­ den erneut zu bündeln. Mit der daraus entstan­ denen Sammlung der „Legenda aurea" schuf er eines der am weitest verbreiteten religiösen Er­ bauungsbücher des späten Mittelalters. Es fiel in einer äußerst kirchenfrommen Zeit auf einen fruchtbaren Boden und beeinflusste die Mentali­ tät aller sozialen Schichten nachhaltig." Zur Popularität der Heiligenpredigten trug auch die bald einsetzende umfangreiche deutschsprachige Überlieferung bei. Die Samm­ lungen der Lebensbeschreibung von Heiligen sind die am häufigsten überlieferte erzählerische Gattung des gesamten deutschsprachigen Mit­ telalters. Es beginnt mit dem „Passional" an der Jahrhundertwende vom 13. zum 14. Jahrhundert, eine Sammlung deutschsprachiger Legenden in Versform aus dem Umfeld des Deutschen Ordens.12 Mit der „Elsässischen Legenda aurea", um 1350 in Straßburg als Übersetzung der „Le­ genda aurea" des Jacobus de Voragine entstan­ den, sowie der aus Nürnberg stammenden und in der zweiten Hälfte des 14. Jh. zusammenge­ stellten Sammlung „Der Heiligen Leben" treten hier zwei in Süddeutschland breit tradierte Ver­ treter stark in den historischen Fokus. Daneben existieren mehrere kleinere und größere Legen­ densammlungen mit geringerer Breitenwirkung. Das Ende der deutschsprachigen Legendenüber­ lieferung lässt sich mit dem Beginn der Refor­ mation um 1520 fassen. Auch die Predigt in deutscher Sprache war entgegen der teilweise existierenden landläufigen Auffassung - ein weit verbreitetes Phänomen im Mittelalter. Während die Feier des Gottesdiens­ tes, also die Liturgie, der lateinischen Sprache vorbehalten blieb, sind spätestens seit der zwei­ ten Hälfte des 12. Jahrhunderts, aber natürlich auch hier stark zunehmend im späten Mittelal­ ter, deutschsprachige Predigten erhalten. Anzu­ nehmen ist der Beginn der deutschsprachigen Predigten im Umkreis der Benediktinermönche und Augustiner-Chorherren zu suchen, da diese Orden neben dem Säkularklerus einen wesentli­ chen Anteil an der Seelsorgetätigkeit im 12. Jahr­ hundert hatten.13 Ein bedeutendes Beispiel für einen mittelalter­ lichen Prediger in deutscher Sprache: Berthold von Regensburg (um 1210 bis 1272). 10 Werner Williams-Krapp: Die deutschen und niederländischen Legendäre des Mittelalters. Studien zu ihrer Überlieferungs-, Text- und Wirkungsgeschichte, Tübingen 1986 (= Texte und Textgeschichte 20), S. 18f. ’’ Ebd., S. 1 bis 6. 12 Kurt Gärtner: Zur Überlieferungsgeschichte des „Passionais". - In: Zeitschrift für deutsche Philologie 104 (1985), S. 35 bis 69. 13 Hans-Jochen Schiewer: Art. Predigt, B. Volkssprachliche Literaturen des Westens, I. Deutsche Literatur. - In: Lexikon des Mit­ telalters 7 (1995), Sp. 174 bis 176; Karin Morvay / Dagmar Grube: Bibliographie der deutschen Predigt des Mittelalters. Ver­ öffentlichte Predigten, München 1974 (= Münchener Texte und Untersuchungen zur deutschen Literatur des Mittelalters 47). 60 Die Heiligenpredigt im Besonderen war an den Festtag des jeweiligen Heiligen gebunden. Dabei wurden sie vornehmlich aus der Woche gesammelt sonntags gehalten, da Heiligenfeste in der Regel von höherem liturgischem Rang waren als die gewöhnlichen Sonntagsfeiern und diese oft verdrängten. Am Sonntag wurden dann oftmals im Anschluss an die herkömmliche Predigt Hinweise wie biografische Kurzangaben zu den Heiligenfesten unter der kommenden Woche gegeben.14 Mit der schier unüberschaubaren und weit­ läufigen Überlieferungslage der Legenden­ sammlungen und Heiligenpredigten stellen sich besondere Herausforderungen ein: „Legenden­ sammlungen zählen zu den ausgesprochen ,offenen' Literaturtypen. Die Geschichte ihrer Über­ lieferung ist in erster Linie durch Änderungen in der Zusammenstellung des Corpus bestimmt. Selten gleicht hier eine Handschrift der anderen; Auslassungen, Zufügungen, Auswechslungen, Umstellungen von Legenden sind die Regel."15 Dies hat Auswirkungen auf die Interpretation der Überlieferungssituation und Textgeschichte. Diese Herausforderungen stellen sich auch bei der näheren Untersuchung des nun darzu­ stellenden Handschriftenfunds mit den „Schwä­ bischen Heiligenpredigten". Zwar ist die Über­ lieferungslage durchaus überschaubar - es sind insgesamt sechs Überlieferungsträger bekannt, von denen eine jedoch nur eine einzige Predigt aus der Reihe der „Schwäbischen Heiligenpre­ digten" enthält. Dennoch bleibt die beschrie­ bene Überlieferungscharakteristik der Legen­ dentradition eine Grundkonstituente, der sich auch die Beschäftigung der „Schwäbischen Hei­ ligenpredigten" zu stellen hat. Die nun als neu vorzustellende Handschrift dient sowohl in der Herangehensweise an die Texte als auch über­ lieferungskritisch als zentrale beziehungsweise als „Leithandschrift", da sie den ältesten und umfangreichsten Überlieferungsträger darstellt. Die Backnanger Predigthandschrift Äußerliche Beschreibung Die Handschrift Stuttgart (S), Württember­ gische Landesbibliothek, cod. theol. et. philos. 2° 64 ist eine Papierhandschrift, die nach der No­ tiz auf fol. 68v unten im Jahr 1393 geschrieben wurde.16 Der Buchblock beträgt 25,5 x 17 cm, der Schriftraum 21,5 x 14 cm. Die Handschrift besteht aus 77 einspaltig beschriebenen Blättern, jedoch waren es ursprünglich mehr, die Hand­ schrift ist an verschiedenen Stellen defekt, es las­ sen sich ursprünglich circa 100 Blätter vermuten. Blatt 1 ist bis auf einen kleinen Rest oben links herausgerissen.17 Der inzwischen ausgelöste hintere Spiegel (zur Verstärkung eingeklebtes Pergamentblatt) überliefert auf einem Doppelblatt eine kom­ mentierte Fassung einer lateinischen Grammatik, nämlich der Ars minorâes Aelius Donatus (* um 320, + um 380)’8, geschrieben auf Pergament in einer frühgotischen Minuskel mit starker Nähe zur karolingischen Schrift, aus der ersten Hälfte des 12. Jahrhunderts. Der ebenfalls inzwischen ausgelöste vordere Spiegel besteht aus einem Blatt einer liturgischen Handschrift, geschrieben auf Pergament in einer frühgotischen Minuskel um 1200. Dabei handelt es sich um das Fragment eines liturgischen Buches, nämlich eines Breviers, das vollständig sämtliche Texte des klösterlichen beziehungs- 14 Werner Williams-Krapp: Mittelalterliche deutsche Heiligenpredigtsammlungen und ihr Verhältnis zur homiletischen Praxis. In: Die deutsche Predigt im Mittelalter. Internationales Symposium am Fachbereich Germanistik der Freien Universität Berlin vom 3. bis 6. Oktober 1989. Hg. von Volker Mertens und Hans-Jochen Schiewer, Tübingen 1992, S. 352 bis 360, hier S. 352 f. 15 Die Elsässische „Legenda Aurea" Bd. II: Das Sondergut. Hg. von Konrad Kunze, Tübingen 1983 (= Texte und Textgeschichte 10), S. XI. 16 Anno domini milesimo CCCLxxxxiii ante diem festum purificationis sancte marie virginis (1. Februar 1 393). Vgl. den handschriftlichen Katalog in der Handschriftenabteilung der WLB Stuttgart. Ausführlicher: Leuze (wie Anm. 9); auch WilliamsKrapp (wie Anm. 10), S. 18; Konrad Kunze: Art. „Schwäbische Heiligenpredigten". - In: 2VL 8 (1992), Sp. 907 bis 909; Madeleine Boxler: „ich bin ein predigerin und appostlorin". Die deutschen Maria-Magdalena-Legenden des Mittelalters (1 300 bis 1550). Untersuchungen und Texte, Bern/Berlin u.a. 1996 (= Deutsche Literatur von den Anfängen bis 1700 22), S. 223. Textprobe zu Maria Magdalena: Ebd., S. 409f. 17 Eine genaue Lagebeschreibung bietet Leuze (wie Anm. 9), Nr. 3. 18 Vgl. Axel Schönberger: Die Ars minor des Aelius Donatus. Lateinischer Text und kommentierte deutsche Übersetzung einer antiken Elementargrammatik aus dem 4. Jahrhundert nach Christus, Frankfurt 2008 (Bibliotheca Romanica et Latina 6), S. 20 bis 46, und allgemein zu den mittelalterlichen Kommentaren Geoffrey L. Burshill-Hall: Medieval Donatus Commentaries. - In: Historiographia linguistica 8 (1981), S. 69 bis 97. 61 weise stiftischen Stundengebets beinhaltet. Das Fragment in der vorliegenden Handschrift über­ liefert Texte zur Matutin, also zum Morgengebet, die am Nikolaustag (6.12.) gebetet wurden. Oft lässt sich anhand von liturgischen Texten deren Herkunft nachweisen, da viele Orden, Diözesen oder Reformverbünde eigene liturgische Cha­ rakteristiken ausgebildet haben. Allerdings ist das Stundengebet zu Nikolaus noch zu wenig erforscht, als dass hier eine Zuordnung zum Bei­ spiel zur Liturgie des Marbacher Augustiner­ chorherrenstifts, und damit im 12. Jahrhundert eine Lokalisierung des Fragments nach Back­ nang, gelingen könnte. Noch im Mittelalter wurde die Handschrift in Holzdeckel mit ursprünglich weißem Halbleder eingebunden, nachträglich wurde mit braunem Leder nachgearbeitet. Dieser Einband ist defekt. Auf dem Vorderdeckel finden sich rechts zwei Abschrägungen für Schließen, die aber wohl nie ausgeführt wurden, zumindest sind keinerlei Reste vorhanden. Anfang des 20. Jahrhunderts wurde der Einband restauriert. Auf fol. 2r findet sich der Besitzstempel der „Bibliotheca Regia Pu­ blica Stuttgartensis", also der Königlichen Öffent­ lichen Bibliothek des 19. Jahrhunderts. Geschrieben wurde die Handschrift von einer Haupthand in einer älteren gotischen Kursive, dabei weist die Schrift „verschiedene Nuancen auf, ist aber wohl doch einer Hand zuzuweisen".19 Auch die Zeilenanzahl variiert bei gleich­ bleibendem Schriftspiegel zwischen 30 und 46 Zeilen. Der Schreiber ist nicht ungeübt, scheint aber auf eine stringent durchgezogene Einheit­ lichkeit in der Texteinrichtung keinen allzu gro­ ßen Wert gelegt zu haben. Schon das Schriftbild spricht dafür, dass es sich um eine Gebrauchs­ handschrift und kein Repräsentationsobjekt ge­ handelt hat. Verschiedene kleinere Stellen auf fol. 67v und 68v wurden von weiteren, meist Die Stuttgarter Handschrift aus dem Backnanger Augustiner-Chorherrenstift. Hier der Text der Heili­ genpredigt für Cäcilia. 19 Leuze (wie Anm. 9), Nr. 4. 62 späteren Händen geschrieben. Der Text ist wei­ testgehend gut leserlich, allerdings sind grund­ sätzlich starke Gebrauchsspuren zu erkennen. Inhaltliche Beschreibung Die Handschrift enthält als Haupttext die „Schwäbischen Heiligenpredigten" als ältesten Überlieferungsträger. Allerdings ist der Text un­ vollständig, da die Handschrift verstümmelt ist: Fol. 1, vordem noch eine Anzahl weiterer Blätter existiert haben müssen, ist bis auf ein kleines Fragment herausgerissen. Der vorliegende Be­ stand beginnt auf dem Blatt-Fragment auf fol. 1 Benutzungseinträge am Ende der Stuttgarter Handschrift. mit der Heiligenpredigt zum Fest Simonis und Judä (28.10.) und reicht dann bis zum 16. Feb­ ruar, dem Festtag zu Juliana (fol. 30v), bricht hier aber aufgrund von Blattverlust mitten im Satz ab, um auf fol. 31 v wieder mitten im Satz mit dem Fest Petrus episcopus (22.2.) fortzuführen. Ein solcher Blattverlust findet sich auch zwischen fol. 34v (Gangolf, 13.5.) und fol. 35v (Veit, 15.6.), der ebenfalls einen Textabbruch und -anschluss mitten im Satz zur Folge hat. Fol. 49v ist nur mit vier Zeilen der Predigt zu Bartholo­ mäus beschrieben, die schon auf fol. 48v beginnt, ansonsten ist die Seite unbeschrieben. Die Pre­ digt zu Gallus (16.10.) hingegen beginnt auf der darauffolgenden Seite (fol. 50r) mitten im Satz, ohne dass ein Blattverlust vorangeht. Die Heili­ genpredigten enden mit dem Fest Crispinus und Crispianus (25.10.), es folgen Predigten für die Sonntage Septuagesimä, Sexagesimä, Estomihi, 2. Sonntag nach Epiphanias, Reminiscere, Oculi, Laetere, Judica, Palmsonntag; den Abschluss bil­ det eine Gründonnerstags-Predigt. Fol. 68v ent­ hält noch die lateinische Benediktionen für die Osterlammweihe (Benedictio agni in Pascha), ebenfalls von der Haupthand geschrieben: Post celebrantem und Deus universae Creator carnis, die Benediktion für die Weihe des Aufbewah­ rungsortes für Fleisch Omnipotens et misericors deus qui necessitati humani generis sowie die Brotweihe Descendat benedicio dei patris et filii et Spiritus sancti super hanc creaturam panis.20 Weiter finden sich auf fol. 68v die Antifon (liturgischer Gesang) Vidi aquam egrendientem de templo,2' und darunter eine Federprobe einer späteren, ungeübten Hand des 1 5. Jahrhunderts: Item ioh honns wolff. Die Wohltäterliste des Stifts Backnang aus der zweiten Hälfte des 15. Jahrhunderts nennt einen Hans Wolff.22 Der Ein­ trag ist nicht näher datiert, aber es bleibt zumin­ dest wahrscheinlich, dass der Wohltäter Wolfs mit dem Schreiber der Federprobe identisch ist. 20 Adolph Franz: Die kirchlichen Benediktionen im Mittelalter Bd. 1, Freiburg i. Br. 1909, S. 584f., 640, 268. 21 Corpus Antiphonalium Officii. Hg. von René-Jean Hesbert. Bd. 3: Invitatoria et antiphonae, Rom 1968 (= Rerum ecclesiasticarum documenta, Series maior 9), Nr. 5403. 22 Necnon Johannis Wolffen, uxoris, puerorum, parentumque suorum et benefactorum omnium. Vgl. Müller (wie Anm. 6), S. 291. Weder das Backanger Lagerbuch von 1393 noch das Cültverzeichnnis um 1450/53 kennen einen Johannes/Hans Wolfs. Vgl. Carsten Kottmann: Die Crundherrschaft des Augustirier-Chorherrenstifts Backnang - das älteste Backnanger Lagerbuch von 1393 und das Gültverzeichnis aus den 1450er-Jahren. - In: Württembergische Stiftskirchen. Insbesondere das Stift St. Pancratius in Backnang. Beiträge .der Backnanger Tagung vom 19. Mai 2001. Hg. von Gerhard Fritz, Backnang 2003 (= Backnanger For­ schungen 5), S. 87 bis 142 (Register S. 150 bis 155). Unter den Kanonikern des Stifts ist ebenfalls niemand dieses Namens be­ kannt, vgl. Gerhard Fritz: Das regulierte Augustiner-Chorherrenstift Backnang vom frühen Mittelalter bis zu seiner Umwand­ lung in ein Säkularstift 1477. - In: Ebd., S. 21 bis,54. 63 Herkunft der Handschrift Neben diesen bisherigen Hinweisen lässt sich die Herkunft der Handschrift aus weiteren Hin­ weisen erschließen. In der letzten Zeile auf fol. 68v wird die dem hl. Guido geweihte Kollegiatkirche in Speyer genannt.23 „Das Stift Backnang war nachweislich demjenigen Archidiakonat des Speyrer Bistums zugeteilt, an dessen Spitze der Propst des Stiftes St. Guido in Speyer als Archidiakon stand."24 Zudem werden auf fol. 67v und 68v in wirtschaftlichen, ja lagerbuchartigen Notizen die Ortsnamen bittenfeit und Rudelsperg genannt. Beide Einträge gehören unmittelbar zusammen und wurden nur aus Platzgründen auf zwei verschiedenen Seiten notiert; sie stam­ men von einer zweiten, zum Hauptschreiber ungefähr gleichzeitigen Hand, die auch zeitnah zur Haupthand des ebenfalls 1393 entstandenen Backnanger Stiftslagerbuchs ist.25 Bei den ge­ nannten Orten handelt es sich um Bittenfeld (Stadt Waiblingen) und Rudersberg (nördlich von Schorndorf). In beiden Orten hatte das Stift Back­ nang zu dieser Zeit erheblichen Besitz,26 und diesen wirtschaftlichen Einfluss dokumentieren auch die beiden Einträge in der vorliegenden Handschrift. Sprachlich weist die Handschrift in den ale­ mannischen Raum (südliches Elsass, Bodensee­ region, Oberrheingebiet), aber mit deutlichen schwäbischen Anklängen. Ebenso ist der Text der „Schwäbischen Heiligenpredigten" im Ale­ mannischen entstanden (s. u.), und diese Text­ geschichte sieht man der Handschrift an. Aber es findet eine sprachliche Anpassung statt, die ins Schwäbische, genauer sogar in den mittleren Neckarraum um Stuttgart und Esslingen weist. Die Hinweise der Handschrift auf die Kirche St. Guido in Speyer, auf die Besitztümer in Bit­ tenfeld und Rudersberg, die wahrscheinliche Nennung des Wohltäters Johannes Wolf und die Marbacher Liturgie in den Fragmenten des vor­ deren Spiegels lassen definitiv den Rückschluss zu, dass sich die Handschrift gegen Ende des 14. Jahrhunderts im Backnanger Augustiner-Chor­ herrenstift befand und auch hier in Gebrauch stand. Mehr noch: Es ist durchaus wahrscheinlich, dass die Handschrift auch in Backnang geschrie­ ben wurde. Damit ist sie die einzige nahezu voll­ ständig erhaltene mittelalterliche Handschrift aus dem Augustiner-Chorherrenstift Backnang. Dies setzt einen organisierten Schreib- und Einband­ betrieb mit Skriptorium und Werkstatt voraus, der bisher höchstens vermutet werden konnte. Predigtsammlung der „Schwäbischen Heiligenpredigten" Weitere Überlieferung Die Kurzpredigtsammlung der „Schwäbischen Heiligenpredigten" 27 wird neben der beschrie­ benen Stuttgarter Handschrift (Sigle: S) noch in fünf weiteren Handschriften überliefert: O: Ottobeuren, Stiftsbibliothek, cod. 0.29, fol. 142r bis 194v.28 84 Texte der „Schwäbischen Heiligenpredigten" sind in dieser oberdeutschen Papierhandschrift aus der ersten Hälfte des 15. Jahrhunderts am Ende einer Sammlung verschie­ denartiger lateinischer Predigten überliefert. M: München, Bayerische Staatsbibliothek, cgm 631, fol. 2ra bis 68va.29 89 Texte, darunter neben Heiligenpredigten auch etliche Sonn- und Festtagspredigten; Papierhandschrift, datiert auf das Jahr 1473. Die Handschrift stammt aus Westbayern und wurde in einem westmittel­ bairischen Dialekt mit starker alemannischer 23 Nouerint vniversi Officii domine prepositi ecclesie sancti Widonis spirensis plebeno. Vgl. Urkunden zur Geschichte der Stadt Speyer. Hg. von Alfred Hilgard, 1885, S. 556 et passim. 24 Leuze (wie Anm. 9), Nachtrag. Vgl. Franz Xaver Remling: Geschichte der Bischöfe zu Speyer Bd. 1, Mainz 1852, S. 139f. und Franz Xaver Glasschröder: Die Speyerer Bistumsmatrikel des Bischofs Mathias Ramung. - In: Mitteilungen des Historischen Vereins der Pfalz 28 (1907), S. 75 bis 126, hier S. 104. 25 HStAS H 102/6 Bd. 1. Eine Abb. des Backnanger Lagerbuchs findet sich bei Kottmann (wie Anm. 22), S. 136f. 26 Vgl. Ebd., S. 107 bis 109 (A [90] - [104], Bittenfeld) u. 101, 118 (A [20], A [21], A [195], Rudersberg). 27 Grundsätzlich dazu Kunze (wie Anm. 16). 28 Hermann Hauke: Die mittelalterlichen Handschriften in der Abtei Ottobeuren. Kurzverzeichnis, Wiesbaden 1974, S. 40; Boxler (wie Anm. 16), S. 223. 29 Karin Schneider: Die deutschen Handschriften der Bayerischen Staatsbibliothek München. Cgm 501 bis 690. Editio altera, Wiesbaden 1978 (= Catalogus codicum manun scriptorum Bibliothecae Monacensis 5,4), S. 276 bis 279; Boxler (wie Anm. 16), S. 221; Julia Offermann: „Sie aber begriffen nichts davon, und der Sinn der Rede war ihnen verborgen, und sie verstan­ den nicht, was damit gesagt war." Quinquagesimalpredigten über Lk. 18,31 bis 43 von Gregor bis Luther, Berlin 2010, Bd. 1, S. 215, Bd. 2, S. 16 bis 18. 64 /+$+ Die Münchner Handschrift der „Schwäbischen Heiligenpredigten''. Hier der Text der Heiligenpredigt für Cäcilia. Färbung geschrieben, was auf die verwendete Vorlage hinweist. B1: Berlin, Staatsbibliothek - Preußischer Kul­ turbesitz, mgq 496, fol. 212r bis 284v.30 Die in der zweiten Hälfte des 15. Jahrhunderts entstan­ dene geistliche Sammelhandschrift stammt aus dem südostdeutsch-alemannischen Grenzgebiet, vermutlich aus dem Bodenseeraum. Die Papier­ handschrift enthält neben dem Paradisus animae dt. (fol. 116r bis 211 v), Sündenfall und Erlösung (fol. 285r bis 301 v) und weiteren erbaulichen Texten 57 Predigten, allesamt Heiligenpredigten. Über die Entstehung ist nichts bekannt, aller­ dings befand sie sich Anfang des 16. Jahrhunderts im Konstanzer Raum. Die Handschrift besteht aus vier verschiedenen Faszikeln, die ursprüng­ lich nicht zusammen gehörten und jeweils wohl auch ungebunden waren. Die „Schwäbischen Heiligenpredigten" bilden eines dieser Faszikel. B2: Berlin, Staatsbibliothek - Preußischer Kul­ turbesitz, mgq 1111, 1 r bis 118r.31 Diese auf Papier geschriebene Handschrift aus dem 15. Jahrhun­ dert überliefert 72 Predigttexte (darunter zwei Sonntagspredigten). Die Handschrift stammt aus dem schwäbischen Sprachraum, vermutlich aus der Ulmer Region. Neben den „Schwäbischen Heiligenpredigten" sind kleinere Wund-, Wasser-, Wurm- und Weidesegen überliefert. Der Textbe­ stand weist große Übereinstimmung mit B1 auf, wobei B2 auf eine Textstufe zurückgehen muss, wie sie B1 repräsentiert. Ro: Rottenburg, Bibliothek des Priestersemi­ nars, cod. 11, fol. 288ra bis rb.32 Die Papier­ handschrift ist datiert auf die Jahre 1463/64. Geschrieben wurde sie zweispaltig in einem schwäbischen Dialekt und stammt aus dem Au­ gustiner-Chorherrenstift St. Mauritius in Ehingen (heute zu Rottenburg am Neckar); allerdings wird die Handschrift hier nicht entstanden sein. Aus der Sammlung der „Schwäbischen Heili­ genpredigten" ist lediglich der Text zu Konrad enthalten; des Weiteren die Texte zu Verena und Gallus aus dem „Bebenhauser Legendär" sowie vor allem der komplette Text der „Elsässischen Legenda aurea". 30 Hermann Degering: Kurzes Verzeichnis der germanischen Handschriften der Preußischen Staatsbibliothek. Bd. II: Die Hand­ schriften in Quartformat, Leipzig 1926 (= Mitteilungen aus der Preußischen Staatsbibliothek VIII), S. 89f.; Karl Firsching: Die deutschen Bearbeitungen der Kilianslegende unter besonderer Berücksichtigung deutscher Legendarhandschriften des Mittel­ alters, Würzburg 1973 (= Quellen und Forschungen zur Geschichte des Bistums und Hochstifts Würzburg 26), S. 108; Werner Williams-Krapp: Überlieferung und Gattung. Zur Gattung „Spiel" im Mittelalter. Mit einer Edition von „Sündenfall und Erlö­ sung" aus der Berliner Handschrift mgq 496, Tübingen 1980 (= Untersuchungen zur deutschen Literaturgeschichte 28), S. 33f.; Bertram Söller, das Traktat „Paradisus animae" des Pseudo-Albertus Magnus im deutschen Mittelalter. Überlieferungs­ geschichte -Wirkungsgeschichte - Textedition der vntugent-Version aus dem 15. Jahrhundert, Diss. (masch.) Würzburg 1987, S. 124 bis 130; Boxler (wie Anm. 16), S. 214. 31 Degering (wie Anm. 30) S. 187; Firsching (wie Anm. 30), S. 108; Williams-Krapp (wie Anm. 10), S. 18; Boxler (wie Anm. 16), S.214. 32 Handschriftenarchiv der Berlin-Brandenburgischen Akademie der Wissenschaften: Rottenburg, Bibliothek des Priesterseminars, cod. 11, beschr. von A. Hauber, Tübingen 1913, 6 Bll.; Heribert Hummel: Die Handschriften der Stiftsbibliothek St. Moriz zu Ehingen in der Seminarbibliothek Rottenburg, Rottenburg/Neckar 1963, S. 17 f.; Die „Elsässische Legenda Aurea". Bd. I: Das Normalcorpus. Hg. von Ulla Williams und Werner Williams-Krapp, Tübingen 1980 (= Texte und Textgeschichte 3), S. XXII; Die Elsässische „Legenda Aurea". Bd. II: Das Sondergut. Hg. von Konrad Kunze, Tübingen 1983 (= Texte und Textgeschichte 10), S. LXIII; Williams-Krapp (wie Anm. 10), S. 44f.; Bpxler (wie Anm. 16), S. 223. 65 Herkunft und Verwendung Die „Schwäbischen Heiligenpredigten" ent­ standen als Sammlung wohl im 14. Jahrundert Drei der Textzeugen stammen aus dem schwäbi­ schen Raum, der einzige bairische Textzeuge (die Münchner Handschrift M) zeigt deutlich schwä­ bische Relikte. Für eine Entstehung im südost­ alemannischen Raum deutet die Auswahl einer Reihe von Heiligen aus diesem Raum (Otmar, Ulrich, Afra, Gallus) hin. Allerdings finden sich zum Beispiel die häufig im Südwesten vorzufin­ denden Heiligen Arbogast (21.7.) und Fides (6.10) nicht im Bestand der „Schwäbischen Hei­ ligenpredigten". Verena (1.9.) und Magnus (6.9.), deren ebenfalls stark im Südwesten gedacht wur­ de, finden sich aus einem anderen Grund nicht im Bestand: Hier findet sich eine Textlücke, die sich durch die gesamte Überlieferung zieht. Verwendung fand die Sammlung vorwiegend im schwäbischen Gebiet, wie aus der geografi­ schen Verteilung der Überlieferung hervorgeht. Der schwäbische Raum befindet sich gleich einem „Niemandsland" (Konrad Kunze) zwischen den Hauptverbreitungsgebieten der „Elsässischen Legenda aurea" im Westen (Elsass, Oberrhein) und der Nürnberger Sammlung „Der Heiligen Leben" im Osten (Franken, Westbayern, Tirol). Im Norden dieses schwäbischen Überlieferungsva­ kuums breitete sich das „Bebenhauser Legendär" aus, im Süden davon die „Schwäbischen Heili­ genpredigten". Die Stuttgarter Handschrift aus Backnang hat es allerdings entgegen dieser Sys­ tematik nördlich der Überlieferungsträger des „Bebenhauser Legendars" verschlagen. Als eine Schnittstelle zeigt sich die Rottenburger Hand­ schrift, die zwar hauptsächlich die „Elsässische Legenda aurea" enthält, aber eben auch einzel­ ne Predigten aus dem „Bebenhauser Legendär" und den „Schwäbischen Heiligenpredigten".33 Textbestand Der Textbestand der „Schwäbischen Heiligen­ predigten"34 setzt sich nicht nur aus den Predig­ ten zu den Eigenlesungen an den Heiligentagen (proprium de sanctis) zusammen, sondern ist um Predigten zu Fest- und Sonntagslesungen erwei­ tert. Wie weit diese Erweiterung ursprünglich reichte, kann nicht gesagt werden. Die älteste Handschrift S bietet den umfangreichsten Heili­ genbestand, überliefert aber lediglich einige wenige Sonn- und Festtagspredigten aus der Advents-, Weihnachts-, Vorfasten-, Fasten- und Pfingstzeit sowie zwei Kirchweihpredigten. Die verhältnismäßig junge Handschrift M, reduziert um einige Heiligenpredigten, bietet dafür aber die Sonn- und Festtagspredigten des gesamten Kirchenjahrs sowie ebenso die beiden Kirch­ weihpredigten. Im Folgenden wird als Textbestand der „Schwäbischen Heiligenpredigten" der in der ältesten Handschrift S überlieferte Bestand als Ausgangslage betrachtet. Demnach überliefert S 111 Predigten, O 84, M 89, B1 5 7, B2 72 Pre­ digten und Ro schließlich nur eine Predigt. O, M, B1 und B2 sind nicht einfach um eine bestimmte Anzahl von Predigten gekürzte Fas­ sungen von S, sondern überliefern ihrerseits einige wenige Predigten, die sich nicht in S finden (25.3.: Annunciatio Marie / Mariä Ver­ kündigung, 26.6.: Johannes und Paulus sowie aufgrund eines Defekts der Handschrift 14 Hei­ ligenpredigten zwischen dem 28.8. und dem 9.10.). Somit ist als Gesamtzahl für die „Schwä­ bischen Heiligenpredigten" von 105 überliefer­ ten Predigten auszugehen.35 Die Handschrift S beginnt die Heiligenpredig­ treihe mit Simon und Juda (28.10.), was insoweit ungewöhnlich ist, als dass das Kirchenjahr in aller Regel mit dem Fest Andreas (30.11.) beginnt.36 Mit Silvester (31.12.) beginnt M und führt die Reihe fort bis Pantaleon (27.7.), setzt mit Simon und Juda (28.10.) fort und reicht dann bis Erzbischof von Canterbury Thomas (29.12.), gefolgt vom Fest Siebenschläfer (Septem dormientium, 27.6., folgt direkt auf Abdon und Sen­ nen, 30.7.) bis Ursula (21.10.). B1 und B2 bieten über weite Strecken eine identische Reihe, aller­ dings in verschiedener Reihenfolge und mehr Predigttexten in B2. B2 passt die Reihenfolge nicht an das Kirchenjahr, sondern an das Kalen­ derjahr an und beginnt mit Silvester (31.12.) und führt diese Reihe durch bis Thomas, dem Erzbi- 33 Konrad Kunze: Papierheilige. Zum Verhältnis von Heiligenkult und Legendenüberlieferung um 1400. - In: Jahrbuch der Oswald von Wolkenstein-Gesellschaft 4 (1986/87), S. 53 bis 65, hier S. 61 und Karte S. 65. 34 Zu Details s. Tabelle im Anhang. 35 Kunze (wie Anm. 16) ging von ca. 110, Williams-Krapp (wie Anm. 10), S. 18, von ca. 120 Predigten aus. 36 Vgl. die Bestandstabelle am Ende des Beitrags. 66 Die Heiligen Cäcilia und Valerian in einem Gemälde von Lelio Orsi (1508/1511 bis 1587). 67 schofvon Canterbury am 29.12. B1 weisteine weit komplexere Reihe auf: Fabian und Sebastian (20.1.) bis Assumptio Marie (Mariä Himmelfahrt, 1 5.8.), Clemens (23.11.) bis Nikolaus (6.12.), Hieronymus (30.09.) bis Remigius (1.10.), Cos­ mas und Damian (27.9.) bis zu Erzengel Micha­ el (29.9.), Lukas (18.10) bis Ursula (21.10.), Simon und Juda (28.10) bis Cäcilia (22.11.), nochmals Remigius (1.10) bis Gallus (16.10) und schließlich Lucia (13.12.) bis Thomas dem Apostel am 21.12. Ro überliefert nur die Predigt zu Konrad am 26.11. Zwei Textlücken im Bestand, die sich nicht auf eine einzelne Handschrift beziehen, sondern in der Gesamtüberlieferung zu beobachten sind, finden sich zwischen Gangolf (13.5.) und Vitus (15.6.) sowie zwischen Exaltatio crucis (Kreuzeserhö­ hung, 14.8.) und Matthäus dem Apostel (21.9.). Frage der Vorlage Der bisherigen Forschung nach ist der Bestand der „Schwäbischen Heiligenpredigten" dem des „Bebenhauser Legendars" sehr ähnlich.37 Dies kann hier nicht bestätigt werden. Gegenüber dem „Bebenhauser Legendär" fehlen den „Schwäbischen Heiligenpredigten" so charakte­ ristische Heiligenpredigten wie die zu Ottilia (13.12.), Jodocus (13.12.), Maria Aegyptica (9.4.), Helena (15.4.), Processus und Martianus (2.7.), Christopherus (25.7.), Prothus und Jacintus (11.9.) und Lambrecht (1 7.9.).38 Zudem ent­ halten die „Schwäbischen Heiligenpredigten" etliche Heiligenfeste, die das „Bebenhauser Legendär" nicht kennt. Auch der Vergleich ein­ zelner Predigttexte in beiden Legendären weist nach, dass von einer Abhängigkeit nicht ausge­ gangen werden kann.39 Damit entfällt als Vorlage für die „Schwäbischen Heiligenpredigten" auch das in mehreren Handschriften, vornehmlich aus dem 12. und 13. Jahrhundert, erhaltene lateini­ sche Kurzlegendar, das Konrad Kunze als mögli­ che Vorlage für das „Bebenhauser Legendär" aus­ gemacht hat.40 Die Vorlagenfrage der „Schwäbischen Heili­ genpredigten" ist demnach wieder offen und kann hier nicht geklärt werden. Allerdings würde eine Beantwortung dieser Frage auch für die Ge­ schichte des Stifts Backnang von großem Interes­ se sein, da sich darin klösterliche Beziehungs­ gefüge und literarische Verbindungslinien zeigen könnten, die bisher kaum oder gar nicht bekannt waren. Nutzung der Predigtsammlung im Stift Backnang In der zweiten Hälfte des 14. Jahrhunderts hat­ te das Augustiner-Chorherrenstift Backnang eine ernsthafte Krise zu bewältigen, die im Nachhinein durchaus als existenzbedrohend anzusehen ist. In einem größeren Streit um die Kompetenzen zwi­ schen Propst und Konvent gingen letztlich die Grafen von Württemberg, die ursprünglich nur als Schlichter beauftragt worden waren, als heimlicher Sieger hervor.41 Die Jahre und Jahrzehnte danach hatte das Stift seine herausragende Stellung, die es noch im 12. und 13. Jahrhundert eingenom­ men hatte, eingebüßt. Auch stand es nicht mehr in enger Verbindung zum Marbacher AugustinerChorherrenstift, das zuvor einen nicht geringen Einfluss auf die Entwicklung in Backnang ge­ nommen hatte. Backnang hatte zudem Mitte des 13. Jahrhunderts die Stellung als Grablege der Markgrafen von Baden an das Zisterzienserin­ nenkloster Lichtenthai verloren, womit natürlich auch eine Einbettung in landes- und auch reichs­ politische Belange abhandenkam. Überregiona­ le, kulturelle oder politische Impulse gingen vom Stift wohl nicht mehr aus. Eine bedeutendere ökonomische Stellung - aber vor allem als Erbe der eigenen Geschichte - war dem Stift dennoch im mittleren Neckarraum geblieben.42 37 Kunze (wie Anm. 16). 38 Vgl. die Handschrift m des „Bebenhauser Legendars" (München, Bayerische Staatsbibliothek, cgm 257); Firsching (wie Anm. 30), S. 104f. Zur Münchner Handschrift s. Karin Schneider: Die deutschen Handschriften der Bayerischen Staatsbibliothek München. Cgm 201 bis 350, Wiesbaden 1970 (= Catalogus codicum manu scriptorum Bibliothecae Monacensis V,2), S. 159f. 39 Verglichen wurden in beiden Legendären die Texte zu Andreas (30.11.), Barbara (4.12.), Nikolaus (6.12.), Kilian (8.7.) und Maria Magdalena (22.7.). Vgl. Firsching (wie Anm. 30), S. 105f.; Boxler (wie Anm. 16), S. 409f., 461 bis 464. 40 Vgl. Firsching (wie Anm. 30), S. 106f.; Konrad Kunze: Studien zur Legende der heiligen Maria Aegyptica, Berlin 1969; ders.: Die Legende der heiligen Maria Aegyptica, Berlin 1978 (= Texte des späten Mittelalters und der frühen Neuzeit 28); ders.: Die Hauptquelle des Märterbuches. - In: Zeitschrift für deutsche Philologie 88 (1969), S. 45 bis 57, hier S. 46f. 41 Fritz (wie Anm. 22), S. 39f.; Kottmann (wie Anm. 22), S. 90. 42 Vgl. Ebd. und Gerhard Fritz: Sozial- und Wirtschaftsgeschichte des Stifts Backnang vom späten 14. bis zur Mitte des 15. Jahr­ hunderts. Auswertung des Lagerbuchs von 1393 und des Güitverzeichnisses um 1450/53. - In: BJb 18, 2010, S. 56 bis 94. 68 Der Stiftshof Backnang mit Blick auf die Stiftskirche, von der einzelne Bauteile noch aus der Zeit des ehemaligen Augustiner-Chorherrenstifts stammen. Die Nutzung der „Schwäbischen Heiligen­ predigten" im Stift Backnang ist vor diesem Hin­ tergrund zu sehen. Und vor diesem Hintergrund ist die Entdeckung der Handschrift, die nach Backnang verortet werden kann, eine große Überraschung. Während das Chorherrenstift in den ersten Jahrhunderten seines Bestehens, also vor allem im 12. und 13. Jahrhundert, stark von Marbach und damit lateinisch-liturgisch geprägt war, setzte sich - sicher auch vor dem Hinter­ grund der „Krise" im 14. Jahrhundert - die deut­ sche Sprache nicht nur als Verwaltungs- und Amtssprache durch. Die erste deutschsprachige Urkunde stammt aus dem Jahr 1286.43 Deutsch als Urkundensprache setzte sich demnach in den letzten Jahren des 13. Jahrhunderts durch und war im 14. Jahrhundert schon die Regel.44 Auch im Bereich der Frömmigkeit gewann die deut­ sche Sprache immer mehr die Überhand. Diese zunehmende Veränderung zeigte sich unter an­ derem daran, dass Blätter aus lateinischen Hand­ schriften des 12. und frühen 13. Jahrhundert wohl nicht mehr gebraucht und als Einbandmaterial wiederverwendet wurden: So die beiden Frag­ mente in den Spiegeln der hier neu vorgestellten Handschrift mit den „Schwäbischen Heiligen­ predigten", als auch die Einbandblätter des Back­ nanger Stiftslagerbuchs von 1501.45 Dass sich nun eine deutsche Handschrift nach Backnang verorten lässt, zudem eine mit Heili­ genpredigten, also mit Texten, die sich zur Vor­ bereitung für die Verkündigung in deutscher Sprache verwenden ließen, sagt viel über die Frömmigkeit im Stift und darüber hinaus am Ende des 14. Jahrhunderts aus - Einsichten, die sich in der Forschung bisher nicht ergaben. So wurden die „Schwäbischen Heiligenpredigten" aller Wahrscheinlichkeit nach für den Prediger als eine Art Handbuch angelegt, das dem Predi­ ger in aller Kürze eine Materialfülle bot, an Hand derer er seine deutsche Predigt entwerfen konn­ te. Es wird davon ausgegangen, dass die Samm­ lung der „Schwäbischen Heiligenpredigten" „nicht [...] als Musterpredigtbuch, an dem sich der Prediger auch aufbaumäßig für seine An­ sprache orientieren konnte, sondern eher als ein 43 Württembergisches Urkundenbuch (WUB). Hg. von dem Königlichen Staatsarchiv in Stuttgart, Bd. IX, Stuttgart 1907, Nr. 3563. 44 Z.B. WUB IX, Nr. 4014; X, Nr. 4631; XI, Nr. 5035; Württembergische Regesten von 1301 bis 1500. Hg. vom Königlichen Haus- und Staatsarchiv Stuttgart, 3 Bde., Stuttgart 1916 bis 1940 (Urkunden und Akten des Königlichen Württembergischen Haus- und Staatsarchivs 1,1,1-1,1,3), passim. 45 HStAS H 102/6 Bd. 7; vgl. Kottmann (wieAnm. 1), S. 107 bis 112. 69 Der Backnanger Stadtturm, in dessen unterem Teil sich mit dem Gotischen Chor ein Überrest der ehemaligen Michaelskirche aus dem 13. Jahrhundert befindet. in überschaubarem Umfang gestaltetes Nach­ schlagewerk [verwendet wurde], das dazu dien­ te, die zentralen Ereignisse im Leben eines Hei­ ligen bereitzustellen, um die herum sich dann eine Heiligenpredigt gestalten ließ".46 Die Verwendung der „Schwäbischen Heili­ genpredigten" als „Predigt-Handbuch" (Werner Williams-Krapp) deutet eine Verwendung nicht nur innerhalb der Mauern des AugustinerChorherrenstifts Backnang an. Sicher wurde die Handschrift auch vom Pfarrer der Kirche St. Michael, also der Backnanger Stadtkirche, ver­ wendet. Es spricht viel dafür, dass die hier beschriebene Handschrift auch in Backnang geschrieben wurde. Die Sammlung der „Schwä­ bischen Heiligenpredigten" wurde aber sicher nicht hier zusammengestellt. Dagegen sprechen die deutlichen Hinweise wie die Sprache sowie der Heiligenbestand, die die Sammlung definitiv in den alemannischen Sprachraum, am ehesten sogar in die westliche Bodenseeregion bezie­ hungsweise Oberrheingebiet, verorten lassen. Damit sind literarische Verbindungen von Back­ nang in diese Gegend belegbar, und diese Ver­ bindung zeigt auch die starke Verflechtung Backnangs in das alemannische, das heißt süd­ westdeutsche theologisch-literarische Netzwerk der Zeit: „Die Dynamik des Literaturbetriebs im 14. Jahrhundert hat ein Zentrum im aleman­ nischen Raum, der in dieser Zeit zu einem kul­ turellen Knotenpunkt und zu einer kulturellen Drehscheibe wird. Oberrhein und Bodensee­ raum werden zu einem ,Silicon valley' der Lite­ ratur im 14. Jahrhundert."47 Auch das schwäbi­ sche Gebiet mit dem Hof der württembergischen Grafen in Stuttgart sowie den Reichsstädten Ess­ lingen und Ulm entwickelte eine beträchtliche Literaturproduktion, die sowohl von klerikaler als auch von weltlicher Seite große Unterstützung und Förderung erfuhr.48 Inwieweit die Kompilationsleistung, also die Kombination von Heiligen- sowie Sonn- und Festtagspredigten von Backnang ausgeht, ist zum momentanen Stand der Forschung nicht klar. Klar ist aber, dass sich diese Kombination in der Überlieferung ein weiteres Mal findet, sogar erheblich erweitert (M). Sicher ist auch, dass im Augustiner-Chorherrenstift deutsch-geistliche Literatur nicht nur rezipiert wurde, sondern auch Impulse für die weitere Überlieferung von Back­ nang ausgingen. Die genauen Abhängigkeiten und Verbindungslinien sind noch aufzuzeigen, aber schon jetzt kann eindeutig gesagt werden, dass Backnang in der Überlieferung der „Schwä­ bischen Heiligenpredigten" eine Schlüsselrolle einnimmt. 46 Williams-Krapp (wie Anm. 10), S. 19. Vgl. auch ders.: Mittelalterliche deutsche Heiligenpredigtsammlungen und ihr Verhältnis zur homiletischen Praxis. - In: Mertens / Schiewer (wie Anm. 14), S. 325 bis 360, hier S. 356f. 47 Nigel F. Palmer / Hans-Jochen Schiewer: Literarische Topographie des deutschsprachigen Südwestens im 14. Jahrhundert. - In: Zeitschrift für deutsche Philologie 122 (2003), Sonderheft, S. 178 bis 202, hier S. 182 f. 48 Ein Beispiel findet sich bei Carsten Kottmann: Das buch der ewangely und epistel. Untersuchungen zur Überlieferung und Cebrauchsfunktion südwestdeutscher Perikopenhandschriften, Münster 2009 (= Studien und Texte zum Mittelalter und zur frühen Neuzeit 14), S. 76 bis 82. Vgl. Henrike Lähnemann: Margarethe von Savoyen in ihren literarischen Beziehungen. - In: „Encomia-Deutsch". Sonderheft der deutschen Sektion der International Courtly Literature Society. Hg. von Ingrid Kasten und Andrea Sieber, Berlin 2002, S. 158 bis 173. 70 Tabelle: Heiligenbestand der „Schwäbischen Heiligenpredigten"49 Heiligenfest/Sonn- oder Festtag S M B1 B2 Simonis et/ude ap. - 28.10. Ir 36rb 268v 80r Omnium sanctorum -1.11. 2r 37rb 270v 83v Commemoratio animarum -2.11. 2r 37va 271v 84v Martini ep. cf. - 11.11. 2v 37vb 271v 85r Briccii ep. cf. - 13.11. 3v 38rb 273v 87v Othmari abb. -16.11. 4r 39ra 274v 89r Elisabeth lantgravie -19.11. 4v Cecilie v. m. - 22.11. 4v 39va 275v 90v Clementis pp. m. - 23.11. 5v 40rb 251v 93r Katharine v. m. - 25.11. 6r 40vb 252v 84v Conradi ep. - 26.11. 7r Andree apost. - 30.11. 7r 41 rb 1. Sonntag im Advent 8r 2ra Barbare v. m. - 4.12. 10r Nicolai ep. cf. - 6.12. Ro 288ra‘rb 254r 96v 41vb 255r 98r 10v 42 ra 255v 98v Lucie v. m. - 13.12. 12v 43va 281 r 103v Thome apost. -21.12. 14r 44 rb 283r 106r Nativitas Domini - 25. 12. 15v 6vb Stephani protom. -26. 12. 17r 8vb 111r lohannis ap. et ev. - 27. 12. 17r 9rb 112r Innocentium m. - 28. 12. 18r 11ra 115r Thome ep. m. - 29.12. 18v 45vb 116v Silvestri pp. cf. - 31.12. 19v 12ra 1r Circumcisio Domini - 1.1. 19v 12vb Epiphania Domini - 6. /. 20v 13va Pauli primi heremite - 10.1. 21r Felicis in pincis -14.1. 21v Mauri et Macharii cf. - 15.1. 21v Marcelli pp. m. - 16.1. 22r Anthonii cf. - 17.1. 22r Prisce v. m. - 18.1. 22v Fabiani et Sebastiani m. - 20.1. 22v 14vb 212r 2r Agnetis v. m. - 21.1. 23v 1 5vb 213r 4r Vincentii m. - 22.1. 24v Emerentiane v. m. - 23.1. 25r Timothei apost. -24.1. 25r 17ra 49 S = Stuttgart, WLB, cod. theol. et. philos. 2° 64; M = München, BSB, cgm 631; Bl = Berlin, SB-PK, mgq 496; B2 = Berlin, SB-PK, mgq 1111; Ro = Rottenburg/N., B des Priesterseminars, cod. 11. Die Handschrift Ottobeuren, StifsB, cod. 0.29 konnte noch nicht eingesehen werden und fehlt daher in dieser Auflistung. 71 Heiligenfest/Sonn- oder Festtag S M B1 B2 Conversio Pauli ap. - 25.1. 25r 1 7rb 215r 6v Purificatio Marie v. - 2.2. 26r 19ra 216r 8r Brigide v. - 1.2. 26v 18ra Blasii ep. m. - 3.2. 27r 19vb 217r 9v Agathe v. - 5.2. 28r 20vb 218v 11V Scholastice v. - 10.2. 29v Valentini m. - 14.2. 30r 22vb 221r 14v Juliane v. m. - 16.2. 30v Kathedra Petri-22.2. 31 r 31r Matthie ap. - 24.2. 31r 23va 221v 15v Gregorii pp. - 12.3. 31v 25ra 223v 17v Benedicti abb. -21.3. 32r 25va 224v 19r 26ra 225r 19v Annunciatio Marie v. - 25.3. Tiburtii m. - 14.4. 32v 27ra Georgii m. - 23.4. 32v 27rb 227r 22r Marei evang. -25.4. 33r 27vb 228r 23r Vitalis m. - 28.4. 33r Jacobi ap. - 1.5. 33v 28rb 229v 24v Philippi ap. -1.5. 33v 28vb 228v 23v Inventio crucis -3.5. 34r 29ra 230r 25v Johannis ante portam latinam - 6.5. 34v 29vb Cordiani m. - 10.5. 34v Pancratii m. - 12.5. 34v Gangolfi m. - 13.5. 34v Viti m. - 15.6. 35r 30ra 231r 26v Marcelliani m. - 18.6. 35v Gervasii m. (?) - 19.6. 35v Albani m. - 21.6. 35v Decem milium m. - 22.6. 35v 30va 232v 28r Nativitas Johannis bapt. - 24.6. 35v 30vb 232v 28v 31vb 234v 30v Johannis et Pauli m. - 26.6. Petri et Pauli apost. - 29.6. 36v, 37 v Petri apost. - 29.6. 36v 32 ra 235r 31v Udalrici ep. - 4.7. 37v 33ra 236v 33r Kiliani m. - 8.7. 37v 33ra 237r 33v Septem fratrum filiorum Felicitatis -10.7. 38r Margarete v. m. - 13.7. 38r 33rb 237v 34r Alexii cf. -17.7. 39r 34rb 239r 36r Marie Magdalene -22.7. 39r 34vb 240r 37r Apollinaris ep. m. -23.7. 40r 72 Ro Heiligentest/Sonn- oder Festtag s Christine v. m. - 24.7. 40r Jacobi apost. -25.7. M B1 B2 40r 3 5 rb 241v 39r Pantaleonis m. -27.7. 40v 35vb 242v 40r Felicis pp. - 29.7. 44r Abdon et Sennen m. - 30.7. 45r Septem dormientium m. -27.6. 41v 46rb 243v 41r Pfingstfest 42r 94 vb Trinitatis 43r 96ra Ad vincula Petri -1.8. 44r 46vb 245r 42v Stephani pp. -2.8. 44v Inventio Stephani protom. -3.8. 45r Oswaldi regis - 5.8. 45v 47va 246r 44r Sixti pp. m. - 6.8. 45v Afre m. - 7.8. 46r 47vb 247r 44v Cyriaci m. - 8.8. 46r Laurentii m. - 10.8. 46v 48rb 248r 46r Ypoliti m. - 13.8. 47r 49ra 249r 47v Assumtio Marie v. - 15.8. 47v 49va 250r 48v Bartholomei apost. - 24.8. 48v 51 ra 51v 52 ra 53v Augustini ep. cf. -28.8. Hermetis m. -28.8. 54v Pelagii m. - 28.8. 54v Decollatio Johannis bapt. - 29.8. 52va 55r Egidii abb. -1.9. 53rb 56r Nativitas Marie v. - 8.9. 53vb 57v Exaltatio crucis -14.8. 54vb 59v Matthei apost. et. ev. - 21.9. 55vb 61 r Mauricii et soc. m. -22.9. 56vb 63r Cosme et Damiani m. -27.9. 57vb 260v 65r Michaelis archangeli - 29.9. 58vb 261v 67r Jeronymi pb. cf. -30.9. 59va 257r 68v Remigio ep. - 1.10. 60rb 258v, 277r 70r Dionysii ep. -9.10. 60va 277r 70v Calli abb. cf. -16.10. 50r 61 va 278v 72v Luce evang. - 18.10. 50v 62 va 265r 75v Ursule m. (Undecim millium v. m.) -21.10. 51r 63 ra 265v 76v Kirchweihe, allg. (Zachäusgeschichte, Lk. 19,1 bis 10) 52r 64vb Ro 73 Heiligenfest/Sonn- oder Festtag S M Kirchweihfest „dieser Kirche" (Mein Haus soll ein Bethaus sein, Jes. 56,7) 53 v 66vb Severini ep. - 23.10. 55r Crispini et Crispiniani m. - 25.10. 55r Sonntag Septuagesima (1. Kor. 9,24 ff.; Mt. 20,1 ff.) 55v 72 va Sonntag Sexagesima (Lk. 8,4 ff.) 56v 73 rb Sonntag Quinquagesima (Lk. 18,31 bis 43) 58r 74 va Sonntag nach Invocavit (Mt. 4,1 ff.) 59r 76ra Sonntag Reminiscere (Mt. 15,21) 60v 77va Sonntag Ocu1i (Lk. 11,14 ff.) 61r 78ra Sonntag Laetere (Joh. 6,1 ff.) 61v 78vb Sonntag Judica (Hebr. 9,1 ff.) 62r Palmsonntag (Mt. 21,5) 63v 81ra Gründonnerstag (Lk. 22,15) 64 v 82rb 74 B1 B2 Ro
https://openalex.org/W4283752014
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English
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The Impact of a Digital Artificial Intelligence System on the Monitoring and Self-management of Nonmotor Symptoms in People With Parkinson Disease: Proposal for a Phase 1 Implementation Study
JMIR research protocols
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College of Experts Non drug approaches grant pre-proposal reviewer form This form should be completed by the College of Expert reviewer for a non drug approaches grant pre-proposal. The information you provide is maintained as a record of the decision-making process and to give feedback to applicants. If you have any questions, please contact the Research team by emailing researchapplications@parkinsons.org.uk. Application reference (Internal use) NDA-21-15 Reviewer reference (Internal use) Principal applicant Dr Edward Meinert Dr Camille Carroll Application title The impact of a digital portal on the monitoring and self-management of nonmotor symptoms in people with Parkinson’s Please do remember to provide a score for your evaluation at the end of this review. Importance ● How important are the questions, or gaps in knowledge, that are being addressed? Is there a good rationale for pursuing these? Is success likely to lead to significant new understanding? ● Is the research relevant to Parkinson's? ● Does the proposal realistically set out the ultimate potential health or social gain to people with Parkinson's? ● How important is it to do the work now? ● Is there similar or complementary research underway elsewhere? Are the proposals competitive? The application provides a strong case for the need for this research, highlighting the anticipated increase in prevalence of Parkinson’s by 2016, and highlighting how non-motor symptoms in particular are a troubling, yet often overlooked, aspect of the condition. Coupled with the ongoing COVID-19 pandemic, an investigation into a novel method of remote NMS monitoring to improve clinical intervention and quality of life is more important than ever. I do believe the application would benefit from further insight into the novelty of the NMS Assist application – what novelty does it provide over similar approaches? Application reference (Internal use) NDA-21-15 Reviewer reference (Internal use) Principal applicant Dr Edward Meinert Dr Camille Carroll Application title The impact of a digital portal on the monitoring and self-management of nonmotor symptoms in people with Parkinson’s Please do remember to provide a score for your evaluation at the end of this review. Importance ● How important are the questions, or gaps in knowledge, that are being addressed? Is there a good rationale for pursuing these? Is success likely to lead to significant new understanding? Is the research rele ant to Parkinson's? This form should be completed by the College of Expert reviewer for a non drug approaches grant pre-proposal. The information you provide is decision-making process and to give feedback to applicants. If you have any questions, please contact the Research team by emailing researchapplications@parkinsons.org.uk. Application reference (Internal use) NDA-21-15 Reviewer reference (Internal use) Principal applicant Dr Edward Meinert Dr Camille Carroll Application title The impact of a digital portal on the monitoring and self-management of nonmotor symptoms in people with Parkinson’s ● Does the proposal realistically set out the ultimate potential health or social gain to people with Parkinson's? The application provides a strong case for the need for this research, highlighting the anticipated increase in prevalence of Parkinson’s by 2016, and highlighting how non-motor symptoms in particular are a troubling, yet often overlooked, aspect of the condition. Coupled with the ongoing COVID-19 pandemic, an investigation into a novel method of remote NMS monitoring to improve clinical intervention and quality of life is more important than ever. I do believe the application would benefit from further insight into the novelty of the NMS Assist application – what novelty does it provide over similar approaches? College of Experts Non drug approaches grant pre-proposal reviewer form This form should be completed by the College of Expert reviewer for a non drug approaches grant pre-proposal. The information you provide is maintained as a record of the decision-making process and to give feedback to applicants. If you have any questions, please contact the Research team by emailing researchapplications@parkinsons.org.uk. Application reference (Internal use) NDA-21-15 Reviewer reference (Internal use) Principal applicant Dr Edward Meinert Dr Camille Carroll Application title The impact of a digital portal on the monitoring and self-management of nonmotor symptoms in people with Parkinson’s Please do remember to provide a score for your evaluation at the end of this review. Importance ● How important are the questions, or gaps in knowledge, that are being addressed? Is there a good rationale for pursuing these? Is success likely to lead to significant new understanding? ● Is the research relevant to Parkinson's? ● Does the proposal realistically set out the ultimate potential health or social gain to people with Parkinson's? ● How important is it to do the work now? ● Is there similar or complementary research underway elsewhere? Are the proposals competitive? The application provides a strong case for the need for this research, highlighting the anticipated increase in prevalence of Parkinson’s by 2016, and highlighting how non-motor symptoms in particular are a troubling, yet often overlooked, aspect of the condition. Coupled with the ongoing COVID-19 pandemic, an investigation into a novel method of remote NMS monitoring to improve clinical intervention and quality of life is more important than ever. I do believe the application would benefit from further insight into the novelty of the NMS Assist application – what novelty does it provide over similar approaches? College of Experts Non drug approaches grant pre-proposal reviewer form This form should be completed by the College of Expert reviewer for a non drug approaches grant pre-proposal. The information you provide is maintained as a record of the decision-making process and to give feedback to applicants. If you have any questions, please contact the Research team by emailing researchapplications@parkinsons.org.uk. Application reference (Internal use) NDA-21-15 Reviewer reference (Internal use) Principal applicant Dr Edward Meinert Dr Camille Carroll Application title The impact of a digital portal on the monitoring and self-management of nonmotor symptoms in people with Parkinson’s Please do remember to provide a score for your evaluation at the end of this review. Importance ● How important are the questions, or gaps in knowledge, that are being addressed? Is there a good rationale for pursuing these? Is success likely to lead to significant new understanding? ● Is the research relevant to Parkinson's? ● Does the proposal realistically set out the ultimate potential health or social gain to people with Parkinson's? ● How important is it to do the work now? ● Is there similar or complementary research underway elsewhere? Are the proposals competitive? The application provides a strong case for the need for this research, highlighting the anticipated increase in prevalence of Parkinson’s by 2016, and highlighting how non-motor symptoms in particular are a troubling, yet often overlooked, aspect of the condition. Coupled with the ongoing COVID-19 pandemic, an investigation into a novel method of remote NMS monitoring to improve clinical intervention and quality of life is more important than ever. I do believe the application would benefit from further insight into the novelty of the NMS Assist application – what novelty does it provide over similar approaches? College of Experts Non drug approaches grant pre-proposal reviewer form College of Experts Non drug approaches grant pre-proposal reviewer form This form should be completed by the College of Expert reviewer for a non drug approaches grant pre-proposal. The information you provide is maintained as a record of the decision-making process and to give feedback to applicants. If you have any questions, please contact the Research team by emailing researchapplications@parkinsons.org.uk. Parkinson’s UK is the operating name of the Parkinson’s Disease Society of the United Kingdom. A company limited by guarantee. Registered in England and Wales (948776). Registered office: 215 Vauxhall Bridge Road, London SW1V 1EJ. A charity registered in England and Wales (258197) and in Scotland (SC037554). Environment and people The applicants have allocated contingency time to areas which are often prone to delay, such as ethics approval. The methods and study design appear to be well thought out. t would be beneficial to provide more detail on planned recruitment. Will you recruit participants from a wide range of ages, with a wide range of symptoms and at various stages of their Parkinson’s journey? The stratification of participants may provide interesting insight into who can most benefit from this intervention. The application mentions that responses to remote monitoring that trigger additional service support will be measured – further details of this process would be beneficial. Justification of resources requested ● Is the number of staff appropriate for the work described? Are the reasons for purchasing major items of equipment clearly set out? ● Are the funds requested appropriate for the work proposed and does the scientific potential justify funding on the scale requested? Is the project value for money? As a significant amount of funding (£50,000) will be dedicated to the subcontracting of software development on this already implemented solution, it would be beneficial for the applicants to provide some further detail of the types of additional development that are envisioned. It would also be beneficial to show when this software development is anticipated to take place within Appendix A (Gantt Chart) Environment and people Members of the research team are well placed to deliver this work, possessing a good balance of technical and clinical expertise, in addition to representation of people with Parkinson’s and their caregivers. Both lead applicants have an impressive track record of research grants and publications in relevant areas. Parkinson’s UK is the operating name of the Parkinson’s Disease Society of the United Kingdom. A company limited by guarantee. Registered in England and Wales (948776). Registered office: 215 Vauxhall Bridge Road, London SW1V 1EJ. A charity registered in England and Wales (258197) and in Scotland (SC037554). It is particularly interesting to see that Dr Meineart has interests in clinical artificial intelligence – will this be integrated into NMS Assist? I believe this could be an excellent source of novelty. It is particularly interesting to see that Dr Meineart has interests in clinical artificial intelligence – will this be integrated into NMS Assist? I believe this could be an excellent source of novelty. Research plans ● Will the proposed plan of work achieve the objectives outlined in the proposal? ● Are the methodological plans realistic, given the aims of the research and the resources? ● Are the methods and study designs appropriate and free of flaws? I believe the research team have the ability to achieve the proposed objectives within the time frame outlined in the proposal. The applicants have allocated contingency time to areas which are often prone to delay, such as ethics approval. The methods and study design appear to be well thought out. It would be beneficial to provide more detail on planned recruitment. Will you recruit participants from a wide range of ages, with a wide range of symptoms and at various stages of their Parkinson’s journey? The stratification of participants may provide interesting insight into who can most benefit from this intervention. The application mentions that responses to remote monitoring that trigger additional service support will be measured – further details of this process would be beneficial. Justification of resources requested It is particularly interesting to see that Dr Meineart has interests in clinical artificial intelligence – will this be integrated into NMS Assist? I believe this could be an excellent source of novelty. I believe the research team have the ability to achieve the proposed objectives within the time frame outlined in the proposal. Please let us know whether this is apparent in this pre proposal application This work is of clear importance, however, I do feel the application would benefit from further insight into the novelty of this work, and evidence-based potential benefits to quality of life / clinical intervention / costs that may result. Additionally, further insight into anticipated pathway to clinical integration (should the trials be successful) would be beneficial. These elements should form part of the full application. Evidence of potential impact and clear pathway for benefit to people with Parkinson’s in the near future 2 With these awards we are focused on delivering outcomes that make a difference to people with the condition. That means that we're looking for projects that, if successful, have a clear pathway to be made available to people affected by Parkinson's. ● Please provide constructive comments/suggestions suitable to be fed back to the applicant to help form a full application. These should highlight specific weaknesses and positive aspects of the application. ●Very strong, internationally competitive team, containing all relevant disciplines. ●Very good value for money. ●Clear and well written pre-proposal. ●Plain English summary accurately reflects the research proposal and is realistic about potential outcomes and timescales involved. Exceptional 10 Excellent quality research 9 Very good, bordering on excellent 8 Potentially fundable ●Relevant research questions; likely to result in advancement in the scientific understanding of Parkinson’s, or benefit for people affected by Parkinson’s by addressing the priority research areas for improving everyday life. ●Good quality and appropriate methods and research design. ●Competent and appropriate research team containing all key disciplines. ●Good value for money. ●Plain English summary accurately reflects the research proposal and is realistic about potential outcomes and timescales involved. Good quality research 7 Above average quality research 6 Acceptable quality 5 Not fundable (without significant changes) ●Research questions are not directly relevant to the scientific understanding of Parkinson’s or do not address the priority research areas for people affected by Parkinson’s. ●Inappropriate methods and research design of only modest or poor quality. ●Applicants without relevant research experience or key disciplines not represented. ●Poor value for money. ●Plain English summary is unclear, does not accurately reflect the research proposal and is unrealistic about the potential outcomes and timescales involved. Borderline quality research 4 Below acceptable quality 3 Definitely not fundable ●Irrelevant research questions. ●Poor/flawed/duplicative methods and research design. ●Key skills missing from the research team. ●Very poor value for money. ●Unclear application. ●Plain English summary is unclear, does not accurately reflect the research proposal and is unrealistic about the potential outcomes and timescales involved. Many identified flaws 2 Serious weaknesses or major concerns 1 Project Grants - Parkinson’s UK College of Experts scoring system The listed characteristics are for guidance only. The characteristics are general statements on the overall quality of the application in each funding category. They are not a checklist of minimum criteria for the funding category. Please send your completed review to researchapplications@parkinsons.org.uk Please send your completed review to researchapplications@parkinsons.org.uk Funding category Characteristics for Scientific Members Rating scale Highly fundable ●Very important research questions; likely to result in advancement in the scientific understanding of Parkinson’s or significant benefit for people affected by Parkinson’s by addressing the priority research areas for improving everyday life. ●Excellent and appropriate methods and research design. ●Very strong, internationally competitive team, containing all relevant disciplines. ●Very good value for money. ● Please provide constructive comments/suggestions suitable to be fed back to the applicant to help form a full application. These should highlight specific weaknesses and positive aspects of the application. ● Please provide constructive comments/suggestions suitable to be fed back to the applicant to help form a full application. These should highlight specific weaknesses and positive aspects of the application. ● Please provide constructive comments/suggestions suitable to be fed back to the applicant to help form a full application. These should highlight specific weaknesses and positive aspects of the application. The applicants should be commended for their concise, clearly written application with clear research questions, objectives and methods. As a significant amount of funding (£50,000) will be dedicated to the subcontracting of software development on this already implemented solution, it would be beneficial for the applicants to provide some further detail of the types of additional development that are envisioned. It would also be beneficial to show when this software development is anticipated to take place within Appendix A (Gantt Chart) t is particularly interesting to see that Dr Meineart has interests in clinical artificial intelligence – will this be integrated into NMS Assist? I believe this could be an excellent source of novelty. Further detail on how NMS Assist differs from other existing approaches would be beneficial – what are the novel aspects? t would be beneficial to provide more detail on planned recruitment. Will you recruit participants from a wide range of ages, with a wide range of symptoms and at various stages of their Parkinson’s journey? The stratification of participants may provide interesting insight into who can most benefit from this intervention. The application mentions that responses to remote monitoring that trigger additional service support will be measured – further details of this process would be beneficial. This work is of clear importance, however, I do feel the application would benefit from further insight into the novelty of this work, and evidence-based potential benefits to quality of life / clinical intervention / costs that may result. Additionally, further insight into anticipated pathway to clinical integration (should the trials be successful) would be beneficial. These elements should form part of the full application. Any other comments Score: 7/10 3 Please send your completed review to researchapplications@parkinsons.org.uk Funding category Characteristics for Scientific Members Rating scale Highly fundable ●Very important research questions; likely to result in advancement in the scientific understanding of Parkinson’s or significant benefit for people affected by Parkinson’s by addressing the priority research areas for improving everyday life. ●Excellent and appropriate methods and research design. ● Please provide constructive comments/suggestions suitable to be fed back to the applicant to help form a full application. These should highlight specific weaknesses and positive aspects of the application. ●Clear and well written pre-proposal. ●Plain English summary accurately reflects the research proposal and is realistic about potential outcomes and timescales involved. Exceptional 10 Excellent quality research 9 Very good, bordering on excellent 8 Potentially fundable ●Relevant research questions; likely to result in advancement in the scientific understanding of Parkinson’s, or benefit for people affected by Parkinson’s by addressing the priority research areas for improving everyday life. ●Good quality and appropriate methods and research design. ●Competent and appropriate research team containing all key disciplines. ●Good value for money. ●Plain English summary accurately reflects the research proposal and is realistic about potential outcomes and timescales involved. Good quality research 7 Above average quality research 6 Acceptable quality 5 Not fundable (without significant changes) ●Research questions are not directly relevant to the scientific understanding of Parkinson’s or do not address the priority research areas for people affected by Parkinson’s. ●Inappropriate methods and research design of only modest or poor quality. ●Applicants without relevant research experience or key disciplines not represented. ●Poor value for money. ●Plain English summary is unclear, does not accurately reflect the research proposal and is unrealistic about the potential outcomes and timescales involved. Borderline quality research 4 Below acceptable quality 3 Definitely not fundable ●Irrelevant research questions. ●Poor/flawed/duplicative methods and research design. ●Key skills missing from the research team. ●Very poor value for money. ●Unclear application. ●Plain English summary is unclear, does not accurately reflect the research proposal and is unrealistic about the potential outcomes and timescales involved. Many identified flaws 2 Serious weaknesses or major concerns 1 Project Grants - Parkinson’s UK College of Experts scoring system The listed characteristics are for guidance only. The characteristics are general statements on the overall quality of the Funding category Funding category Not fundable (without significant changes) Definitely not fundable Project Grants - Parkinson’s UK College of Experts scoring system Project Grants - Parkinson’s UK College of Experts scoring system The listed characteristics are for guidance only. The characteristics are general statements on the overall quality of the application in each funding category. They are not a checklist of minimum criteria for the funding category. 4 Parkinson’s UK top 10 priority research areas for improving everyday life For more information on the priority research areas and the full list, please visit our website. For more information on the priority research areas and the full list, please visit our website. 5 College of Experts Non drug approaches grant pre-proposal reviewer form This form should be completed by the College of Expert reviewer for a non drug approaches grant pre-proposal. The information you provide is maintained as a record of the decision-making process and to give feedback to applicants. If you have any questions, please contact the Research team by emailing researchapplications@parkinsons.org.uk. Application reference (Internal use) NDA-21-15 Reviewer reference (Internal use) Principal applicant Dr Edward Meinert Dr Camille Carroll Application title The impact of a digital portal on the monitoring and self-management of nonmotor symptoms in people with Parkinson’s Please do remember to provide a score for your evaluation at the end of this review. Importance ● How important are the questions, or gaps in knowledge, that are being addressed? Is there a good rationale for pursuing these? Is success likely to lead to significant new understanding? ● Is the research relevant to Parkinson's? ● Does the proposal realistically set out the ultimate potential health or social gain to people with Parkinson's? ● How important is it to do the work now? ● Is there similar or complementary research underway elsewhere? Are the proposals competitive? The monitoring and assessment of non-motor symptoms is notoriously difficult to assess and has considerable impact on People with Parkinson’s (PwP) quality of life. The proposed study aims to assess the benefits of a digital tool that has been developed to support the monitoring and self-management of non-motor symptoms, as well as providing a means of accessing resources to aid management of these. As well as potentially improving the quality of life of PwP, a health economic assessment is proposed to understand better the potential cost benefits of such a system. A fuller consideration of any competing technologies would have been useful. College of Experts Non drug approaches grant pre-proposal reviewer form This form should be completed by the College of Expert reviewer for a non drug approaches grant pre-proposal. The information you provide is decision-making process and to give feedback to applicants. If you have any questions, please contact the Research team by emailing researchapplications@parkinsons.org.uk. Parkinson’s UK top 10 priority research areas for improving everyday life Application reference (Internal use) NDA-21-15 Reviewer reference (Internal use) Principal applicant Dr Edward Meinert Dr Camille Carroll Application title The impact of a digital portal on the monitoring and self-management of nonmotor symptoms in people with Parkinson’s ● How important is it to do the work now? The monitoring and assessment of non-motor symptoms is notoriously difficult to assess and has considerable impact on People with Parkinson’s (PwP) quality of life. The proposed study aims to assess the benefits of a digital tool that has been developed to support the monitoring and self-management of non-motor symptoms, as well as providing a means of accessing resources to aid management of these. As well as potentially improving the quality of life of PwP, a health economic assessment is proposed to understand better the potential cost benefits of such a system. A fuller consideration of any competing technologies would have been useful. Environment and people Both of the applicants have highly relevant and extensive research experience in their respective fields, as evidenced by many peer reviewed publications to date and are therefore well placed to deliver the work proposed. They are supported by experienced technologists and health professionals. Both of the applicants have highly relevant and extensive research experience in their respective fields, as evidenced by many peer reviewed publications to date and are therefore well placed to deliver the work proposed. They are supported by experienced technologists and health professionals. The aims and objectives of the proposed research are clearly stated and justified. These are detailed in the respective work packages and summary work plan; they are well justified and scheduled in the context of a realistic timescale. A clear methodology has been adopted to ensure rigorous investigation can be achieved. The aims and objectives of the proposed research are clearly stated and justified. These are detailed in the respective work packages and summary work plan; they are well justified and scheduled in the context of a realistic timescale. A clear methodology has been adopted to ensure rigorous investigation can be achieved. The aims and objectives of the proposed research are clearly stated and justified. These are detailed in the respective work packages and summary work plan; they are well justified and scheduled in the context of a realistic timescale. A clear methodology has been adopted to ensure rigorous investigation can be achieved. Justification of resources requested ● Is the number of staff appropriate for the work described? Are the reasons for purchasing major items of equipment clearly set out? ● Are the funds requested appropriate for the work proposed and does the scientific potential justify funding on the scale requested? Is the project value for money? A good balance between research staff, administrative support and consultancy has been proposed to achieve the most effective use of resources. I believe that the work proposed does justify funding as requested and, importantly, is realistic in what is required to achieve a successful outcome in the timescale proposed. It is, therefore, good value for money. Evidence of potential impact and clear pathway for benefit to people with Parkinson’s in the near future With these awards we are focused on delivering outcomes that make a difference to people with the condition. That means that we're looking for projects that, if successful, have a clear pathway to be made available to people affected by Parkinson's. Please let us know whether this is apparent in this pre proposal application Environment and people Parkinson’s UK is the operating name of the Parkinson’s Disease Society of the United Kingdom. A company limited by guarantee. Registered in England and Wales (948776). Registered office: 215 Vauxhall Bridge Road, London SW1V 1EJ. A charity registered in England and Wales (258197) and in Scotland (SC037554). Both of the applicants have highly relevant and extensive research experience in their respective fields, as evidenced by many peer reviewed publications to date and are therefore well placed to deliver the work proposed. They are supported by experienced technologists and health professionals. Research plans ● Will the proposed plan of work achieve the objectives outlined in the proposal? ● Are the methodological plans realistic, given the aims of the research and the resources? ● Are the methods and study designs appropriate and free of flaws? The aims and objectives of the proposed research are clearly stated and justified. These are detailed in the respective work packages and summary work plan; they are well justified and scheduled in the context of a realistic timescale. A clear methodology has been adopted to ensure rigorous investigation can be achieved. Justification of resources requested ● Is the number of staff appropriate for the work described? Are the reasons for purchasing major items of equipment clearly set out? ● Are the funds requested appropriate for the work proposed and does the scientific potential justify funding on the scale requested? Is the project value for money? ● Are the timescale and scheduling realistic? Both of the applicants have highly relevant and extensive research experience in their respective fields, as evidenced by many peer reviewed publications to date and are therefore well placed to deliver the work proposed. They are supported by experienced technologists and health professionals. Research plans ● Will the proposed plan of work achieve the objectives outlined in the proposal? ● Are the methodological plans realistic, given the aims of the research and the resources? ● Are the methods and study designs appropriate and free of flaws? The aims and objectives of the proposed research are clearly stated and justified. These are detailed in the respective work packages and summary work plan; they are well justified and scheduled in the context of a realistic timescale. A clear methodology has been adopted to ensure rigorous investigation can be achieved. Evidence of potential impact and clear pathway for benefit to people with Parkinson’s in the near future With these awards we are focused on delivering outcomes that make a difference to people with the condition. That means that we're looking for projects that, if successful, have a clear pathway to be made available to people affected by Parkinson's. 2 If the outcome of this project is that the digital tool is effective in supporting the monitoring and self-management of non-motor symptoms, it will be of considerable benefit to PwP in the short to medium term. If the outcome of this project is that the digital tool is effective in supporting the monitoring and self-management of non-motor symptoms, it will be of considerable benefit to PwP in the short to medium term. If the outcome of this project is that the digital tool is effective in supporting the monitoring and self-management of non-motor symptoms, it will be of considerable benefit to PwP in the short to medium term. If the outcome of this project is that the digital tool is effective in supporting the monitoring and self-management of non-motor symptoms, it will be of considerable benefit to PwP in the short to medium term. ● Please provide constructive comments/suggestions suitable to be fed back to the applicant to help form a full application. These should highlight specific weaknesses and positive aspects of the application. A well justified and compelling proposal that gives confidence a thorough investigation into the benefits of the digital technology will be rigorously investigated. A fuller consideration of any competing technologies would have been useful. Please send your completed review to researchapplications@parkinsons.org.uk Funding category Characteristics for Scientific Members Rating scale Highly fundable ●Very important research questions; likely to result in advancement in the scientific understanding of Parkinson’s or significant benefit for people affected by Parkinson’s by addressing the priority research areas for improving everyday life. ●Excellent and appropriate methods and research design. ●Very strong, internationally competitive team, containing all relevant disciplines. ●Very good value for money. ●Clear and well written pre-proposal. ●Plain English summary accurately reflects the research proposal and is realistic about potential outcomes and timescales involved. Exceptional 10 Excellent quality research 9 Very good, bordering on excellent 8 3 Potentially fundable ●Relevant research questions; likely to result in advancement in the scientific understanding of Parkinson’s, or benefit for people affected by Parkinson’s by addressing the priority research areas for improving everyday life. ●Good quality and appropriate methods and research design. Evidence of potential impact and clear pathway for benefit to people with Parkinson’s in the near future ●Competent and appropriate research team containing all key disciplines. ●Good value for money. ●Plain English summary accurately reflects the research proposal and is realistic about potential outcomes and timescales involved. Good quality research 7 Above average quality research 6 Acceptable quality 5 Not fundable (without significant changes) ●Research questions are not directly relevant to the scientific understanding of Parkinson’s or do not address the priority research areas for people affected by Parkinson’s. ●Inappropriate methods and research design of only modest or poor quality. ●Applicants without relevant research experience or key disciplines not represented. ●Poor value for money. ●Plain English summary is unclear, does not accurately reflect the research proposal and is unrealistic about the potential outcomes and timescales involved. Borderline quality research 4 Below acceptable quality 3 Definitely not fundable ●Irrelevant research questions. ●Poor/flawed/duplicative methods and research design. ●Key skills missing from the research team. ●Very poor value for money. ●Unclear application. ●Plain English summary is unclear, does not accurately reflect the research proposal and is unrealistic about the potential outcomes and timescales involved. Many identified flaws 2 Serious weaknesses or major concerns 1 Project Grants - Parkinson’s UK College of Experts scoring system The listed characteristics are for guidance only. The characteristics are general statements on the overall quality of the application in each funding category. They are not a checklist of minimum criteria for the funding category. Project Grants - Parkinson’s UK College of Experts scoring system Project Grants - Parkinson’s UK College of Experts scoring system The listed characteristics are for guidance only. The characteristics are general statements on application in each funding category. They are not a checklist of minimum criteria for the fund Parkinson’s UK top 10 priority research areas for improving everyday life 8Sleep 9Dexterity 10Urinary problems For more information on the priority research areas and the full list, please visit our website. For more information on the priority research areas and the full list, please visit our website. 5 Non drug approaches pre-proposal review form for lay grant reviewers ay review of a Non drug approaches pre-proposal review form for lay grant reviewers ay review of a Thank you for agreeing to review a Parkinson’s UK research grant application. With these awards we are focused on delivering outcomes that make a difference to people with Parkinson’s. That means that we're looking for projects that, if successful, have a clear pathway to be made available to people affected by Parkinson's. Find out more about the scheme Application reference NDA 21-15 Principal applicant Dr. Edward Meinert Dr Camille Carroll Application title The impact of a digital portal on the monitoring and self management of non-motor symptoms in people with Parkinson’s. 1. Do you think that the proposed project is important to people affected by Parkinson’s? Yes We want to know if the project is important to people affected by Parkinson’s. And whether it is likely to deliver impact or improvement to the lives of people affected by Parkinson’s. Has the potential to make assessments more timely, thorough, relevant and cost effective in a non intrusive way. 2. Does the proposed project include a clear pathway to being made available to people affected by Parkinson's? Yes Does involve Plymouth Parkinson’s service. Mention of PPI but unclear about recruitment, ease of access and time commitment etc. 3. Do you think the applicants should be invited to submit a full application? Yes Full application will need to address recruitment issues, time commitment and how PPI involvement will be taken into account. Non drug approaches pre-proposal review form for lay grant reviewers y Thank you for agreeing to review a Parkinson’s UK research grant application. With these awards we are focused on delivering outcomes that make a difference to people with Parkinson’s. That means that we're looking for projects that, if successful, have a clear pathway to be made available to people affected by Parkinson's. Find out more about the scheme We want to know if the project is important to people affected by Parkinson’s. And whether it is likely to deliver impact or improvement to the lives of people affected by Parkinson’s. Has the potential to make assessments more timely, thorough, relevant and cost effective in a non intrusive way. 2. Does the proposed project include a clear pathway to being made available to people affected by Parkinson's? Yes Does involve Plymouth Parkinson’s service. Mention of PPI but unclear about recruitment, ease of access and time commitment etc. We want to know if the project is important to people affected by Parkinson’s. And whe deliver impact or improvement to the lives of people affected by Parkinson’s. deliver impact or improvement to the lives of people affected by Parkinson s. Has the potential to make assessments more timely, thorough, relevant and cost effective in a non intrusive way. 2. Does the proposed project include a clear pathway to being made available to people affected by Parkinson's? Yes Does involve Plymouth Parkinson’s service. Mention of PPI but unclear about recruitment, ease of access and time commitment etc. 2. Does the proposed project include a clear pathway to being made available to people affected by Parkinson's? Yes Does involve Plymouth Parkinson’s service. Mention of PPI but unclear about recruitment, ease of access and time commitment etc. 3. Do you think the applicants should be invited to submit a full application? Yes Full application will need to address recruitment issues, time commitment and how PPI involvement will be taken into account. minimum criteria for the funding category. Please tick the box next to the score which best reflects your views. Funding category Characteristics Rating scale Highly fundable ●Clear and well written proposal. ●Very important research questions; likely to result in significant benefit for people affected by Parkinson’s. ●Strong evidence of meaningful and well-planned patient and public involvement, with activities integrated at relevant points throughout the project. Non drug approaches pre-proposal review form for lay grant reviewers Exceptional 10 Excellent quality research 9 Very good, bordering on excellent 8 Potentiall y fundable ●Relevant research questions; likely to result in benefit for people affected by Parkinson’s. ●All key aspects of application are clearly presented. ●Some evidence of patient and public involvement, with activities well planned and integrated at relevant points. Good quality research 7 Above average quality research 6 X Acceptable quality 5 Not fundable (without significan t changes) ●Research questions are not directly relevant to Parkinson’s or are unlikely to result in benefit for people affected by Parkinson’s. ●Key elements of the application are unclear. ●Limited evidence of patient and public involvement with unclear plans. Borderline quality research 4 Below acceptable quality 3 Definitely not fundable ●Irrelevant research questions. ●Unclear application. ●No or limited evidence of appropriate patient and public involvement in the research. Many identified flaws 2 Serious weaknesse s or major concerns 1 Parkinson’s UK top 10 priority areas for improving everyday life For more information on the priority research areas and the full list, please visit our website. 2 Please email the completed review forms to: researchapplications@parkinsons.org.uk Once we have shortlisted the pre-proposal applications based on lay reviews, we will let you know which applications have been invited to submit a full application. Pre-proposal applications which are not reviewed favourably by the lay grant reviewers, will not be invited to submit a full application. When we have received the full applications, we will invite you to review the same applications as you reviewed in the pre-proposal stage. 1 Balance and falls 2 Stress and anxiety 3 Uncontrollable movements 4 Personalised treatments 5 Dementia 6 Mild thinking and memory problems 7 Monitoring symptoms 8 Sleep 9 Dexterity 10 Urinary problems Please tick here if you would be happy for us to use anonymised versions of your comments for future training and feedback purposes. X 1 Balance and falls 2 Stress and anxiety 3 Uncontrollable movements 4 Personalised treatments 5 Dementia 6 Mild thinking and memory problems 7 Monitoring symptoms 8 Sleep 9 Dexterity 10 Urinary problems Please tick here if you would be happy for us to use anonymised versions of your comments for future training and feedback purposes. X Please tick here if you would be happy for us to use anonymised versions of your comments for future training and feedback purposes. Non drug approaches pre-proposal review form for lay grant reviewers X Please tick here if you would be happy for us to use anonymised versions of your comm or future training and feedback purposes. Please email the completed review forms to: researchapplications@parkinsons.org.uk Once we have shortlisted the pre-proposal applications based on lay reviews, we will let you know which applications have been invited to submit a full application. Pre-proposal applications which are not reviewed favourably by the lay grant reviewers, will not be invited to submit a full application. When we have received the full applications, we will invite you to review the same applications as you reviewed in the pre-proposal stage. If you have any other questions, we are always happy to help. 3 College of Experts Non drug approaches grant pre-proposal reviewer form This form should be completed by the College of Expert reviewer for a non drug approaches grant pre-proposal. The information you provide is maintained as a record of the decision-making process and to give feedback to applicants. If you have any questions, please contact the Research team by emailing researchapplications@parkinsons.org.uk. Application reference (Internal use) NDA-21-15 Reviewer reference (Internal use) Principal applicant Dr Edward Mienert Application title The impact of a digital portal on the monitoring and self-management of nonmotor symptoms in people with Parkinson’s Please do remember to provide a score for your evaluation at the end of this review. Importance • How important are the questions, or gaps in knowledge, that are being addressed? Is there a good rationale for pursuing these? Is success likely to lead to significant new understanding? • Is the research relevant to Parkinson's? • Does the proposal realistically set out the ultimate potential health or social gain to people with Parkinson's? • How important is it to do the work now? • Is there similar or complementary research underway elsewhere? Are the proposals competitive? While the design of this study fits within the standard framework for usability, it studies only a single platform: the NMS Assist web system. This is one among a very large number of potential web-based data entry systems which could be programmed for the task of collecting data on non-motor symptoms. Because technology moves fast, any single system like this programmed today, will likely be obsolete in a year or even less. Please email the completed review forms to: researchapplications@parkinsons.org.uk Therefore, the main criticism I would raise is that from this research, not much can be learned in about the enduring principles, if any, for creating systems which use digital data capture to improve the lives of those with College of Experts Non drug approaches grant pre-proposal reviewer form This form should be completed by the College of Expert reviewer for a non drug approaches grant pre-proposal. The information you provide is maintained as a record of the decision-making process and to give feedback to applicants. If you have any questions, please contact the Research team by emailing researchapplications@parkinsons.org.uk. Application reference (Internal use) NDA-21-15 Reviewer reference (Internal use) Principal applicant Dr Edward Mienert Application title The impact of a digital portal on the monitoring and self-management of nonmotor symptoms in people with Parkinson’s Please do remember to provide a score for your evaluation at the end of this review. Importance • How important are the questions, or gaps in knowledge, that are being addressed? Is there a good rationale for pursuing these? Is success likely to lead to significant new understanding? • Is the research relevant to Parkinson's? • Does the proposal realistically set out the ultimate potential health or social gain to people with Parkinson's? • How important is it to do the work now? • Is there similar or complementary research underway elsewhere? Are the proposals competitive? While the design of this study fits within the standard framework for usability, it studies only a single platform: the NMS Assist web system. This is one among a very large number of potential web-based data entry systems which could be programmed for the task of collecting data on non-motor symptoms. Because technology moves fast, any single system like this programmed today, will likely be obsolete in a year or even less. Therefore, the main criticism I would raise is that from this research, not much can be learned in about the enduring principles, if any, for creating systems which use digital data capture to improve the lives of those with Parkinson's. This is an observational study and does not address a particular digital intervention, on a small, selective cohort. These shortcomings mean that the research does not seem like a cost-effective way of leading to improved outcomes for people with Parkinson's. If you have any questions, please contact the Research team by emailing researchapplications@parkinsons.org.uk. Please email the completed review forms to: researchapplications@parkinsons.org.uk Evidence of potential impact and clear p Parkinson’s in the near future With these awards we are focused on deliv to people with the condition. That means t Environment and people • Have the applicants established a high-quality track record in the field, including publication history? • Are the applicants uniquely placed to deliver the work? • Where the proposal embarks on work in a field new to the applicants, or is a first funding application, is there a firm foundation to take the work forward? • How well does the work fit with other relevant research pursued by the applicants? The team have extensive experience in running observational technology studies such as this and the environment at Plymouth appears to be well set up to support the research. Research plans • Will the proposed plan of work achieve the objectives outlined in the proposal? • Are the methodological plans realistic, given the aims of the research and the resources? • Are the methods and study designs appropriate and free of flaws? As a small cohort study the design appears sound. However, there are no sample size computations included and therefore it is hard to decide whether the quantitative part of the study would really be effectively powered. More problematic however, is that the study is purely observational, so it is unlikely to be able to address the stated question of whether the app really is leading to improved self-management, and/or prompting adjustment in care. Justification of resources requested • Is the number of staff appropriate for the work described? Are the reasons for purchasing major items of equipment clearly set out? • Are the funds requested appropriate for the work proposed and does the scientific potential justify funding on the scale requested? Is the project value for money? • Are the timescale and scheduling realistic? Given the scale of the proposal, the costs and timescales associated with organizing quite a large team for this project are certainly to be expected. However, the substantial cost for the limited reward does seem excessive. Evidence of potential impact and clear pathway for benefit to people with Parkinson’s in the near future With these awards we are focused on delivering outcomes that make a difference to people with the condition. That means that we're looking for projects that, if As a small cohort study the design appears sound. Please email the completed review forms to: researchapplications@parkinsons.org.uk However, there are no sample size computations included and therefore it is hard to decide whether the quantitative part of the study would really be effectively powered. More problematic however, is that the study is purely observational, so it is unlikely to be able to address the stated question of whether the app really is leading to improved self-management, and/or prompting adjustment in care. Justification of resources requested • Is the number of staff appropriate for the work described? Are the reasons for purchasing major items of equipment clearly set out? • Are the funds requested appropriate for the work proposed and does the scientific potential justify funding on the scale requested? Is the project value for money? • Are the timescale and scheduling realistic? Given the scale of the proposal, the costs and timescales associated with organizing quite a large team for this project are certainly to be expected. However, the substantial cost for the limited reward does seem excessive. Evidence of potential impact and clear pathway for benefit to people with Parkinson’s in the near future With these awards we are focused on delivering outcomes that make a difference to people with the condition. That means that we're looking for projects that, if Please email the completed review forms to: researchapplications@parkinsons.org.uk • How important is it to do the work now? • Is there similar or complementary research underway elsewhere? Are the proposals competitive? While the design of this study fits within the standard framework for usability, it studies only a single platform: the NMS Assist web system. This is one among a very large number of potential web-based data entry systems which could be programmed for the task of collecting data on non-motor symptoms. Because technology moves fast, any single system like this programmed today, will likely be obsolete in a year or even less. Therefore, the main criticism I would raise is that from this research, not much can be learned in about the enduring principles, if any, for creating systems which use digital data capture to improve the lives of those with Parkinson's. This is an observational study and does not address a particular digital intervention, on a small, selective cohort. These shortcomings mean that the research does not seem like a cost-effective way of leading to improved outcomes for people with Parkinson's. Environment and people • Have the applicants established a high-quality track • Are the applicants uniquely placed to deliver the wo • Where the proposal embarks on work in a field new is there a firm foundation to take the work forward? • How well does the work fit with other relevant resea The team have extensive experience in running observa environment at Plymouth appears to be well set up to Research plans • Will the proposed plan of work achieve the objectiv • Are the methodological plans realistic, given the aim • Are the methods and study designs appropriate and As a small cohort study the design appears sound. How included and therefore it is hard to decide whether the effectively powered. More problematic however, is that unlikely to be able to address the stated question of w self-management, and/or prompting adjustment in care Justification of resources requested • Is the number of staff appropriate for the work desc items of equipment clearly set out? • Are the funds requested appropriate for the work pr funding on the scale requested? Is the project value • Are the timescale and scheduling realistic? Given the scale of the proposal, the costs and timescale for this project are certainly to be expected. However, seem excessive. Justification of resources requested successful, have a clear pathway to be made available to people affected by Parkinson's. Please let us know whether this is apparent in this pre proposal application As this is designed as a feasibility study, it is quite early in the research for firm conclusions to be drawn about the applicability of the new NMS Assist platform. This is to be expected, but if the research does not show a positive benefit from this platform, then the research would largely have been wasted as it is so highly specific. Feedback to applicants • Please provide constructive comments/suggestions suitable to be fed back to the applicant to help form a full application. These should highlight specific weaknesses and positive aspects of the application. Any other comments Please let us know whether this is apparent in this pre proposal application As this is designed as a feasibility study, it is quite early in the research for firm conclusions to be drawn about the applicability of the new NMS Assist platform. This is to be expected, but if the research does not show a positive benefit from this platform, then the research would largely have been wasted as it is so highly specific. Feedback to applicants • Please provide constructive comments/suggestions suitable to be fed back to the applicant to help form a full application. These should highlight specific weaknesses and positive aspects of the application. Any other comments Please send your completed review to researchapplications@parkinsons.org.uk Funding category Characteristics for Scientific Members Rating scale Highly fundable • Very important research questions; likely to result in advancement in the scientific understanding of Parkinson’s or significant benefit for people affected by Parkinson’s by addressing the priority research areas for improving everyday life. • Excellent and appropriate methods and research design. • Very strong, internationally competitive team, containing all relevant disciplines. • Very good value for money. • Clear and well written pre-proposal. • Plain English summary accurately reflects the research proposal and is realistic about potential outcomes and timescales involved. Exceptional Excellent quality research Very good, bordering on excellent Please send your completed review to researchapplications@parkinsons.org.uk Potentially fundable • Relevant research questions; likely to result in advancement in the scientific understanding of Parkinson’s, or benefit for people affected by Parkinson’s by addressing the priority research areas for improving everyday life. • Good quality and appropriate methods and research design. • Competent and appropriate research team containing all key disciplines. • Good value for money. • Plain English summary accurately reflects the research proposal and is realistic about potential outcomes and timescales involved. Good quality research Above average quality research Acceptable quality Not fundable (without significant changes) • Research questions are not directly relevant to the scientific understanding of Parkinson’s or do not address the priority research areas for people affected by Parkinson’s. • Inappropriate methods and research design of only modest or poor quality. • Applicants without relevant research experience or key disciplines not represented. • Poor value for money. • Plain English summary is unclear, does not accurately reflect the research proposal and is unrealistic about the potential outcomes and timescales involved. Borderline quality research 4 Below acceptable quality Definitely not fundable • Irrelevant research questions. • Poor/flawed/duplicative methods and research design. Project Grants - Parkinson’s UK College of Experts scoring system The listed characteristics are for guidance only. The characteristics are general statements on the overall quality of the application in each funding category. They are not a checklist of minimum criteria for the funding category. Please let us know whether this is apparent in this pre proposal application • Key skills missing from the research team. • Very poor value for money. • Unclear application. • Plain English summary is unclear, does not accurately reflect the research proposal and is unrealistic about the potential outcomes and timescales involved. Many identified flaws Serious weaknesses or major concerns Project Grants - Parkinson’s UK College of Experts scoring system The listed characteristics are for guidance only. The characteristics are general statements on the overall quality of the application in each funding category. They are not a checklist of minimum criteria for the f di t Not fundable (without significant changes) Definitely not fundable Parkinson’s UK top 10 priority research areas for improving everyday life 1 Balance and falls 2 Stress and anxiety 3 Uncontrollable movements 4 Personalised treatments 5 Dementia 6 Mild thinking and memory problems 7 Monitoring symptoms 8 Sleep 9 Dexterity 10 Urinary problems For more information on the priority research areas and the full list, please visit our website. For more information on the priority research areas and the full list, please visit our website. For more information on the priority research areas and the full list, please visit our website. Lay review of a ● Do you think the proposed research would benefit people affected by Parkinson’s, and if yes, is this potential benefit well communicated to you in the application? ● How relevant do you think this area of research is to the priorities and needs of people affected by Parkinson’s? We are moving into a phase of computer aided clinical management, and I think it would be beneficial for our care to investigate the effectiveness of an assessment tool such as NMS Assist. The non-motor symptoms of PD is an important field. The entire care team, including carers are included which is very important. Clear, easy to understand and relatively jargon free. 2. How important is this research to you as a person affected by Parkinson’s? Please consider how important you think this area of research is to you both personally and to the wider Parkinson’s community, including carers. ● Do you think the proposed research would benefit people affected by Parkinson’s, and if yes, is this potential benefit well communicated to you in the application? ● How relevant do you think this area of research is to the priorities and needs of people affected by Parkinson’s? We are moving into a phase of computer aided clinical management, and I think it would be beneficial for our care to investigate the effectiveness of an assessment tool such as NMS Assist. The non-motor symptoms of PD is an important field. The entire care team, including carers are included which is very important. Clear, easy to understand and relatively jargon free. 2. How important is this research to you as a person affected by Parkinson’s? Please consider how important you think this area of research is to you both personally and to the wider Parkinson’s community, including carers. ● Do you think the proposed research would benefit people affected by Parkinson’s, and if yes, is this potential benefit well communicated to you in the application? ● How relevant do you think this area of research is to the priorities and needs of people affected by Parkinson’s? We are moving into a phase of computer aided clinical management, and I think it would be beneficial for our care to investigate the effectiveness of an assessment tool such as NMS Assist. The non-motor symptoms of PD is an important field. The entire care team, including carers are included which is very important. Clear, easy to understand and relatively jargon free. Thank you for agreeing to review a Parkinson’s UK project grant pre-proposal application. Below each question, we have provided some key prompts for you to consider when commenting on each aspect of the application. Please feel free to make any additional comments which you think are relevant. Your comments help us and the members of the College of Experts to understand exactly what you think about an application; and why you may or may not want to support it or think it’s important to people affected by Parkinson’s. It is therefore very important that you elaborate wherever possible. Your anonymised review form will be sent to applicants for their information. General comments that could be considered vague and unspecific do not give the scientific reviewers a clear idea of what you think of the proposed research. For more guidance please refer to the lay grant reviewer training pack and briefing document. If you have any other questions, we are always happy to help. Please call us on 020 7963 3950 or email researchapplications@parkinsons.org.uk. y q , y ppy p researchapplications@parkinsons.org.uk. Application reference NDA-21-15 Principal applicant Camille Carroll Application title The impact of a digital portal on the monitoring and self-management of non- motor symptoms in people with Parkinson’s Plain English title Lay reviewer reference 1. What did you think of the plain English summary? Excellent Good ✠ Acceptable ○ Poor ○ It is really useful if lay grant reviewer’s comment on the language used by researchers to explain their work in the plain English summary, as this feedback will help ensure that their future applications are accessible to those without a scientific background. ● Was the purpose of the proposed research clear? ● Did the summary help you carry out your review? If not, why not? ● Was the background to the research summarized adequately? ●Is the language used clear and understandable? Are the scientific terms and jargon well explained? If not, which terms need explanation? The impact of a digital portal on the monitoring and self-management of non- motor symptoms in people with Parkinson’s 1 Clear, easy to understand and relatively jargon free. 2. How important is this research to you as a person affected by Parkinson’s? Please consider how important you think this area of research is to you both personally and to the wider Parkinson’s community, including carers. 2. How important is this research to you as a person affected by Parkinson’s? 2 2 5. Do you have any additional comments about the application? If you have any additional thoughts about the ways in which the application could be improved please add them below. Outcomes of the NMS assessment on service support and resources on patient activation management are clear and obtainable. Well thought out study. Please tick here if you would be happy for us to use anonymised versions of your comments for future training and feedback purposes. Score The listed characteristics are for guidance only. The characteristics are general statements on the overall quality of the application in each in funding category. They are not a checklist of minimum criteria for the funding category. Please tick the box next to the score which best reflects your views. 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With these awards we are focused on delivering outcomes that make a difference to people with Parkinson’s. That means that we're looking for projects that, if successful, have a clear pathway to be made available to people affected by Parkinson's. Find out more about the scheme Application reference NDA-21-15 Principal applicant Dr Edward Meinert Dr Camille Carroll Application title The impact of a digital portal on the monitoring and self-management of nonmotor symptoms in people with Parkinson's 1. Do you think that the proposed project is important to people affected by Parkinson’s? Yes  No We want to know if the project is important to people affected by Parkinson’s. And whether it is likely to deliver impact or improvement to the lives of people affected by Parkinson’s. The project focuses on non-motor symptoms. The app allows PwP a certain amount of self-monitoring/control of their symptoms. It allows their clinicians to view their data and propose changes. 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That means that we're looking for projects that, if successful, have a clear pathway to be made available to people affected by Parkinson's. Non drug approaches pre-proposal review form for lay grant reviewers Find out more about the scheme Application reference NDA-21-15 Principal applicant Dr Edward Meinert Dr Camille Carroll Application title The impact of a digital portal on the monitoring and self-management of nonmotor symptoms in people with Parkinson's 1. Do you think that the proposed project is important to people affected by Parkinson’s? Yes  No We want to know if the project is important to people affected by Parkinson’s. And whether it is likely to deliver impact or improvement to the lives of people affected by Parkinson’s. The project focuses on non-motor symptoms. The app allows PwP a certain amount of self-monitoring/control of their symptoms. It allows their clinicians to view their data and propose changes. It also allows the PwP to flag concerns to their care teams. Having this ability would be very good for the PwP but I doubt that clinicians/care team would have the time to review every patients' data or be willing to have patients contact them. 2. Does the proposed project include a clear pathway to being made available to people affected by Parkinson's? Yes No  There is no clear pathway to being made available as it stops at the point that if successful it goes to a larger trial which will take yet more time and yet more money. 3. Do you think the applicants should be invited to submit a full application? Yes  No Yes but we need a fixed being-made-available date not just a call for yet more work. We want it now. Can it be speeded up? Score q y pp g g y y criteria for the funding category. Please tick the box next to the score which best reflects your views. 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Many identified flaws 2 Serious weaknesses or major concerns 1 Parkinson’s UK top 10 priority areas for improving everyday life For more information on the priority research areas and the full list, please visit our website. 1 Balance and falls 2 Stress and anxiety 3 Uncontrollable movements 4 Personalised treatments 5 Dementia 6 Mild thinking and memory problems 7 Monitoring symptoms 8 Sleep 9 Dexterity 10 Urinary problems Please tick here if you would be happy for us to use anonymised versions of your comments for future training and feedback purposes.  Please email the completed review forms to: researchapplications@parkinsons.org.u Once we have shortlisted the pre-proposal applications based on lay reviews, we will let you know which applications have been invited to submit a full application. Pre-proposal applications which are not reviewed favourably by the lay grant reviewers, will not be invited to submit a full application. 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https://openalex.org/W3174608275
https://www.mdpi.com/1996-1073/14/13/3753/pdf?version=1624427110
English
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Optimization of Air Cooling System Using Adjoint Solver Technique
Energies
2,021
cc-by
12,835
energies energies   Keywords: adjoint method; heat sink; shape optimization; heat transfer; CFD; pseudo-transient solver Article Grzegorz Czerwi´nski † and Jerzy Wołoszyn *,† and Jerzy Wołoszyn *,† Grzegorz Czerwi´nski † Department of Power Systems and Environmental Protection Facilities, Faculty of Mechanical Engineering and Robotics, AGH University of Science and Technology, Mickiewicz 30 Av., 30-059 Krakow, Poland; gczerwin@agh.edu.pl † These authors contributed equally to this work. Abstract: Air cooling systems are currently the most popular and least expensive solutions to maintain a safe temperature in electronic devices. Heat sinks have been widely used in this area, allowing for an increase in the effective heat transfer surface area. The main objective of this study was to optimise the shape of the heat sink geometric model using the Adjoint Solver technique. The optimised shape in the context of minimal temperature value behind the heat sink is proposed. The effect of radiation and trapezoidal fin shape on the maximum temperature in the cooling system is also investigated. Simulation studies were performed in Ansys Fluent software using the Reynolds— averaged Navier–Stokes technique. As a result of the simulation, it turned out that not taking into account the radiation leads to an overestimation of temperatures in the system—even by 14 ◦C. It was found that as the angle and height of the fins increases, the temperature value behind the heat sink decreases and the heat source temperature increases. The best design in the context of minimal temperature value behind the heat sink from all analysed cases is obtained for heat sink with deformed fins according to iteration 14. The temperature reduction behind the heat sink by as much as 25 ◦C, with minor changes in heat source temperature, has been achieved.   Citation: Czerwi´nski, G.; Wołoszyn, J. Optimization of Air Cooling System Using Adjoint Solver Technique. Energies 2021, 14, 3753. https:// doi.org/10.3390/en14133753 Academic Editor: Andrea Lazzaretto Received: 21 May 2021 Accepted: 17 June 2021 Published: 23 June 2021   Citation: Czerwi´nski, G.; Wołoszyn, J. Optimization of Air Cooling System Using Adjoint Solver Technique. Energies 2021, 14, 3753. https:// doi.org/10.3390/en14133753 Academic Editor: Andrea Lazzaretto Received: 21 May 2021 Accepted: 17 June 2021 Published: 23 June 2021 1. Introduction With the rapid development of technology and semiconductor engineering, the trend is towards the miniaturization of electronic devices and the resulting increase in power den- sity. The operating temperature range for most electronic devices is between 85–100 ◦C [1,2]. Above this limit, a decrease in the reliability of electronic equipment is observed, character- ized by unstable operation, shortened lifespan associated with various types of mechanical failures, or slowed computing speed. Received: 21 May 2021 Accepted: 17 June 2021 Published: 23 June 2021 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. To date, many experimental studies have been conducted on convective heat trans- fer in a heat sink cooling system. The first studies by Starner and McManus [3], and Chaddock [4] aimed to illustrate the phenomenon of natural convection and its effect on heat transfer in heat sinks with rectangular fins. Subsequent experiments performed by Leung [5–9], on the other hand, were focused on different geometrical parameters of the fins, their array and the orientation of the heat sink. Among the techniques used in studies of heat sinks with rectangular fins were empirical correlations. Vollaro [10] determined the value of the optimal spacing between the fins of a heat sink aligned in the vertical direction. Baskaya [11] investigated the dependence of the fin spacing on a horizontal plate. On the other hand, Harahap and Setio [12] empirically determined the relationships between the geometric dimensions of the heat sink regardless of its orientation. 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/). An important topic addressed in experimental research on air cooling systems is the effect of the radiation process, as described in articles by Edwars and Chaddock [13], Azarkish [14], and Rao [15], among others. It has been shown in these publications that the https://www.mdpi.com/journal/energies Energies 2021, 14, 3753. https://doi.org/10.3390/en14133753 Energies 2021, 14, 3753 2 of 24 heat flux transferred by radiation accounts for 25–40% of the total heat flux exchanged in the system. heat flux transferred by radiation accounts for 25–40% of the total heat flux exchanged in the system. 1. Introduction Numerical simulations are increasingly being used to model heat and mass trans- fer phenomena. The aim of the research is often to improve the thermal comfort of buildings [16]. It turns out that modern wind tower designs [17] with a moistened pad provides a large space for airflow inside the column and thus make it possible to increase efficiency at higher wind velocities. Furthermore, numerical simulations make it possi- ble to investigate the influence of structures, such as the exhaust grille, deflecting-ring, and heat exchanger, on the obtained characteristics of the airflow passing through the air conditioner [18]. In addition, CFD analysis is used: to assess the capacity for microbial decontamination of indoor air [19]; to investigate the effect of the position of the air cleaner on the dispersion and removal of particles in an indoor environment [20] and to determine the optimal installation position of the forced air duct during drilling operation [21]. p p g g p [ ] CFD analysis is also often used to simulate the phenomenon of heat removal from electronic devices. In addition to studying the velocity and temperature fields around the heat sink, optimising the system’s geometric model can be done efficiently. Tati and Mehrtash [22] performed numerical simulations by varying the position of the heat sink to determine the Nusselt number for different values of Rayleigh numbers. The authors determined correlations for the Nusselt number by considering the angle contained be- tween the base surface and the horizontal surface for two ranges of the modified Rayleigh number. Kim [23], on the other hand, investigated the airflow and heat transfer char- acteristics of vertical heat sinks. It turned out that the most important factors affecting the heat sink optimization are the length of the fins, the difference between the heat sink temperature and the ambient temperature, and the thermophysical properties of the fluid. It was noted that fin height is not a key parameter in heat sink optimization. Kulkarni and Dotihall [24] also undertook a numerical analysis of an air cooling system. They evaluated heat sink with various fin geometries with Custom pin fin, Slanted mirror, Fluted, Zigzag and staggered array configurations. In terms of maximum temperature, the heat sink with Slanted mirror geometry is the most favourable. 1. Introduction Then, multi-objective parametric optimization was performed to minimize entropy generation. The research conducted showed that the air-cooled forced convection has a high potential for use in the thermal management of electronic packages. The importance of the topic of efficient power management of electronic compo- nents is evidenced by the growing number of publications on topology optimization of fin shapes [30] or heat exchangers [31] using both liquid [32] and air [33] cooling methods. Topology optimization has been performed using either genetic optimization approaches [34] or surrogate modelling-based Bayesian optimization [35]. For this type of optimization, the interpolation scheme such as modified Solid Isotropic Material with Penalization (SIMP) [36], as well as the algorithm based on the method of moving asymp- totes (MMA) [37–39] and the globally convergent version of MMA (GCMMA) [37,40,41] are often used. Based on the literature review, it was found that the vast majority of previous research neglect to conduct comparative studies, focusing on the occurrence of natural convection and radiation in the system or only the phenomenon of natural convection. The numerical calculations of cooling systems have consisted of comparing heat sinks in different fin orientations or fin shapes, and too little attention has been paid to the trapezoidal fins. There are also limited researches using the Adjoint Solver. Compared to the standard topological optimization methods described above, this technique does not interfere with the topology of the fins but only modifies their shape. Therefore, the authors conduct research to fill these research gaps. The objective of this work was to optimise the shape of the heat sink using the Adjoint Solver technique to minimise the average temperature in a specified volume behind the heat sink. To achieve this goal: the authors obtain differential temperature fields for a good understanding of the effect of radiation as a heat transfer mechanism; the design of the experiments (DOE) was used to evaluate the effect of trapezoidal fin modifications on the temperature distribution behind the heat sink and the heat source temperature; a comparative analysis for simple, trapezoidal and optimise by Adjoint fins was performed; the authors also investigated the effect of the power dissipated by the element on the temperature in the cooling system. The flowchart of the conducted research is presented in Figure 1. 1. Introduction A system with Zigzag fins is characterized by the occurrence of the highest maximum temperature value on the heat sink surface, which is an undesirable phenomenon. On the other hand, although the lowest maximum temperature is observed on the Custom pin fin, the significant pressure drop precludes their use. A comparative analysis of pin fins was also performed by Maji [25]. His main goal was to reduce the pressure drop in the cooling system using this type of fins. He examined geometric models that differed in the number of fins, their shape, and the size of the holes formed in them. The results showed that pin fins having holes have better heat removal properties than the basic solution. In contrast, as the number of holes and their size increases, the Nusselt number also increases and the pressure drop in the system decreases. Using modified geometric models, an increase in the heat transport rate of up to 40.5% was noted. Shadlaghani [26], on the other hand, investigated different fin configurations with perforations to obtain the highest possible heat transfer coefficient for the heat sink. Trian- gular, rectangular and trapezoidal perforations with constant volume were analyzed. As a result of the simulation, it was found that the fins with triangular inserts have the highest heat transfer rate compared to the other solutions. Al.-Sallami [27] investigated eleven designs of a standard heat sink, including various modifications of the heat sink by using circular perforations, rectangular notches and slot perforations. Simulation results showed the advantage of fins having slots instead of circular perforations. Their use significantly lowered the fin base temperature below its critical temperature with less fan power and further saved material. Simulations by Saraiya [28] considered heat transfer in a cooling system using a steady-state heat sink with natural convection. Based on the parametric study, a range of parameters was established to perform the Design of Experiments (DOE). The response surfaces were determined using data obtained from a computer experiment based on Optimal Space Filling (OSF). A Multi-objective Genetic Algorithm (MOGA) was used to optimize the parameters. The maximum heat transfer from the above analysis Energies 2021, 14, 3753 3 of 24 occurs at the optimum value of fin length −20 mm, fin height −25 mm, spacing −14.8 mm and fin thickness −2.14 mm. A study by Patel and Matawala [29] presents an experimental and numerical analysis of a heat sink. 1. Introduction In the beginning, a proprietary geometric model was developed, which was subjected to CFD analysis in order to investigate the effect of radiation on the temperature values in the system. Then, by carrying out the design of the experiments, the influence of the trapezoidal fins was determined. Furthermore, the geometric model of the heat sink was optimised using the Adjoint Solver technique. The final stage of the research was to compare the performance of the three geometric models of the heat sink under variable power conditions. 4 of 24 Energies 2021, 14, 3753 Figure 1. The flowchart of the conducted research. Figure 1. The flowchart of the conducted research. Figure 1. The flowchart of the conducted research. 2. Research Object Cutting plane parameters by which modified geometric models of the heat sink were ob- tained. No. 1 2 3 4 5 6 7 8 9 w [mm] 55 55 55 45 45 45 35 35 35 α [◦] 30 45 60 30 45 60 30 45 60 2. Research Object The simulation of the cooling system of an electronic component is performed by the use of the simplified geometric model. The heat sink and the heat source is placed in the housing. The influence of other electronic components was neglected in the study. The geometric model of the cooling system is shown in Figure 2. Figure 2. Geometric model of the cooling system with names of individual parts. Figure 2. Geometric model of the cooling system with names of individual parts. The dimensions of the system are shown in projections perpendicular (Figure 3) as well as parallel (Figure 4) to the flow direction. Figure 3. Domain dimensions in front view. Figure 3. Domain dimensions in front view. Figure 3. Domain dimensions in front view. 5 of 24 Energies 2021, 14, 3753 Figure 4. Domain dimensions in side view. It was decided to investigate the effect of modifications in the geometric model of the fins on the temperature distribution behind the heat sink and the heat source temperature. The study was performed using the DOE technique. The heat sink shape modification was determined by two parameters shown schematically in Figure 5. These are the distance of the top surface of the heat sink from the lowest point of intersection of the cutting plane with the fin (w) and the angle of inclination of the cutting plane with respect to the XY plane (α). The values of the geometric parameters adopted in the simulation are shown in Table 1. Figure 4. Domain dimensions in side view. Figure 4. Domain dimensions in side view. It was decided to investigate the effect of modifications in the geometric model of the fins on the temperature distribution behind the heat sink and the heat source temperature. The study was performed using the DOE technique. The heat sink shape modification was determined by two parameters shown schematically in Figure 5. These are the distance of the top surface of the heat sink from the lowest point of intersection of the cutting plane with the fin (w) and the angle of inclination of the cutting plane with respect to the XY plane (α). The values of the geometric parameters adopted in the simulation are shown in Table 1. Figure 5. Example geometric model of a trapezoidal heat sink. Figure 5. Example geometric model of a trapezoidal heat sink. Table 1. 3.1. Mathematical Model 3.1. Mathematical Model Heat transport in the developed cooling system is an issue described as a function of spatial coordinates. This is also a problem coupled with fluid flow. Coupling involves the conduction heat transfer from the heat source to the heat sink and to a lesser extent to the base. The heat is then transferred to the outside through natural convection and radiation. An additional assumption can be made to simplify the mathematical model. The fluid should be considered Newtonian—its thermophysical properties in the form of conductivity, specific heat, viscosity are constant, and there are no chemical reactions or heat generation. Furthermore, the flow is assumed to be a steady-state. The following partial differential equations are solved for the problem formulated in this way: p q p y The continuity equation can be written for the fluid: The continuity equation can be written for the fluid: ∂vx ∂x + ∂vy ∂y + ∂vz ∂z = 0, (1) (1) The momentum equations are as follows: The momentum equations are as follows: ρ ∂vx ∂t + vx ∂vx ∂x + vy ∂vx ∂y + vz ∂vx ∂z  = −∂p ∂x + µ ∂2vx ∂x2 + ∂2vx ∂y2 + ∂2vx ∂z2 ! , (2) (2) Energies 2021, 14, 3753 6 of 24 ρ ∂vy ∂t + vx ∂vy ∂x + vy ∂vy ∂y + vz ∂vy ∂z  = −∂p ∂y + µ ∂2vy ∂x2 + ∂2vy ∂y2 + ∂2vy ∂z2 ! , (3) ρ ∂vz ∂t + vx ∂vz ∂x + vy ∂vz ∂y + vz ∂vz ∂z  = −∂p ∂z + µ ∂2vz ∂x2 + ∂2vz ∂y2 + ∂2vz ∂z2 ! + ρ gz, (4) (3) ρ  ∂t + x ∂x + y ∂y + z ∂z  ∂y + µ ∂x2 + ∂y2 + ∂z2 ! , ( ) ρ ∂vz ∂t + vx ∂vz ∂x + vy ∂vz ∂y + vz ∂vz ∂z  = −∂p ∂z + µ ∂2vz ∂x2 + ∂2vz ∂y2 + ∂2vz ∂z2 ! 3.1. Mathematical Model + ρ gz, (4) There is heat transfer in the flow, hence energy equation to be considered is: vx ∂T ∂x + vy ∂T ∂y + vz ∂T ∂z = λ ρ · c ∂2T ∂2x + ∂2T ∂2y + ∂2T ∂2z  , (5) (5) The calculated Rayleigh number value Ra = 2.85 × 108 for the characteristic dimension Lrad = √ S where S is the contact area of the heat sink and the fluid. The determined value of the Ra number does not indicate the existence of a fully turbulent flow. Many sources [42,43] give a critical Ra number of 109. However, in his work, Dixit [44] further distinguished transient flow in the Ra number range from 106 to 108. Any 2-equation turbulence model is a strong simplification of the real phenomenon of chaotic turbulence. However, failure to use an appropriate turbulence model, in this case, could lead to an incorrect description of the phenomenon under study. Turbulence model based on k (turbulent kinetic energy) and ε (rate of dissipation of turbulent kinetic energy) was adopted in works [45,46]. This model allows to correctly solved a cooling system using natural convection due to the lack of stability of the solution of the governing equations. Thus, in this study, the RANS turbulence modelling technique was used and—to close the Reynolds-averaged Navier–Stokes equations—the realizable model k-ε was assumed. The system is also affected by radiation, which results in the use of the Surface to Surface model [47]. It is suitable for modelling the radiation in an enclosure without the participating media. Despite its limitations, this model is widely used for simple circuits. p p g p y p In this study, optimization of the base shape of heat sink fins using the Adjoint Solver module is undertaken. This is a module of the Ansys Fluent package for nonparametric freeform shape optimization. Its principle of operation is based on the search for a geometric model satisfying the assumed optimization requirements. y g p q In the initial stage of optimisation, the shape and dimensions of the geometric model are adopted by the designer. The first calculations for solving the continuity, momentum and energy equations are carried out traditionally. After determining the velocity fields and the pressure distribution, the objective function J is defined to be optimised with respect to the design parameters c [48]. 3.1. Mathematical Model Theoretically, the value of c represents the vector of all parameters that define the problem, including the locations of the grid nodes [49]. Thus, the objective function depends not only on the design parameters but also on the flow condition q(c): q(c) = [vx, vy, vz, p, T], (6) J = J(q(c), c), (7) (6) (7) The equations describing heat and mass transfer can be written in a simplified form [50]: (8) R(q(c), c) = 0, (8) As the state of the flow q(c) and the control variables c change, a change in the selected optimisation objective is also observed [51]: δ J = δJ δqδ q + δJ δc δ c, (9) (9) The first term of the above equation relates to the change in a flow state, controlled by the steady-state RANS equations, in response to a change in geometry or other boundary conditions. The Adjoint technique provides a mechanism for replacing this term with an expression that depends only on c. This replacement is achieved by exploiting the Energies 2021, 14, 3753 7 of 24 linearization of the equations describing heat and mass transfer. The optimisation should operate between defined flow field limitations: flow and design variables. Consequently, when the flow field changes, the residual variable (δ R) should remain zero [52]. linearization of the equations describing heat and mass transfer. The optimisation should operate between defined flow field limitations: flow and design variables. Consequently, when the flow field changes, the residual variable (δ R) should remain zero [52]. 3.1. Mathematical Model δ R = δR δq δ q + δR δc δ c = 0, (10) (10) In the next step, we need to obtain The Lagrange Multipliers for transforming the constrained optimization problem into an unconstrained optimization problem [53]: In the next step, we need to obtain The Lagrange Multipliers for transforming the constrained optimization problem into an unconstrained optimization problem [53]: L( f, c, λ) = J(q, c) −λTR(q, c), (11) (11) In Equation (11), the parameter λ is called the vector of adjoint variables [48]: λ = h v∗ x, v∗ y, v∗ z, p∗, T∗i , (12) (12) Combining Equations (9) and (11) together, we get: Combining Equations (9) and (11) together, we get: Combining Equations (9) and (11) together, we get: δ J = δJT δq δ q + δJT δc δ c −λT δR δq δq + δR δc δc  = δJT δq −λT δR δq  δ q + δJT δc −λT δR δc δc  δ c, (13) (13) The value of λ is taken in such a way as to eliminate the influence of flow variables. This provides the adjoint equation to be solved [54]: The value of λ is taken in such a way as to eliminate the influence of flow variables. This provides the adjoint equation to be solved [54]: δJT δc = λT δR δc δ c, (14) (14) It is possible to obtain the dependence between cost function and design variables by inserting Equation (14) into Equation (13): δ J = δJT δq −λT δR δq  δ q, (15) (15) In the next step, the new shape of the geometric model is obtained by shifting the cell nodes using control nodes [55]. Subsequently, the flow analysis is repeated. Achieving the assumed value of a given objective function enables the optimisation to be successfully completed. This optimization method has been successfully applied, for example, in the field of aerodynamics [56,57] and in solving problems coupled with heat transfer [58]. Hence, it seems fully reasonable to use this tool to optimize the shape of the heat sink. 3.2. Validation of the Adopted Model To check the validity of the adopted mathematical model, the results of the nu- merical simulation were compared with the results of the experiment performed by Ruhul [59]. The exact boundary conditions, thermophysical properties of solid mate- rials and air were assumed during validation. Furthermore, the same simulation do- main, operating conditions and heat generation from the semiconductor element equal to qm_heatsource = 2,970,000 W/m3 were also assumed. The role of mass forces, in this case, is played by a gravitational force in line with the direction of the z-axis, with opposite direction and a value of gy = −9.81 m/s2. The factor to verify both methods was the aver- age temperature of the individual channels, i.e., the space between the ribs. The resulting temperature distribution is shown in Figure 6. 8 of 24 Energies 2021, 14, 3753 Figure 6. Dependence of average temperature of channels on their location [59]. Figure 6. Dependence of average temperature of channels on their location [59]. After analyzing the obtained distribution, it was found that the absolute error of the temperature does not exceed 2 ◦C. Therefore, with a relative percentage error of less than 3% obtained, the model is used in subsequent calculation steps. 3.3. Computational Conditions • a pressure inlet type boundary condition was assumed on surface A; relative pressure value 0 Pa, surface temperature 40 ◦C, flow direction normal to the surface; • a pressure outlet type boundary condition was assumed on surface C; relative pressure value 0 Pa, surface temperature 40 ◦C, flow direction normal to the surface; • a pressure inlet type boundary condition was assumed on surface A; relative pressure value 0 Pa, surface temperature 40 ◦C, flow direction normal to the surface; • a pressure outlet type boundary condition was assumed on surface C; relative pressure value 0 Pa, surface temperature 40 ◦C, flow direction normal to the surface; Energies 2021, 14, 3753 9 of 24 • a wall type boundary condition was assumed on surfaces B, D, E, F; stationary wall, no slip (v = 0); • a wall type boundary condition was assumed on surfaces B, D, E, F; stationary wall, no slip (v = 0); • a wall type boundary condition was assumed on surfaces B, D, E, F; stationary wall, no slip (v = 0); • the emissivity value of the outer surface of the heat sink is equal to 0.9; • the emissivity value of the outer surface of the heat source is equal to 0.3 y q • the emissivity value of the upper base surface is equal to 0.9; y pp q • the emissivity value of the surfaces B, D, E is equal to 0.9. • the emissivity value of the surfaces B, D, E is equal to 0.9. The design assumption is to receive a heat flux whose value is Pheat = 80 W. Hence, we proceeded to determine the solid_heatsource Vb element volume and calculated the heat generation value (Equation (16)). qm_heat = Pheat Vb = 417,558 W m3 , (16) (16) Material Properties and Solver Settings Material Properties and Solver Settings The table (Table 2) gives the assumed properties of copper, glass fibre epoxy laminate (fr-4) and heat source material. The values of specific heat, density and thermal conductivity were determined [37]. Table 2. Determination of material properties of individual system components. 3.3. Computational Conditions Appropriate boundary conditions are required to solve the governing equations. Also, computational conditions must be adopted, and a grid developed. The airflow takes place by natural convection, so the presence of positive gauge pressure at the inlet and negative pressure at the outlet, which could force the air to move, has been neglected. g The location where the boundary conditions are adopted is shown in Figure 7. The air inlet and outlet surfaces (A and C) are characterized only by the atmospheric pressure value, with no set velocity value. This makes it possible to reproduce the natural convection. The remaining surfaces were characterized as walls with air movement between them. It was also assumed that the system was tested at an ambient temperature of 40 ◦C; hence both at the inlet and outlet domain surface, the temperature was equal to 40 ◦C. To represent the radiation, they were assigned emissivity values. The boundary conditions can be described as follows: Figure 7. Nomenclature of external planes of the domain. Figure 7. Nomenclature of external planes of the domain. 3.3. Computational Conditions Part of Domain Material Density Specific Heat Thermal Conductivity ρ [kg/m3] cp [J/(kg·K)] [W/(m·K)] solid_heat sink copper 8978 381 387.6 solid_base fr-4 381 1300 795 solid_heatsource component 387.6 0.35 10 The properties of the air, which is the working medium of the cooling system, were also assumed, i.e., the specific heat c = 1006.43 J/(kgK), thermal conductivity λ = 0.0242 W/(mK) and dynamic viscosity µ = 1.7894 × 10−5. Given the fact of negligible pressure changes, the density of air is described by the incompressible-ideal-gas model and depends only on temperature according to the Ideal Gas Equation. Thus, the existence of a fully compressible flow can be neglected. g In a further step, the operating conditions in the domain were determined. They are characterized by a reference pressure poper = 101,325 Pa, an average temperature Toper = 313.15 K in the domain defined by inlet and outlet conditions, and an operating density ρoper = 1.126 kg/m3. p The Pseudo Transient option [37] was used in the calculations. Thus, the pseudo time step for air ∆tair = 0.37 s and copper ∆tcopper = 255 s was adopted in the developed cooling system model—as they have the lowest value among the time step values calculated for the other materials. Calculations were performed using a coupled pressure-based solver. A Warped-Face Gradient Correction function was used to assist in solving the equations for polyhedral grids. The interpolation scheme for pressure was adopted as Body Force Weighted, while First Order Upwind was adopted for momentum equation, turbulence kinetic energy equation, turbulence kinetic energy dissipation equation. In contrast, the Second Order Upwind scheme was chosen for the energy equation. The values of the under-relaxation parameters for pressure and momentum are 0.4; for density, mass forces, and turbulent viscosity are 1; for energy, turbulence kinetic energy and turbulence kinetic energy dissipation is 0.75. The maximum number of iterations equal to 800 or RMS (root mean square) of the residues equal to RMS = 10−5 were used as convergence criteria for the iterative solution scheme. Calculations were performed in ANSYS FLUENT software. Further, numerical parameters were adopted to control the solution of the equations of the optimization algorithm. An important parameter is the Courant number. As it increases, it is possible to obtain faster convergence of the solution; however, this may mean getting an unstable solution. 3.3. Computational Conditions Obtaining a convergent solution was possible for a Courant number Energies 2021, 14, 3753 10 of 24 10 of 24 value of 10. Residual Minimization was used to achieve faster convergence of the solution. A convergence criterion value of 10−4 was set for each equation, and then the calculations were initialized. The number of iteration steps was set to 150. After 23 iteration steps, convergence was obtained for all equations (Figure 8). value of 10. Residual Minimization was used to achieve faster convergence of the solution. A convergence criterion value of 10−4 was set for each equation, and then the calculations were initialized. The number of iteration steps was set to 150. After 23 iteration steps, convergence was obtained for all equations (Figure 8). Figure 8. Residual graphs for optimization equations. Figure 8. Residual graphs for optimization equations. 3.4. Adopted Numerical Grid Therefore, it was Energies 2021, 14, 3753 11 of 24 decided to investigate the mass flow rate between the inlet and outlet and the total heat transfer rate between the surfaces. The results are shown in Figure 10. decided to investigate the mass flow rate between the inlet and outlet and the total heat transfer rate between the surfaces. The results are shown in Figure 10. Figure 10. Effect of the grid on the balance of streams. Determination of the balance of individual rates for each grid allows, as it were, to confirm the fact that for grids 4 and 5, the greatest loss in mass and heat flow is observed. Hence, to obtain a correct numerical solution, it was decided to choose grid 3 (Figure 11). It is also a trade-off between the number of mesh elements and the accuracy of the solution. Figure 10. Effect of the grid on the balance of streams. Determination of the balance of individual rates for each grid allows, as it were, to confirm the fact that for grids 4 and 5, the greatest loss in mass and heat flow is observed. Hence, to obtain a correct numerical solution, it was decided to choose grid 3 (Figure 11). It is also a trade-off between the number of mesh elements and the accuracy of the solution. Determination of the balance of individual rates for each grid allows, as it were, to confirm the fact that for grids 4 and 5, the greatest loss in mass and heat flow is observed. Hence, to obtain a correct numerical solution, it was decided to choose grid 3 (Figure 11). It is also a trade-off between the number of mesh elements and the accuracy of the solution. Determination of the balance of individual rates for each grid allows, as it were, to confirm the fact that for grids 4 and 5, the greatest loss in mass and heat flow is observed. Hence, to obtain a correct numerical solution, it was decided to choose grid 3 (Figure 11). It is also a trade-off between the number of mesh elements and the accuracy of the solution. Figure 11. Polyhexcore grid. To select an appropriate turbulence model, the value of the y+ parameter had to be determined. A preliminary numerical simulation resulted in the dependence of the y+ parameter on the heat sink length (Figure 12). 3.4. Adopted Numerical Grid Five poly-hexcore grids were created for the geometric model (Figure 2) differing in settings such as element size on the heat sink surface and heat source element (local_1), maximum mesh size on the surface (maximum size), and minimum mesh size (minimum size). The characteristics of all grids were collected and presented in Table 3. Table 3. Characteristics of numerical grids subjected to CFD analysis. No. Local_1 Minimum Size Maximum Size Number of Elements min OQ [mm] [mm] [mm] [-] [-] 1 3.0 2.50 25 486,382 0.1129 2 3.5 1.17 25 379,152 0.1017 3 4.5 2.50 25 342,902 0.1011 4 6.0 2.50 50 312,676 0.1499 5 7.0 2.50 60 286,189 0.1500 Table 3. Characteristics of numerical grids subjected to CFD analysis. The grid independence study was examined based on parameters such as the temper- ature behind the heat sink (point-temperature) and the average temperature of the heat source (heatsource-temperature). The results are shown in Figure 9. The grid independence study was examined based on parameters such as the temper- ature behind the heat sink (point-temperature) and the average temperature of the heat source (heatsource-temperature). The results are shown in Figure 9. Figure 9. Effect of the grid on the temperature value. Figure 9. Effect of the grid on the temperature value. Figure 9 shows that the highest point temperature from all meshes was achieved for mesh no. 5. However, the temperature differences between meshes at a specific point does not exceed 0.1 K, while for the temperature of the heat source, 0.8 K. Therefore, it was Figure 9. Effect of the grid on the temperature value. Figure 9 shows that the highest point temperature from all meshes was achieved for mesh no. 5. However, the temperature differences between meshes at a specific point does not exceed 0.1 K, while for the temperature of the heat source, 0.8 K. Therefore, it was Figure 9 shows that the highest point temperature from all meshes was achieved for mesh no. 5. However, the temperature differences between meshes at a specific point does not exceed 0.1 K, while for the temperature of the heat source, 0.8 K. Therefore, it was Figure 9 shows that the highest point temperature from all meshes was achieved for mesh no. 5. However, the temperature differences between meshes at a specific point does not exceed 0.1 K, while for the temperature of the heat source, 0.8 K. 3.4. Adopted Numerical Grid Its value is in the range of 20 to 130. Hence it is possible to apply a turbulence model that uses the wall functions—k-ε Realizable. Figure 11. Polyhexcore grid. To select an appropriate turbulence model, the value of the y+ parameter had to be determined. A preliminary numerical simulation resulted in the dependence of the y+ parameter on the heat sink length (Figure 12). Its value is in the range of 20 to 130. Hence it is possible to apply a turbulence model that uses the wall functions—k-ε Realizable. To select an appropriate turbulence model, the value of the y+ parameter had to be determined. A preliminary numerical simulation resulted in the dependence of the y+ parameter on the heat sink length (Figure 12). Its value is in the range of 20 to 130. Hence it is possible to apply a turbulence model that uses the wall functions—k-ε Realizable. 12 of 24 Energies 2021, 14, 3753 Figure 12. Value of the y+ parameter along the length of the heat sink. Figure 12 Value of the y+ parameter along the length of the heat sink Figure 12. Value of the y+ parameter along the length of the heat sink. 4. Results and Discussion of the Numerical Calculations 4.1. Effect of Radiation on the Solution 4. Results and Discussion of the Numerical Calculations 4.1. Effect of Radiation on the Solution To develop the analysis of the results, the coordinates of the measurement point behind the heat sink need to be defined (x = 0.2125 m, y = 0.03 m, z = −0.5 m). The results of the analysis are presented as a velocity vector field in the planes parallel to the direction of motion and as a temperature field (Figure 13). The temperature scale applies to the solid domain, and the velocity scale applies to the air domain. The highest temperature value in the system of about 353 K is noted near the heat source. Slightly lower values (around 350 K) are observed on the walls of the heat sink. The base of the system—near the inlet, outlet and on the sides—is much cooler, and its temperature reaches values close to the ambient temperature. However, it can be noticed that just behind the heat sink, the area gets much hotter. This is a negative phenomenon, especially in the case when it would be decided to place another electronic component behind the heat sink. The temperature difference in the system causes natural convection. The air, starting from the inlet, flows past the heat sink, where its temperature increases. This effect reduces the air density and causes it to move towards the outlet in the direction of the z-axis. The described phenomenon can be seen in the velocity vector field. There is an increase in air velocity near the heat sink compared to its velocity at the inlet. However, it is also a fact that the velocity also increases at the outlet from the domain. Thus, the air heats up from the base wall of the system behind the heat sink. Furthermore, air streams further away from the heat sink get hotter, mainly due to the existence of the radiation phenomenon. Figure 13. Vector velocity field around the heat sink and temperature distribution of the solid type domain. Figure 13. Vector velocity field around the heat sink and temperature distribution of the solid type domain. Energies 2021, 14, 3753 13 of 24 13 of 24 One of the convergence criteria besides the adopted residuum values was the temper- ature behind the heat sink and the average temperature of the heat source (Figure 14). It can be concluded that the calculation resulted in a stable value of both temperatures after 600 iterations. Figure 14. 4. Results and Discussion of the Numerical Calculations 4.1. Effect of Radiation on the Solution Dependence of the temperature behind the heat sink (point temperature) and the average temperature of the heat source during the calculation. Figure 14. Dependence of the temperature behind the heat sink (point temperature) and the average temperature of the heat source during the calculation. The CFD analysis was also performed for the case without radiation in the system under study, and the results obtained from both analyses were compared in the form of differential temperature field (Figure 15). The temperature range varied from 313 K to 367 K for the calculation assuming only natural convection. In contrast, the temperature range varied from 313 K to 355 K for the calculation assuming natural convection and radiation. The most significant differences in the temperature field are most noticeable behind the heat sink and on its surface. It can be seen (Figure 15) that the temperature difference equal 14 K and 22 K for the heat source and air behind the heat sink, respectively. Failure to account for the radiation phenomenon in the system under study leads to an overestimation of the temperature value due to the numerical model simplification. Figure 15. Temperature distribution and the difference between simulation assuming convection and both convection and radiation. Figure 15. Temperature distribution and the difference between simulation assuming convection and both convection and radiation. Energies 2021, 14, 3753 14 of 24 14 of 24 . Effect of Fin Shape on the Maximum Temperature in the Cooling System 4.2. Effect of Fin Shape on the Maximum Temperature in the Cooling System The dependence of temperature versus section plane angle (α) and parameter (w) is shown in Figures 16 and 17. The functions were determined by performing an approxima- tion with second-order polynomials. The temperature behind the heat sink and the heat source temperature for the simulation before modifying the geometric model was 330.35 K and 353.15 K, respectively. Figure 16. Temperature dependence behind the heat sink depending on fin shape. Figure 16. Temperature dependence behind the heat sink depending on fin shape. Figure 17. Temperature dependence of the heat source depending on the case under study. Figure 17. Temperature dependence of the heat source depending on the case under study. A trapezoidal fin shape reduced the temperature value behind the heat sink for each of the cases studied. It decreases with increasing angle of the section plane (α) and increasing (w) parameter. 4. Results and Discussion of the Numerical Calculations 4.1. Effect of Radiation on the Solution The maximum values of parameters (α) and (w) allow to decrease the value of the temperature behind the heat sink compared to the initial design by 1.5 K; however, they cause the heat source temperature to increase by 6 K. Thus, the excessive inclination of the fins negatively affects the heat source temperature value, which is related to the decrease of the heat transfer surface between the air and the heat sink. The best design in the context of minimal temperature value behind the heat sink is obtained for w = 55 mm and angle α = 60◦. The best design in the context of minimal heat source temperature is obtained for w = 35 mm and angle α = 30◦. However, finding the single best design solution requires optimization with two objective functions and is a trade-off between the Energies 2021, 14, 3753 15 of 24 15 of 24 temperature behind the heat sink and the heat source, and such studies have not been conducted in this research. temperature behind the heat sink and the heat source, and such studies have not been conducted in this research. 4.3. Use of Adjoint Solver Technique for Heat Sink Shape Optimization The average temperature in the volume defined by the coordinates of the Box Re- gion was assumed to be minimized. This is important because of the need to keep the temperature of the air flowing to other electronic components behind the heat sink as low as possible. A volume 0.09 m long, 0.02 m wide, and 0.02 m high are consistent with the location of the control point used to monitor the temperature behind the heat sink Tp (Figure 18). The initial value of the average temperature in the given volume is equal to 338.7 K. Figure 18. Volume behind the heat sink whose average temperature is minimized. Figure 18. Volume behind the heat sink whose average temperature is minimized. In the next step, we evaluated the influence of the geometric model of the heat sink on the value of the adopted optimization criterion. To this end, log10 (Shape Sensitivity Magnitude) field was prepared on the surfaces of the heat sink, the electronic component and the base. Based on this, it was found that changing the shape of the back of the fins at the bottom base would have the greatest effect on reducing the temperature values behind the heat sink (Figure 19). The sensitivity of a variable to the value of mass forces exerted on a numerical grid cell can be similarly represented (Figure 20). The shape change area in the optimization process was assumed to be a cuboid encompassing the boundaries of the entire heat sink, taking into account the possibility of changing the shape of the geometric model near the edges of the fins as shown on the log10 field (Figure 19). Figure 19. Sensitivity of heat sink shape expressed by the log10 function. Figure 19. Sensitivity of heat sink shape expressed by the log10 function. Energies 2021, 14, 3753 16 of 24 16 of 24 Figure 20. Sensitivity of the optimization criterion to mass forces. Figure 20. Sensitivity of the optimization criterion to mass forces. For the current calculations, the number of control nodes was increased to perform more accurate changes to the geometric model. For the x-direction, 40 points were de- termined; for the y-direction and z-direction, 30 points were determined. A Freeform Scale Factor, which allows the magnitude of the attempted design change to be adjusted, equal to 2, was also assumed. 4.3. Use of Adjoint Solver Technique for Heat Sink Shape Optimization After the initial calculation, the percentage value of the expected improvement of the optimization criterion was determined. The numerical grid transformed in the given iterative step was then modified. In the next step, calculations were carried out in ANSYS Fluent (taking into account the radiation phenomenon), and the optimization was performed again. In this way, 21 iterations were implemented, during which the geometric model of the heat sink was optimized to reduce the temperature behind the heat sink. Figure 21 shows the temperature trend behind the heat sink, both the approximate temperature determined in the optimization process (orange) and the actual temperature verified in ANSYS Fluent (blue). The change in the optimization criterion under study is presented over successive iterations. Figure 21. Temperature change behind the heat sink for 21 iterations. Figure 21. Temperature change behind the heat sink for 21 iterations. From Figure 21, the positive effect of performing the optimization can be observed. The set temperature value has decreased from about 340 K to 315 K. Thus, it can be concluded that it corresponds to the inlet and outlet conditions. Hence further improvement is practically impossible. With each iteration, a decrease in the minimum orthogonal quality Energies 2021, 14, 3753 17 of 24 17 of 24 of the generated division grid is observed, which negatively affects the calculations and limits the number of possible iterations in the optimization process (Figure 22). of the generated division grid is observed, which negatively affects the calculations and limits the number of possible iterations in the optimization process (Figure 22). Figure 22. Change in minimum orthogonal quality over successive iterations. Figure 22. Change in minimum orthogonal quality over successive iterations. The trend of the maximum temperature of the heat source was also investigated (Figure 23). Its value also decreased, however, not as much as the air temperature behind the heat sink. Over the iterations, the value of the studied temperature changed by a maximum of about 0.3 K. Hence, modifying the geometric model to lower the temperature behind the heat sink does not adversely affect the thermal performance of the heat source. An inverse relationship between both temperatures was observed during the numerical simulation of heat sinks with trapezoidal fins. Figure 23. Change in maximum temperature of electronic component. Figure 23. Change in maximum temperature of electronic component. 4.3. Use of Adjoint Solver Technique for Heat Sink Shape Optimization Figure 24 shows the change in the shape of the geometric model in selected optimisa- tion steps and the temperature distribution on the heat sink surface and on the system base plane. The observed differences correspond to the projections included in the sensitivity fields (Figures 19 and 20). In the initial iterations, the geometric model of the heat sink does not change significantly. In the 8th iteration, a bending of the back part of the fins can be observed. In the 16th iteration, this change deepens and additionally begins to include Energies 2021, 14, 3753 18 of 24 18 of 24 also the fins located on the outer side. The modification of the geometrical model also decreases the temperature of the base behind the heat sink. Although up to the 8th iteration, the value of this temperature practically does not change, in subsequent iterations, it is already below 320 K. Additionally, a differential temperature distribution was developed between the 1st and 21st iteration steps (Figure 25). The results are presented on a plane parallel to the base plane, offset by 0.03 m according to the OY-axis direction. From this, it can be concluded that the optimisation performed not only contributed to lowering the temperature just behind the heat sink (by about 25 K) but also made it possible to reduce it in a much larger area behind the heat source. In these areas, the temperature difference is as much as 10 K. This makes it possible, in practice, to place another component that would require appropriate temperature conditions during its operation. Furthermore, a reduction in temperature values in any volume (not only in the immediate vicinity of the heat sink) can be given as an objective in this type of optimisation, depending on the design assumptions. Figure 24. Change in heat sink shape and temperature distribution. Figure 24. Change in heat sink shape and temperature distribution. Energies 2021, 14, 3753 19 of 24 Figure 25. Differential temperature distribution between the first and last iteration. Figure 25. Differential temperature distribution between the first and last iteration. 4.4. Effect of Power Dissipated by an Element on the Temperature in the Cooling System In this study, the temperature dependence of the heat source and the temperature behind the heat sink versus the power generated by the electronic component was de- termined. 4.3. Use of Adjoint Solver Technique for Heat Sink Shape Optimization The characteristics were plotted for three geometric models: the base model (Figure 2), the trapezoidal heat sink for parameter w = 55 mm and α = 60◦, and the optimal shape (solution for 16 iterations). The assumed value of the heat generation in the electronic component is shown in Table 4. Table 4. Assumed value of the heat generation in the electronic component. Power [W] Heat Generation [W/m3] 30 156,584 40 208,779 50 260,974 60 313,169 70 365,364 80 417,558 90 469,753 100 521,948 110 574,143 120 626,337 Table 4. Assumed value of the heat generation in the electronic component. Summary results for all heat sink shape cases in terms of the temperature dependence behind the heat sink versus heat flux are shown in Figure 26. A similar relationship, however, in this case for the heat source temperature, is presented in Figure 27. Summary results for all heat sink shape cases in terms of the temperature dependence behind the heat sink versus heat flux are shown in Figure 26. A similar relationship, however, in this case for the heat source temperature, is presented in Figure 27. Summary results for all heat sink shape cases in terms of the temperature dependence behind the heat sink versus heat flux are shown in Figure 26. A similar relationship, however, in this case for the heat source temperature, is presented in Figure 27. Summary results for all heat sink shape cases in terms of the temperature dependence behind the heat sink versus heat flux are shown in Figure 26. A similar relationship, however, in this case for the heat source temperature, is presented in Figure 27. Figure 26. Temperature dependence behind the heat source versus heat flux. Figure 26. Temperature dependence behind the heat source versus heat flux. Energies 2021, 14, 3753 20 of 24 Figure 27. Temperature dependence of the heat source versus heat flux. Figure 27. Temperature dependence of the heat source versus heat flux. By analyzing Figures 26 and 27 it can be observed that the temperature value increases with the increase of generated power. The temperature value behind the heat sink is highest for the basic heat sink, while the cooling system using the trapezoidal fins has lower temperature values for the entire power range. Notably, the temperature behind the heat sink decreases significantly for the geometric model of the heat sink optimized in Adjoint Solver. 4.3. Use of Adjoint Solver Technique for Heat Sink Shape Optimization For the entire power range, a nearly constant value of this temperature is observed equally to the inlet and outlet conditions. The temperature dependence of the heat source follows a similar pattern; however, higher values are recorded for trapezoidal fin shapes. For powers lower than 80 W, significant reductions in source temperature were achieved. We decided to compare three geometric models: a basic heat sink, a heat sink with trapezoidal fins (w = 55 mm, α = 60◦) and a heat sink optimised using the Adjoint Solver technique for a heat source power of 70 W. The results were presented in a plane parallel to the XZ plane for y = 0.03 m. A velocity vector field was used for comparison, and the temperature values were plotted on it (Figure 28). Figure 28. Comparison of flow direction and temperature field for three geometric models of a heat sink. Figure 28. Comparison of flow direction and temperature field for three geometric models of a heat sink. From Figure 28 we conclude that the direction of the velocity vectors behind the basic heat sink and the heat sink with trapezoidal fins is similar. In the case of the heat sink optimised with the Adjoint Solver technique, the airflow is divided into two parts at the Energies 2021, 14, 3753 21 of 24 21 of 24 back of the domain. This results in a lower temperature of the airflow in the central part of the system behind the heat sink, compared to the other cases. It can also be noted that the temperature values for the systems with the basic heat sink and the heat sink with trapezoidal fins have a similar distribution. Hence, we decided to compare the temperature change behind the heat sink for the three geometric models. Consequently, a chart (Figure 29) was made basic on a centre line of the velocity field plane (for constant values of x = 0.2125 m, y = 0.03 m and a change in value of z value from −0.4 m to −1.2 m). Figure 29. Comparison of the temperature dependence behind the heat sink. Figure 29. Comparison of the temperature dependence behind the heat sink. In comparing the basic and the trapezoidal fins heat sinks, it is found that the tem- perature change in the backside is similar. 4.3. Use of Adjoint Solver Technique for Heat Sink Shape Optimization Lower temperature values are observed behind the basic heat sink, but in the latter part, its average value is already higher. At the outlet of the system, a temperature drop behind the basic heat sink is visible compared to the temperature behind the trapezoidal heat sink. The use of a heat sink with an optimised shape by using the Adjoint Solver technique allows a significant reduction in temperature in the rear part of the system—on average by 5 K. The best design in the context of minimal temperature value behind the heat sink from all analysed cases is obtained for heat sink with deformed fins according to iteration 14 (Figure 24). The further modifications do not improve the solution. 5. Summary and Conclusions This study presents the results of numerical calculations of the problem of cooling electronic components in a confined space. Analyses were performed using the RANS technique taking into account the natural convection and radiation. The authors present the results of optimizing the shape of the geometric model using the Adjoint Solver technique and analyzing the effect of the power dissipated by the electronic component on the temperature in the cooling system. An analysis of the effect of radiation in the cooling process simulation on the solution was also performed, and the effect of the trapezoidal shape of the fins on the maximum temperature in the cooling system was investigated. The major findings of the study are presented in several sections: • The optimisation performed using the Adjoint Solver technique allowed for a reduc- tion of the assumed temperature by 25 K. This did lead to a decrease in the minimum value of the orthogonal grid quality. However, in this case, it is possible to re-generate the grid for the optimised geometric model. • Neglecting the radiation phenomenon in the tested system led to overestimated heat sink temperatures (by nearly 14 K). p y y • Increasing the angle of the fin (α) and increasing the value of the parameter (w) allows a lower value of the air temperature behind the heat sink, which on the other hand, adversely affects the heating of the heat source component. The maximum values of the parameters α and w cause a 1.5 K decrease in the temperature value behind the heat sink and a 6 K increase in the heat source temperature. Energies 2021, 14, 3753 22 of 24 22 of 24 • The proposed optimal fin design ensures that the increase in heat source power does not significantly affect the temperature in the area behind the heat sink. • The proposed optimal fin design ensures that the increase in heat source power does not significantly affect the temperature in the area behind the heat sink. • The heat source temperature for optimal fin design and heat flux up to 80 W is lower than the basic design and trapezoidal fins. • The heat source temperature for optimal fin design and heat flux up to 80 W is lower than the basic design and trapezoidal fins. 5. Summary and Conclusions Despite its ability to investigate heat transport problems, the Adjoint Solver technique combined with Ansys Fluent has some limitations: sensitivity to mesh element size and inability to perform transient calculations, not considering the phase change model or the radiation phenomenon. As a further direction of this research, the authors propose to perform a similar optimisation for a different type of heat sink (for example, with pin fins) and consider the influence of other boundary conditions on the model’s shape optimised using the Adjoint Solver technique. Author Contributions: Conceptualization, G.C.; Investigation, G.C.; Methodology, G.C. and J.W.; Supervision, J.W.; Validation, G.C.; Writing—original draft, G.C. and J.W. Both 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. Informed Consent Statement: Not applicable. Data Availability Statement: Data sharing not applicable. Acknowledgments: This research was supported by national subvention no. 16.16.130.942. Conflicts of Interest: The authors declare no conflict of interest. Abbreviations The following abbreviations are used in this manuscript: c specific heat of fluid [J/(kg·K)], design parameters, gx,y,z gravitational acceleration in a given direction [m/s2], J objective function, k turbulent kinetic energy [m2/s2], L Lagrangian function, p pressure [Pa], q heat generation [W/m3], flow condition function, R residual variable, T temperature [K], t time [s], ∆t pseudo time step [s], vx,vy,vz Cartesian velocity coordinates [m/s], w fins height [mm], x, y, z Cartesian coordinates [m], α angle of inclination [◦], ε rate of dissipation of turbulent kinetic energy [m2/s3], λ fluid thermal conductivity [W/(m·K)], vector of adjoint variables, µ fluid dynamic viscosity [Pa·s], ρ fluid density [kg/m3] c specific heat of fluid [J/(kg·K)], design parameters, gx,y,z gravitational acceleration in a given direction [m/s2], J objective function, k turbulent kinetic energy [m2/s2], L Lagrangian function, p pressure [Pa], q heat generation [W/m3], flow condition function, R residual variable, T temperature [K], t time [s], ∆t pseudo time step [s], vx,vy,vz Cartesian velocity coordinates [m/s], w fins height [mm], x, y, z Cartesian coordinates [m], α angle of inclination [◦], ε rate of dissipation of turbulent kinetic energy [m2/s3], λ fluid thermal conductivity [W/(m·K)], vector of adjoint variables, µ fluid dynamic viscosity [Pa·s], ρ fluid density [kg/m3] 1. 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Innowacyjny proces projektowania i optymalizacji produktów z Adjoint. In Proceedings of the Innowacje w Zarz ˛adzaniu i in˙zYnierii Produkcji, Zakopane, Poland, 1–3 55. Plutecki, Z.; Sattler, P.; Ryszczyk, K. Innowacyjny proces projektowania i optymalizacji produktów z wykorzystaniem solvera Adjoint. In Proceedings of the Innowacje w Zarz ˛adzaniu i in˙zYnierii Produkcji, Zakopane, Poland, 1–3 March 2015; Volume 18. 56. Kalinowski, M.; Szczepanik, M. Aerodynamic Shape Optimization of Racing Car Front Wing; IOP Conference Series: Materials Science and Engineering; IOP Publishing Ltd.: Bristol, UK, 2021; Volume 1037. Adjoint. In Proceedings of the Innowacje w Zarz ˛adzaniu i inzYnierii Produkcji, Zakopane, Poland, 1–3 March 2015; Volume 18. 56. Kalinowski, M.; Szczepanik, M. Aerodynamic Shape Optimization of Racing Car Front Wing; IOP Conference Series: Materials Science and Engineering; IOP Publishing Ltd.: Bristol, UK, 2021; Volume 1037. 56. Kalinowski, M.; Szczepanik, M. 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MGMR: leveraging RNA-Seq population data to optimize expression estimation
BMC bioinformatics
2,012
cc-by
5,519
PROCEEDINGS Open Access © 2012 Rozov et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: RNA-Seq is a technique that uses Next Generation Sequencing to identify transcripts and estimate transcription levels. When applying this technique for quantification, one must contend with reads that align to multiple positions in the genome (multireads). Previous efforts to resolve multireads have shown that RNA-Seq expression estimation can be improved using probabilistic allocation of reads to genes. These methods use a probabilistic generative model for data generation and resolve ambiguity using likelihood-based approaches. In many instances, RNA-seq experiments are performed in the context of a population. The generative models of current methods do not take into account such population information, and it is an open question whether this information can improve quantification of the individual samples Results: In order to explore the contribution of population level information in RNA-seq quantification, we apply a hierarchical probabilistic generative model, which assumes that expression levels of different individuals are sampled from a Dirichlet distribution with parameters specific to the population, and reads are sampled from the distribution of expression levels. We introduce an optimization procedure for the estimation of the model parameters, and use HapMap data and simulated data to demonstrate that the model yields a significant improvement in the accuracy of expression levels of paralogous genes. Conclusions: We provide a proof of principal of the benefit of drawing on population commonalities to estimate expression. The results of our experiments demonstrate this approach can be beneficial, primarily for estimation at the gene level. Conclusions: We provide a proof of principal of the benefit of drawing on population commonalities to estimate expression. The results of our experiments demonstrate this approach can be beneficial, primarily for estimation at th l l Roye Rozov1, Eran Halperin1,2,3*, Ron Shamir1 Roye Rozov1, Eran Halperin1,2,3*, Ron Shamir1 From Second Annual RECOMB Satellite Workshop on Massively Parallel Sequencing Barcelona, Spain. 19-20 April 2012 From Second Annual RECOMB Satellite Workshop on Massively Parallel Sequencing Barcelona, Spain. 19-20 April 2012 * Correspondence: heran@icsi.berkeley.edu 1The Blavatnik School of Computer Science, Tel-Aviv University, Tel Aviv 69978, Israel Full list of author information is available at the end of the article Rozov et al. BMC Bioinformatics 2012, 13(Suppl 6):S2 http://www.biomedcentral.com/1471-2105/13/S6/S2 Rozov et al. BMC Bioinformatics 2012, 13(Suppl 6):S2 http://www.biomedcentral.com/1471-2105/13/S6/S2 Introduction HapMap [3] populations. Such studies complement genomic DNA sequencing by elucidating the link between SNPs and expression. With the rapid decline in the cost of sequencing, RNA- Seq has emerged as a legitimate competitor to mi-croar- rays as a means of assessing global gene expression. Even as arrays currently enjoy a cost advantage, many new applications of information accessible only through sequencing further strengthen the case that sequencing may soon supplant arrays as the technology of choice for transcription analysis. One such application is fine- grained assessment of variation in expression and the sources for such variation, as exemplified by recent large-scale RNA-Seq studies [1,2] of two different Unfortunately, with any new technology come its lim- itations, and several studies have pointed out that RNA- Seq is not immune to bias [4-6]. Perhaps the most widely discussed hurdle to accurate estimation in the case of RNA-Seq is the problem of reads mapped to multiple locations in the target genome (or in the target transcript sequences). These reads, which are called multireads, can stem from either paralogous gene sequences or from different isoforms of the same gene that share exons. Several methods have emerged to address the multi- read problem for paralog and isoform estimation [7-10]. These methods are all based on probabilistic modeling * Correspondence: heran@icsi.berkeley.edu 1The Blavatnik School of Computer Science, Tel-Aviv University, Tel Aviv 69978, Israel Full list of author information is available at the end of the article Rozov et al. BMC Bioinformatics 2012, 13(Suppl 6):S2 http://www.biomedcentral.com/1471-2105/13/S6/S2 Rozov et al. BMC Bioinformatics 2012, 13(Suppl 6):S2 http://www.biomedcentral.com/1471-2105/13/S6/S2 Page 2 of 7 independent, and errorjk is the number of mismatches in the best alignment of rj to Gk. that is optimized by an expectation maximization proce- dure. It has been repeatedly shown that by using such methods one can get better quantification of the expres- sion levels compared to quantification based on naive approaches of read assignment. j This formulation leads to the likelihood of observing the data: L(P; R) = ρ j=1 P(rj|G, P) = ρ  j=1 M  k=1 P(Gk)P(rj|Gk) (1) In many applications, a set of samples is studied. For instance, one may be interested in comparing the expression levels in cases verses controls, or in tissues originating from different organs. RNA-Seq multiread expression estimation As we seek to extend the prevalent generative model of RNA-Seq [7-11], we begin by reviewing the basic elements of that model. Let G = (G1, ...,GM) be the set of M tran- scribed regions considered and P = (P1, ...,PM) be the pro- portions of RNA bases attributed to each transcript out of the total number of transcribed bases in a sequenced sam- ple. Regions may be either genes or transcripts, depending on the level of transcription being investigated. We require P to satisfy ∑gG Pg = 1 and ∀gG, 0 ≤Pg ≤1. For sample i, we denote the set of reads as Ri = (ri1, . . . , riρi), where each rij is mapped to one or more regions in G. The output of a read alignment pro- gram defines the set of accepted regions for the read (in practice only alignments with up to 2 errors are accepted) and provides the number of errors in alignment for each read-region pair. This allows us to calculate P(rij|Gk) as done above for one sample. For convenience we denote P (rij|Gk) = qijk (taken to be zero for all regions not mapped to), which is independent of a and P. The model describes an RNA sequencing experiment where regions in G are randomly chosen according to the distribution P, start positions in these regions are chosen uniformly, and reads of length ℓare generated by copying ℓconsecutive bases from each chosen region to produce a set of reads R = (r1,..., rr). Sequencing is assumed to be error prone, leading to a certain prob- ability of error for each read base. Based on the repeti- tions present in the set of regions and errors in alignment, reads may fail to map to the region from which they originate or may map to additional locations. Thus, we assign a probability of obtaining read rj given that it originated from region Gk, P(rj|Gk) ≡(1 −ε)(ℓ−errorjk)εerrorjk ℓk . In this case we rely on the alignment of rj to Gk to afford us the best match As before, our objective is to estimate P, but in this case we must optimize by estimating P and a together. We begin by writing the likelihood function: L(p1, . . . , pN, α; R) = Pr(p1, . . . , pN|α)Pr(R|p1, . . . Introduction In such cases, it is plausible that the commonality of expression patterns within each of the defined groups of studied samples may be used to improve the quantification results in each of the samples. We demonstrate that by analyzing expression profiles of a population together, one gets expression estimates more accurate than those obtained by estimating individual sample expression levels inde- pendently. We describe and implement a probabilistic model of the sequencing process that incorporates population commonalities, and demonstrate its advan- tages over existing methods in the population setting. (1) This likelihood function is used to estimate P given the read alignments. Typically, one will use expectation maximization to find the P for which the likelihood is maximized. It is assumed that P(rj|Gk) is zero for all regions to which rj is not aligned. Common population extension To estimate expression levels in N individuals from a defined population, we modify the above model by assuming that samples are drawn from a common popu- lation. This is imposed by having P = [(P11, ...,P1M), .., (PN1 ...,PN M)] be probability densities drawn from a common Dirichlet distribution, defined by a set of hyper-parameters specific to the population: ∀i[1, N], pi = (Pi1,..., PiM) ~ Dirichlet (a1,..., aM). RNA-Seq multiread expression estimation , pN) (2) Since expression values are sampled from the Dirichlet distribution, Pr(p1, . . . , pN|α) = N  i=1 P(pi|α) = N  i=1 C(α) M  k=1 Pαk−1 ik (3) Where . In this case we rely C(α) ≡( kαk)  k(αk) (4) C(α) ≡( kαk)  k(αk) (4) on the alignment of rj to Gk to afford us the best match position instead of summing over all possible starting positions. ℓk is the effective length of Gk (i.e., the num- ber of start positions from which a full length read can be derived) as defined in [11],  is taken to be a constant per-base error rate, errors are assumed to be and similar to (1) above, and similar to (1) above, Pr(R|p1, . . . , pN) = N  i=1 ρi  j=1 M  k=1 Pikqijk (5) (5) Rozov et al. BMC Bioinformatics 2012, 13(Suppl 6):S2 http://www.biomedcentral.com/1471-2105/13/S6/S2 Rozov et al. BMC Bioinformatics 2012, 13(Suppl 6):S2 http://www.biomedcentral.com/1471-2105/13/S6/S2 Page 3 of 7 Page 3 of 7 Page 3 of 7 This leads to where This leads to where where This leads to where This leads to This leads to L(p1, . . . , pN, α; R) =  N  i=1 C(α) M  k=1 Pαk−1 ik  ⎡ ⎣ N  i=1 ρi  j=1 M  k=1 Pikqijk ⎤ ⎦ (6) aijk ≡ P(t) ik qijk M k=1 P(t) ik qijk (12) (12) Taking the log, we get M step Given that each qijk is fixed, the above reduces to maximizing M step Given that each qijk is fixed, the above reduces to maximizing log[L(p1, . . . , pN, α; R)] = NlogC(α) + M  k=1 (αk −1) N  i=1 logPik + N  i=1 pi  j=1 (log( M  k=1 Pikqijk)) (7) log[L(p1, . . . RNA-Seq multiread expression estimation , pN, α; R)] = NlogC(α) + M  k=1 (αk −1) N  i=1 logPik + N  i=1 pi  j=1 (log( M  k=1 Pikqijk)) (7) to maximizing NlogC(α(t)) + M  k=1 (α(t) k −1) N  i=1 logPik + N  ρi  M  aijk ∗logPik (13) NlogC(α(t)) + M  k=1 (α(t) k −1) N  i=1 logPik + N  i=1 ρi  j=1 M  k=1 aijk ∗logPik (13) (7) (13) Multi-Genome Multi-Read (MGMR) algorithm We wish to estimate a and p1,...,pn to maximize equa- tion (7) above. For this purpose, we adopt an alternating iterative procedure of estimating a given the current estimate of p1,...,pn and vice-versa until the total change in a becomes sufficiently small (or until a pre-set num- ber of iterations have been executed). Using Lagrange multipliers and differentiating, we see that this is maximized with P(t+1) ik = α(t) k −1 +  j aijk  k (α(t) k −1 +  j aijk) (14) (14) Although for EM-based estimation methods convexity guarantees an optimal solution will be obtained, here (as shall be seen below) we have no such guarantee. Thus, we confine updates to be local by performing only one update for P and one for a. By one MGMR iteration, we refer to one EM-based P update followed by one a update. p Estimating P given a (15) If we assume a is given, we can write the EM steps to find p1,...,pN: E step Letting Match signify a matching between reads and regions, and Match(j) be the region from which read j originates, we get: By using the known bound (x) ≥(ˆx)exp((x −ˆx)(x)) (having(x) = dlog(x) dx ), By using By (x) ≥(ˆx)exp((x −ˆx)(x)) (having(x) = dlog(x) dx ), log[L(P, α; R, Match)] = NlogC(α) + M  k=1 (αk −1) N  i=1 logPik + N  i=1 ρi  j=1 (log(PiMatch(j)qijMatch(j))) (8 we can get a lower bound on F(a): we can get a lower bound on F(a): F(α) ≥N[(  k αk)(  k α(t) k ) − M  k=1 log(αk) + M  k=1 αklog¯P(t) k + Const(P(t)) (16) (8) (16) which leads to which leads to where log¯Pk = 1 N ∗ i logPik. Q(P, α|P(t), α(t)) = EM atch|R,P(t),α(t)[log(L)] (9) (9) N i We maximize this bound with a fixed point iteration similar to EM, noting that for fixed values of P conver- gence is guaranteed, and that for the Dirichlet distribu- tion the maximum is the only stationary point [12]. This leads to the update = NlogC(α(t)) + M  k=1 (α(t) k −1) N  i=1 logPik + N  i=1 ρi  j=1 M  k=1 (logPik + logqijk) ∗ P(t) ik qijk M k=1 P(t) ik qijk (10 = NlogC(α(t)) + M  k=1 (α(t) k −1) N  i=1 logPik + N  i=1 ρi  j=1 M  k=1 aijklogPik + N  i=1 ρi  j=1 M  k=1 aijklogqijk (11 (10) (10) α(t+) k = −1[(  α(t) k ) + log¯P(t) k ] (17) (17) Estimating a given P Given a new estimate for P(t), we can use a fixed point iteration [12] to get a new estimate of a F(α) = N[log(  k αk) −  k log(αk)] F(α) = N[log(  k αk) −  k log(αk)] + M  k=1 (αk −1) N  i=1 logP(t) ik + Const(P(t)) (15) Heuristics/Implementation As we have found F(a) presented in equation (15) is non-convex even in 2 dimensions (Figure 1), we confine updates to be local by allowing only one update for both the a and P estimation steps at each MGMR (11) Rozov et al. BMC Bioinformatics 2012, 13(Suppl 6):S2 http://www.biomedcentral.com/1471-2105/13/S6/S2 Page 4 of 7 Figure 1 A mesh representation of F(a) [equation (15)] showing non-convex behavior. P is a 10 × 2 constant matrix and a is varied on [0:50,0:50]. The case shown is for N = 10, M = 2 (ten samples, two a parameters). Non-convex behavior is demonstrated by the values on the plane defined by a1 = .06 on the range [0,50] on the right. Figure 1 A mesh representation of F(a) [equation (15)] showing non-convex behavior. P is a 10 × 2 constant matrix and a is varied on [0:50,0:50]. The case shown is for N = 10, M = 2 (ten samples, two a parameters). Non-convex behavior is demonstrated by the values on the plane defined by a1 = .06 on the range [0,50] on the right. iteration. For genes with EM expression estimates equal- ing zero in all samples we substitute 10-20 for their values in MGMR to avoid taking the log of zero. For P updates (e.g., equation 14), we avoid potentially negative P values by adding one to each alpha (thus ignoring -1 in the numerator and denominator). We implemented the algorithm in MATLAB, where the inputs required are read-gene map files for each sample as in SEQEM [7], and an initial P estimate matrix. Alphas are initia- lized as an M-length vector of ones. used the resulting distribution of expression values to generate simulated datasets for testing. Real samples were drawn from lymphoblastoid cells of the Yoruba in Ibadan (YRI) population [2,3]. As MGMR requires initial expression estimates, the estimate derived from the method it was being compared with in each case was input to MGMR. Thus, the four initial estimates used were from SEQEM, MGMR(SEQEM) (namely, MGMR initialized by SEQEM’s result), RSEM, and MGMR (RSEM) (namely, MGMR initialized by RSEM esti- mates). We denote these four estimates SEQEM-A, SEQEM-B, MGMR-A and MGMR-B, respectively. We simulated reads by randomly selecting start sites falling within gene boundaries and extracting sequences from those positions. Experimental setup As in [7,9,10], we examined MGMR’s accuracy by com- paring its estimates of known expression levels with those of existing methods, namely SEQEM [7] and RSEM [9,10]. The initial “known” expression levels were esti- mates obtained from RNA-Seq samples; how these esti- mates were obtained is described below. In our case, we had to simulate a population sharing similar expression levels and the same set of gene regions. Our experiment differed in that we sought to use additional information to improve on the estimates of these existing methods. These methods were designed to estimate expression of single samples, and each had specific advantages which we disregarded in our comparison. For example, we ignored both SEQEM’s ability to utilize SNP information and RSEM’s ability to allow estimation on assembled transcripts by using only reference sequences. To derive the sequence set for the SEQEM compari- son, we expanded upon the procedure used in [7]. There, SEQEM was shown to improve estimation of paralogous gene expression on a set of exon sequences from 51 Homo Sapiens chromosome 1 paralogs from the HomoloGene [13] database. We extracted a larger set of sequences containing all HomoloGene paralogs in Homo Sapiens having at least one exon longer than twice the read length used that do not overlap in geno- mic coordinates. We required this minimal length because sequences were sampled from exons, and we needed to ensure enough positions existed for full length reads to be sampled from these exons. 285 such genes remained (for reads of length 35 bp), and these were the genes on which expression was tested and Heuristics/Implementation Read lengths were defined for each experiment, and simulations were repeated multiple times to account for randomness in the sampling process. Simulating data To derive artificial reads, we first estimated expression on real biological samples using one method and then Rozov et al. BMC Bioinformatics 2012, 13(Suppl 6):S2 http://www.biomedcentral.com/1471-2105/13/S6/S2 Rozov et al. BMC Bioinformatics 2012, 13(Suppl 6):S2 http://www.biomedcentral.com/1471-2105/13/S6/S2 Page 5 of 7 computed as  iPi log Pi Qi . Here P is the estimated dis- tribution generated by the tested algorithm and Q is the true distribution. Error measures were averaged over all repetitions per sample, and then over all samples. from which read sequences were derived. The SEQEM- A and SEQEM-B read sets were generated based on randomly selected exons from these genes and the expression levels from the SEQEM-A and SEQEM-B estimates taken on 20 YRI individuals. The read length of 35 bp corresponded to that of the YRI samples, and a coverage level of 20 was chosen, as this was the level at which SEQEM was shown to perform best in [7]. We performed a total of 30 repetitions of read simulations, where each repetition consisted of 20 samples (corre- sponding to the original 20 YRI samples used). computed as  iPi log Pi Qi . Here P is the estimated dis- computed as  iPi log Pi Qi . Here P is the estimated dis- tribution generated by the tested algorithm and Q is the true distribution. Error measures were averaged over all repetitions per sample, and then over all samples. tribution generated by the tested algorithm and Q is the true distribution. Error measures were averaged over all repetitions per sample, and then over all samples. Simulated data with priors based on real estimates - estimating paralogous gene expression To test performance on paralogous gene estimates, we set out to compare independent sample SEQEM esti- mates with MGMR’s common population estimates. Before applying SEQEM, we looked to address one criti- cism of it from [11], where it was suggested that SEQEM’s estimation could be improved by incorpora- tion of transcript length correction. Upon examination, we found the effect of this correction was an increase in accuracy, and thus we maintained it for subsequent tests. This improvement is documented in the appendix. For the RSEM-A and RSEM-B read sets, the transcript set used was also obtained by filtering the HomoloGene database to avoid gene overlaps, but no length filtering was required: reads were now sampled directly from transcripts which all had effective lengths greater than the read length used. 524 transcripts corresponding to 265 genes survived this filtering. For these read sets, we produced 30 repetitions of 74 samples, where each con- sisted of 100 bp reads at a coverage level of 20. In all other respects the sampling process and read generation steps were identical to those performed for the SEQEM- A and SEQEM-B read sets. With this correction in place, we estimated expression levels on the SEQEM-A and SEQEM-B read sets, apply- ing SEQEM and MGMR to each. Outputs were recorded at 1-10, 20, 30, 40, 50 and 100 iterations for MGMR and at 100 iterations for SEQEM. The results are shown in Figure 2. We observed that both error and variance levels dropped sharply within just a few itera- tions for MGMR, and converged to significantly better estimates on average than SEQEM. These trends were consistent across all three error measures [Table 1]. Variance seemed to diminish more with MGMR over time, as might be expected for a method that shares information across samples. Notably, MGMR Conclusion As shown by the 1000 Genomes and HapMap projects, one of the drives of modern genetics and bioinformatics research is to characterize variation in populations. Because of cost and time constraints, such projects have only recently become feasible. In addition to such stu- dies assessing genomic variation and its relation to dis- ease phenotypes based on DNA, it is anticipated that RNA-Seq population studies will also grow in popularity to more directly assign functional significance to variant loci by means of transcription measures. Thus, it becomes essential to accurately measure the expression levels from each individual to characterize such varia- tion. Here, we have shown that for one common study design an unexpected benefit can arise. When indivi- duals in these studies are drawn from the same popula- tion, the estimates made on each can be made more accurate because of the commonalities among popula- tion members. outperformed SEQEM on estimates for samples based on SEQEM-A, where sample estimates were obtained independently (and thus we expect the variation inher- ent in the real samples to be maintained). Error measures Accuracy was assessed by three error measures, the first two of which were applied in [7]: error rate, computed as 1 n  i |Pi −Qi| Qi , χ2 difference, computed as  iPi log Pi Qi , and Kullback-Leibler divergence, as 1 n  i |Pi −Qi| Qi , χ2 difference, computed as  Pi as 1 n  i |Pi −Qi| Qi , χ2 difference, computed as  iPi log Pi Qi , and Kullback-Leibler divergence, as 1 n  i |Pi −Qi| Qi , χ2 difference, computed as  iPi log Pi Qi , and Kullback-Leibler divergence,  iPi log Pi Qi , and Kullback-Leibler divergence, Figure 2 Relative error measured on SEQEM-A and SEQEM-B data sets. MGMR outputs on SEQEM-A and SEQEM-B initializations were compared with SEQEM up to 100 iterations. MGMR outputs were recorded at 1-10, 20, 30, 40, 50 and 100 iterations. The first few iterations have been trimmed to allow a compact presentation. Figure 2 Relative error measured on SEQEM-A and SEQEM-B data sets. MGMR outputs on SEQEM-A and SEQEM-B initializations were compared with SEQEM up to 100 iterations. MGMR outputs were recorded at 1-10, 20, 30, 40, 50 and 100 iterations. The first few iterations have been trimmed to allow a compact presentation. Rozov et al. BMC Bioinformatics 2012, 13(Suppl 6):S2 http://www.biomedcentral.com/1471-2105/13/S6/S2 Page 6 of 7 Table 3 Proportion of genes for which MGMR improves estimates on different data sets SEQEM-A SEQEM-B RSEM-A RSEM-B Proportion 104/285 78/285 126/524 173/524 % 36.5 27.3 24.0 33.0 Proportions of regions (genes for SEQEM and transcripts for RSEM, respectively) for which MGMR has lower relative error on average than each method compared to. Table 1 MGMR vs. SEQEM error at 100 iterations on SEQEM-A and SEQEM-B data sets SEQEM-A sampling SEQEM-B sampling SEQEM MGMR SEQEM MGMR Error SD Error SD Error SD Error SD E 1.27 1 * 10-2 1.03 0.14 1.50 0.70 0.82 6 * 10-3 c2 0.66 2 * 10-3 0.22 4 * 10-3 0.69 0.05 0.27 1 * 10-4 KL 0.29 7 * 10-4 0.14 1 * 10-4 0.18 2 * 10-4 0.17 1 * 10-4 These data sets were derived from SEQEM and MGMR(SEQEM) estimates, respectively, on 20 YRI samples. (E: relative error rate; c2: Chi-squared error; KL: Kullback-Liebler divergence; SD: standard deviation) Table 1 MGMR vs. Error measures SEQEM error at 100 iterations on SEQEM-A and SEQEM-B data sets Table 3 Proportion of genes for which MGMR improves estimates on different data sets Proportions of regions (genes for SEQEM and transcripts for RSEM, respectively) for which MGMR has lower relative error on average than each method compared to. These data sets were derived from SEQEM and MGMR(SEQEM) estimates, respectively, on 20 YRI samples. (E: relative error rate; c2: Chi-squared error; KL: Kullback-Liebler divergence; SD: standard deviation) List of abbreviations 2 List of abbreviations E: relative error rate; χ2: Chi-squared error; KL: Kullback-Liebler divergence; SD: standard deviation; bp: base pair Acknowledgements E.H. is a faculty fellow of the Edmond J. Safra Bioinformatics program at Tel- Aviv University. R.S. was supported in part by the European Community’s List of abbreviations E: relative error rate; χ2: Chi-squared error; KL: Kullback-Liebler divergence; SD: standard deviation; bp: base pair Simulated data with priors based on real samples - estimating transcript level expression RSEM error at 100 iterations on RSEM- A and RSEM-B data sets RSEM-A Sampling RSEM-B Sampling RSEM MGMR RSEM MGMR Error SD Error SD Error SD Error SD E 0.1 1 * 10-3 0.69 1 * 10-3 1.0 1 * 10-4 0.61 1 * 10-3 c2 0.02 6 * 10-4 1.25 0.01 0.02 9 * 10-4 0.58 3 * 10-4 KL 1.5 0.22 0.6 1 * 10-3 0.8 0.11 0.38 6 * 10-4 E: relative error rate; c2: Chi-squared error; KL: Kullback-Liebler divergence; SD: standard deviation Table 2 MGMR vs. RSEM error at 100 iterations on RSEM- A and RSEM-B data sets RSEM-A Sampling RSEM-B Sampling RSEM MGMR RSEM MGMR Error SD Error SD Error SD Error SD E 0.1 1 * 10-3 0.69 1 * 10-3 1.0 1 * 10-4 0.61 1 * 10-3 c2 0.02 6 * 10-4 1.25 0.01 0.02 9 * 10-4 0.58 3 * 10-4 KL 1.5 0.22 0.6 1 * 10-3 0.8 0.11 0.38 6 * 10-4 E: relative error rate; c2: Chi-squared error; KL: Kullback-Liebler divergence; SD: standard deviation Table 2 MGMR vs. RSEM error at 100 iterations on RSEM- A and RSEM-B data sets Simulated data with priors based on real samples - estimating transcript level expression We also sought to examine whether MGMR can improve results in the more challenging setting of esti- mating transcript level expression. Here, we expect esti- mates to be noisier due to low expression values in the real samples, and we must contend with multiread map- pings due to paralogous genes as well as to isoforms of particular genes sharing subsequences as a result of alternative splicing. In anticipation of this challenge, we used a larger set consisting of 74 sample of single-end YRI samples as the real data source and simulated 100 bp reads instead of 35 bp. This was expected to be a dif- ficult case for estimation, as all genes in the set are paralogs and many have multiple isoforms, as described in the section “Simulating data.” A shortcoming of the MGMR approach is that since it assumes commonality among the samples, outlier sam- ples will be attracted towards the common denominator, and thus appear more similar to the group profile than they really are. In particular, if the data are subject to differential expression analysis, MGMR may reduce the number of differentially expressed genes. Once expression estimation was performed on the YRI samples and read sets RSEM-A and RSEM-B were gen- erated, we again performed expression estimates with RSEM and MGMR on each set. In this case, unfortu- nately, we found the results did not exhibit a consistent trend as before and overall appeared inconclusive. These results are summarized in Table 2. It remains to be seen why the error results differ according to the level of esti- mation (gene vs. transcript) performed. We have investigated the efficacy of MGMR in tack- ling two typical experimental settings - inferring expression levels of paralogs at the gene level, and of isoforms (also drawn from a difficult set of paralogs). Although substantial gains were obtained in the first, more inquiry is required to demonstrate a benefit in the latter. It is worth noting that in each case at least a quarter of the regions considered showed improve- ment, as shown in Table 3. With these results, we sub- mit a proof of concept that population structure can aid in estimation of expression levels for RNA-Seq samples. Table 2 MGMR vs. Competing interests The authors declare that they have no competing interests. Authors’ contributions RR and EH developed the method. RS and EH designed the experiments. R implemented the method and performed experiments. RR, EH, and RS analyzed results and wrote the manuscript. All authors read and approved the final manuscript. RR and EH developed the method. RS and EH designed the experiments. RR implemented the method and performed experiments. RR, EH, and RS analyzed results and wrote the manuscript. All authors read and approved the final manuscript. Author details 1 1The Blavatnik School of Computer Science, Tel-Aviv University, Tel Aviv 69978, Israel. 2Molecular Microbiology and Biotechnology Department, Tel- Aviv University, Tel Aviv 69978, Israel. 3International Computer Science Institute, Berkeley, CA, 94704, USA. Acknowledgements Acknowledgements E.H. is a faculty fellow of the Edmond J. Safra Bioinformatics program at Tel- Aviv University. R.S. was supported in part by the European Community’s Page 7 of 7 Page 7 of 7 Rozov et al. BMC Bioinformatics 2012, 13(Suppl 6):S2 http://www.biomedcentral.com/1471-2105/13/S6/S2 Seventh Framework Programme (grant HEALTH-F4-2009-223575 for the TRIREME project) and by the Israel Science Foundation (grant 802/08). This article has been published as part of BMC Bioinformatics Volume 13 Supplement 6, 2012: Proceedings of the Second Annual RECOMB Satellite Workshop on Massively Parallel Sequencing (RECOMB-seq 2012). References 1. Montgomery SB, Sammeth M, Gutierrez-Arcelus M, Lach RP, Ingle C, Nisbett J, Guigo R, Dermitzakis ET: Transcriptome genetics using second generation sequencing in a Caucasian population. Nature 2010, 464:773-777. 1. Montgomery SB, Sammeth M, Gutierrez-Arcelus M, Lach RP, Ingle C, Nisbett J, Guigo R, Dermitzakis ET: Transcriptome genetics using second generation sequencing in a Caucasian population. Nature 2010, 464:773-777. 2. Pickrell JK, Marioni JC, Pai AA, Degner JF, Engelhardt BE, Nkadori E, Veyrieras JB, Stephens M, Gilad Y, Pritchard JK: Understanding mechanisms underlying human gene expression variation with RNA sequencing. Nature 2010, 464:768-772. 2. Pickrell JK, Marioni JC, Pai AA, Degner JF, Engelhardt BE, Nkadori E, Veyrieras JB, Stephens M, Gilad Y, Pritchard JK: Understanding mechanisms underlying human gene expression variation with RNA sequencing. Nature 2010, 464:768-772. 3. Frazer KA et al.: A second generation human haplotype map of over 3.1 million SNPs. Nature 2007, 449:851-861. 4. Robinson MD, Oshlack A: A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol 2010, 11:R25. 5. Bullard JH, Purdom E, Hansen KD, Dudoit S: Evaluation of statistical methods for normalization and differential expression in mRNA-Seq experiments. BMC Bioinformatics 2010, 11:94. 6. Oshlack A, Wakefield MJ: Transcript length bias in RNA-seq data confounds systems biology. Biol Direct 2009, 4:14. 7. Pasaniuc B, Zaitlen N, Halperin E: Accurate estimation of expression levels of homologous genes in RNA-seq experiments. J Comput Biol 2011, 18:459-468. 7. Pasaniuc B, Zaitlen N, Halperin E: Accurate estimation of expression levels of homologous genes in RNA-seq experiments. J Comput Biol 2011, 18:459-468. 8. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L: Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 2010, 28:511-515. 9. Li B, Ruotti V, Stewart RM, Thomson JA, Dewey CN: RNA-Seq gene expression estimation with read mapping uncertainty. Bioinformatics 2010, 26:493-500. 9. Li B, Ruotti V, Stewart RM, Thomson JA, Dewey CN: RNA-Seq gene expression estimation with read mapping uncertainty. Bioinformatics 2010, 26:493-500. 10. Li B, Dewey CN: RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 2011, 12:323. 11. Pachter L: Models for transcript quantification from RNA-Seq. ArXiv e- prints 2011. 12. Minka TP: Estimating a Dirichlet distribution. 2003 [http://research. microsoft.com/\~minka]. References 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: 13. [http://www.ncbi.nlm.nih.gov/homologene/]. doi:10.1186/1471-2105-13-S6-S2 Cite this article as: Rozov et al.: MGMR: leveraging RNA-Seq population data to optimize expression estimation. BMC Bioinformatics 2012 13 (Suppl 6):S2.
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On the Hasse principle for quartic hypersurfaces
Duke mathematical journal
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29,172
O. MARMON AND P. VISHE Abstract. We establish the Hasse principle for smooth projective quartic hypersurfaces of dimension greater than or equal to 28 defined over Q. 1. Introduction Let X ⊂Pn−1 Q be a quartic hypersurface corresponding to the zero locus of a homogeneous quartic polynomial F ∈Z[x1, ..., xn]. Determining whether X contains a rational point is a fundamental question in Diophantine geometry. The variety X is said to satisfy the Hasse principle if X contains a rational point provided that it contains an ad`elic point. In other words, X(AQ) ̸= ∅⇒X(Q) ̸= ∅, where X(AQ) = X(R) × Q p X(Qp) is the set of ad`elic points of X. The aim of this paper is to establish conditions on n under which X satisfies the Hasse principle. 4 4 4 4 A counterexample due to Swinnerton-Dyer [24]: F(x) = 7x4 1 + 8x4 2 −9x4 3 −14x4 4, implies that the Hasse principle cannot be expected to be true for all quartic hypersurfaces. However, this and other known counterexamples are explained by the Brauer-Manin obstruction. By a result of Colliot-Th´el`ene [21, Appendix], the Brauer-Manin obstruction is void for non- singular hypersurfaces in Pn−1 Q provided that n ⩾5. As a result, it is conjectured that the Hasse principle holds for a non-singular quartic hypersurface X defined over Q as long as n ⩾5. A long-standing result by Birch [1] shows that X(Q) ̸= ∅provided that X possesses a non-singular ad`elic point and n −dim Sing(X) ⩾50, where Sing(X) denotes the singular locus of the projective variety X. In particular, this establishes the Hasse principle for non-singular quartic hypersurfaces as soon as n ⩾49 (recall that the empty set is declared to have dimension −1). Birch in fact provides an admissible range of n for a hypersurface of arbitrary degree d, with a bound depending on d. While Birch’s result has been improved significantly in the cubic case over the years, improving upon it when d ⩾4 has turned out to be a much more formidable task. A breakthrough was achieved by Browning and Heath-Brown [2], when they reduced the lower bound for n in the quartic case from 49 to 41. Hanselmann [9] then established the case n = 40. The methodology in [2] has since been generalised by Browning and Prendiville [3], thus improving upon Birch’s bounds for every degree d ⩾5. In the special case of diagonal forms F = a1x4 1 + . . . + anx4 n with all ai ̸= 0, Vaughan [25] shows that 12 variables suffice. ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES O. MARMON AND P. VISHE as soon as P ⩾P0. In particular, this establishes the Hasse principle for non-singular quartic hypersurfaces as soon as n ⩾30, saving 10 variables over the previously best known result. We expect that a more elaborate version of our approach will allow us to save one further variable. We shall devote a follow-up paper dedicated to achieving this improvement. Adapting an idea of Hooley [14], the result can possibly be improved even further under the assumption of a generalised Riemann hypothesis for a class of Hasse-Weil L-functions. It is likely that in the vein of [4] and [5], the methods here can be generalised to obtain the Hasse principle and weak approximation in the number field and function field setting. Moreover, it is likely that the techniques will be able to generalise to the setting of homogeneous polynomials of degree d. 1.1. Key ideas. Let us briefly discuss the key ideas in the proof of Theorem 1.1. To begin with, we replace the counting function N(X, P) by the smoothed version NW (F, P) = X x∈Zn F(x)=0 W(x/P) NW (F, P) = X x∈Zn F(x)=0 W(x/P) for a suitably chosen smooth weight function W : Rn →R⩾0 with compact support. One clearly has N(X, P) ≫NW (F, P) if W is chosen appropriately. The estimation of NW (F, P) proceeds via a variant of the Hardy-Littlewood circle method. In its classical form, this begins by writing (1.1) NW (F, P) = Z 1 0 S(α)dα, (1.1) where S(α) is the generating function where S(α) is the generating function (1.2) S(α) = X x∈Zn W(x/P)e(αF(x)), (1.2) and splitting the unit interval into major and minor arcs as usual. Most modern versions of the circle method start with an application of the Poisson summation formula to estimate S(α). However, in the present setting, if F is a polynomial of degree 4 or more, the bounds for the exponential integral which emerge out of this process turn out to be too large to obtain an admissible bound for the minor arc contribution. This fundamental issue was overcome by Browning and Heath-Brown [2]. They used a point-wise van der Corput differencing to bound the exponential sum S(α) in the minor arcs. Hanselmann [9] further incorporated the averaging trick introduced by Heath-Brown [12] along with the van der Corput differencing to save an extra variable. and splitting the unit interval into major and minor arcs as usual. 1. Introduction where Sing(X) denotes the singular locus of the projective variety X. In particular, this establishes the Hasse principle for non-singular quartic hypersurfaces as soon as n ⩾49 (recall that the empty set is declared to have dimension −1). Birch in fact provides an admissible range of n for a hypersurface of arbitrary degree d, with a bound depending on d. While Birch’s result has been improved significantly in the cubic case over the years, improving upon it when d ⩾4 has turned out to be a much more formidable task. A breakthrough was achieved by Browning and Heath-Brown [2], when they reduced the lower bound for n in the quartic case from 49 to 41. Hanselmann [9] then established the case n = 40. The methodology in [2] has since been generalised by Browning and Prendiville [3], thus improving upon Birch’s bounds for every degree d ⩾5. In the special case of diagonal forms F = a1x4 1 + . . . + anx4 n with all ai ̸= 0, Vaughan [25] shows that 12 variables suffice. ̸ [ ] The main theorem of the present paper records a major improvement in the range of n for which the Hasse principle holds. Let Xns = X \ Sing(X) denote the non-singular locus of X. As in [2], our result takes the quantitative form of a lower bound for the counting function N(X, P) = #{x ∈X(Q) | H(x) ⩽P}, where H(·) is the usual height on Pn−1(Q) defined by H(x1 : ... : xn) = maxi{|xi|}, where x1, ..., xn ∈Z such that gcd(x1, ..., xn) = 1. 1 O. MARMON AND P. VISHE 2 Theorem 1.1. Let X ⊂Pn−1 Q be a quartic hypersurface satisfying n −dim Sing(X) ⩾31. Assume that Xns(AQ) ̸= ∅. Then there exist constants P0 ⩾1 and c > 0 such that N(X, P) ⩾cP n−4 as soon as P ⩾P0. pq(z) ≪N 1, pq(z) = 1 + ON (q/Q)N for |z| ⩽Q−2, pq(z) = 1 + ON (q/Q)N for |z| ⩽Q−2, for any N ⩾0. The proof of Proposition 1.2 will be carried out in Section 2. The functions pq(z) can be viewed as smooth symmetrically placed arcs around points {a/q : gcd(a, q) = 1} of an approximate length O((qQ)−1+θ). Thus, the proposition can be viewed as an exact smooth version of Kloosterman’s circle method. This reinterpretation of the delta symbol method was already implicitly a key idea in [11], [20] and [4] etc. The version stated here suppresses the dependence on the mysterious h(x, y) function appearing in the previous works completely. This is achieved by providing a finer analysis of the functions pq(z) for “small” values of q as well. Moreover, it also allows us to choose Q independent of the degree d of the polynomial F. This is crucial in our work as we choose Q = P 8/5+ε which is significantly less than the natural choice P 2 permitted by the term h(q/Q, F(x)/Q2) arising from the earlier versions. It should be noted that one may also analogously obtain bounds for the derivatives of py(z) with respect to y, which is often necessary to obtain extra cancellations in the q-sum. Given how versatile the delta symbol method has been in its applications, it is likely that our version in Proposition 1.2 will be of independent interest to the readers. Applying Proposition 1.2 to the expression Applying Proposition 1.2 to the expression NW (F, P) = X x∈Zn W(x/P)δ0(F(x)), we obtain the following corollary, which takes the place of the identity (1.1). Proposition 1.3. For any Q, P ⩾1, Proposition 1.3. For any Q, P ⩾1, NW (F, P) = Q X q=1 Z |z|<(qQ)−1+θ pq(z)S(q, z) dz + ON,θ(Q−NθP n), (1.5) (1.5) where (1.6) S(q, z) = q X∗ a=1 S(a/q + z), and pq(z) is a smooth function satisfying (1.3) and (1.4). S(q, z) = q X∗ a=1 S(a/q + z), (1.6) S(q, z) = q X∗ a=1 S(a/q + z), (1.6) and pq(z) is a smooth function satisfying (1.3) and (1.4). The success of our method relies on combining this Kloosterman type circle method with van der Corput differencing process from [2] as well as the averaging procedure in [9]. as soon as P ⩾P0. Most modern versions of the circle method start with an application of the Poisson summation formula to estimate S(α). However, in the present setting, if F is a polynomial of degree 4 or more, the bounds for the exponential integral which emerge out of this process turn out to be too large to obtain an admissible bound for the minor arc contribution. This fundamental issue was overcome by Browning and Heath-Brown [2]. They used a point-wise van der Corput differencing to bound the exponential sum S(α) in the minor arcs. Hanselmann [9] further incorporated the averaging trick introduced by Heath-Brown [12] along with the van der Corput differencing to save an extra variable. main improvement over previous results comes from achieving non-trivial cancellation verages q q X∗ a=1 S(a/q + z) q X∗ a=1 S(a/q + z) in the minor arcs, as pioneered by Kloosterman [17]. Here the ∗over the sum indicates that a and q are co-prime. Let ( in the minor arcs, as pioneered by Kloosterman [17]. Here the ∗over the sum indicates that a and q are co-prime. Let ( δ0(n) = ( 1, if n = 0, 0, otherwise, δ0(n) = ( 1, if n = 0, 0, otherwise, δ0(n) = ( 0, otherwise, ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 3 denote the delta function detecting when an integer n = 0. We begin by rewriting the delta symbol method of Duke, Friedlander and Iwaniec [7] in the following, possibly a little bit more familiar form. denote the delta function detecting when an integer n = 0. We begin by rewriting the delta symbol method of Duke, Friedlander and Iwaniec [7] in the following, possibly a little bit more familiar form. Proposition 1.2. Let Q ⩾1 and let n be an integer. Then, given any θ > 0, one has δ0(n) = Q X q=1 q X∗ a=1 Z |z|<(qQ)−1+θ pq(z)e((a/q + z)n) dz + ON,θ(Q−Nθ), where pq(z) is a smooth function satisfying where pq(z) is a smooth function satisfying where pq(z) is a smooth function satisfying (1.3) pq(z) ≪N 1, and (1.4) pq(z) = 1 + ON (q/Q)N for |z| ⩽Q−2, for any N ⩾0. (1.3) pq(z) ≪N 1, and (1.4) pq(z) = 1 + ON (q/Q)N for |z| ⩽Q−2, for any N ⩾0. as soon as P ⩾P0. We apply van der Corput differencing process to the exponential sum S(q, z) defined in (1.6) rather than to S(α). This approach still allows us to maintain the key feature of the method in [2] 4 O. MARMON AND P. VISHE with regard to the exponential integral arising from the Poisson summation. The resulting exponential sums, however, are of a different nature. In the case where q is squarefree, they may be interpreted as exponential sums on varieties over finite fields which are intersections of a quartic and a cubic hypersurface. To estimate these, Deligne type bounds due to Katz [16] come into play. For squarefull q we are able to recycle the bounds in [2] for the averages of cubic exponential sums. We also need to consider sums of such exponential sums over sparser sets, corresponding to certain dual varieties, as in [10]. The fact that these dual varieties now vary with the parameter h in van der Corput differencing process, provides an additional difficulty over the situation in [10]. 1.2. Acknowledgements. While working on this paper, the first author was supported by the Knut and Alice Wallenberg Foundation. In the course of this work, we have benefited from discussions with Tim Browning, Chris Hall, Nick Katz, Fabien Pazuki, Dan Petersen and Will Sawin. Their help is greatly appreciated. We also thank the anonymous referees for their comments. Their comments/suggestions have improved the overall exposition significantly. 2. Setup of the circle method ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 5 Upon substituting (2.1) for δ0(n) on the right hand side of the above equation, we get δ0(n) = cQ Q2 Q X q=1 q X∗ a=1 eq (an) h  q Q, n Q2  U  n Q2  + ON(Q−N). (2.2) (2.2) The truncated sum over q is due to the fact that U(n/Q2) is non-zero only when |n| < Q2, and that h(x, y) is non-zero only if x ⩽max{1, 2|y|}. Next, we use the Fourier inversion formula to write h (x, y) U (y) = Z R f(t)e(ty)dt, where h (x, y) U (y) = Z R f(t)e(ty)dt, where where f(t) = Z R h(x, z)U(z)e(−tz) dz. f(t) = Z R h(x, z)U(z)e(−tz) dz. Upon using repeated integration by parts, we get Upon using repeated integration by parts, we get |f(t)| = | Z R (−2πit)−j∂j z(h(x, z)U(z))e(−tz) dz| ≪j,N |tx|−j Z |z|<1 x−1 min  1, (|z|/x)−N dz ≪j |tx|−j, ≪j |tx|−j, for any 0 < x ⩽1. Moreover, using Lemma 4.1 of Browning and Vishe [4], which is essentially proved by repeated integration by parts together with the bounds on the derivatives of the function h(x, z), we have f(t) = Z R h(x, z)U(z)e(−tz) = 1 + ON  (1 + |t|)2N+2|x|N . Substituting this back to (2.2), we get Substituting this back to (2.2), we get δ0(n) = 1 Q2 Q X q=1 q X∗ a=1 eq (an) Z t f(t)e(tn/Q2)dt + ON(Q−N). The theorem now ensues upon the change of variable z = t/Q2 and defining pq(z) = f(Q2z) □ The theorem now ensues upon the change of variable z = t/Q2 and defining pq(z) = f(Q2z). □ By using the decay properties of functions pq(z), one may easily derive Proposition 1.2 from Lemma 2.2. To begin the circle method analysis, we consider a smooth weight function ω with a support in a very small region around a non-singular point x0 ∈X(R), a standard choice necessary to gain control over minor arc contribution. The existence of such a point is guaranteed by the assumption that X possesses a non-singular ad`elic point. p p g p To this end, let x0 ∈Rn be a point satisfying F(x0) = 0 and ∇F(x0) ̸= 0, which we will fix from now on. Without loss of generality, we can assume that ∂x1F(x0) ̸= 0. 2. Setup of the circle method We will begin by establishing the proof of Proposition 1.2 which provides a stepping stone in proving the results in this paper. We start by recalling Heath-Brown’s version [11, Thm 1] of the delta symbol method: Lemma 2.1. For any Q > 1 there is a positive constant cQ, and a smooth function h(x, y) defined on (0, ∞) × R, such that δ0(n) = cQ Q2 ∞ X q=1 q X∗ a=1 eq (an) h  q Q, n Q2  (2.1) δ0(n) = cQ Q2 ∞ X q=1 q X∗ a=1 eq (an) h  q Q, n Q2  (2.1) for n ∈Z. Here eq(x) = e2πix/q. The constant cQ satisfies cQ = 1 + ON(Q−N) for any N > 0. Furthermore, we have ∂j yh(x, y) ≪N x−1−j min  1, (|y|/x)−N for all y and j ⩾0 and h(x, y) ̸= 0 only if x ⩽max{1, 2|y|}. for n ∈Z. Here eq(x) = e2πix/q. The constant cQ satisfies cQ = 1 + ON(Q−N) for any N > 0. Furthermore, we have ∂j yh(x, y) ≪N x−1−j min  1, (|y|/x)−N for all y and j ⩾0 and h(x, y) ̸= 0 only if x ⩽max{1, 2|y|}. The following lemma provides the key in proving Proposition 1.2. The main ingredient in the proof here is a very simple yet effective trick which has appeared in a work of Munshi [20], applied to the Lemma 2.1. Lemma 2.2. Let Q ⩾1 and let n be an integer. Then δ0(n) = Q X q=1 q X∗ a=1 Z z pq(z)e((a/q + z)n) dz + ON(Q−N), where pq(z) is a smooth function satisfying where pq(z) is a smooth function satisfying pq(z) ≪N (qQz)−N, and pq(z) = 1 + ON (1 + Q2|z|)2N+2(q/Q)N . pq(z) ≪N (qQz)−N, and pq(z) = 1 + ON (1 + Q2|z|)2N+2(q/Q)N . for any N ⩾0. Proof. Let U : (−1/2, 1/2) →R be a non-negative compactly supported function satisfying R U(x)dx = 1 and U(0) = 1. The starting point of this method is the following simple observation Proof. Let U : (−1/2, 1/2) →R be a non-negative compactly supported function satisfying R U(x)dx = 1 and U(0) = 1. The starting point of this method is the following simple observation δ0(n) = δ0(n)U(n/Q2). δ0(n) = δ0(n)U(n/Q2). 2. Setup of the circle method Let γ(x) := (Q j e−1/(1−|xj|)2, if |x| < 1, 0 otherwise . otherwise . We will use the weight function (2.3) ω(x) := n Y j=1 γ(ρ−1(x −x0)), (2.3) 6 O. MARMON AND P. VISHE where ρ ∈(0, 1] is a parameter to be chosen at a later stage. With this choice of the smooth weight function, our main goal will be to establish the following asymptotic formula: where ρ ∈(0, 1] is a parameter to be chosen at a later stage. With this choice of the smooth weight function, our main goal will be to establish the following asymptotic formula: Theorem 2.3. Let F be a quartic form defined over Q satisfying n −σ ⩾31. Then Nω(F, P) = cF P n−4 + O(P n−4−δ), for a positive constant cF , provided P ≫1 and Xns(AQ) ̸= ∅. Nω(F, P) = cF P n−4 + O(P n−4−δ), for a positive constant cF , provided P ≫1 and Xns(AQ) ̸= ∅. Here, σ = dim Sing(X), where Sing(X) denotes the singular locus of the projective variety X ⊂Pn−1. As a consequence of Theorem 2.3, we have where Sing(X) denotes the singular locus of the projective variety X ⊂Pn−1. As a consequence of Theorem 2.3, we have N(F, P) ⩾Nω(F, P) ⩾cF P n−4. This would conclude the proof of Theorem 1.1. In the remaining section, we devise a strategy to establish Theorem 2.3, via key Proposition 2.7, stated later. To establish Theorem 2.3, we begin by using Proposition 1.3 with Q = P 8/5+ϕ, (2.4) Q = P 8/5+ϕ, (2.4) where 0 < ϕ < 1 is a small parameter to be chosen at a later stage. This choice of Q is concurrent with the one in [2]. It arises from balancing various terms in the bounds coming from van der Corput differencing which are useful for “large” q’s. This choice is much less than that of Birch Q = P 2, since our bounds are significantly better than those of Birch for minor arcs corresponding to large q’s. They are supplemented by bounds from Weyl differencing which are necessary for minor arcs around small q’s. 2.1. Major arcs considerations. The dominating contribution to the main term in (1.5) is expected to occur from small values of q and z. 2. Setup of the circle method More explicitly, given ∆> 0, let SM denote the contribution from the major arcs regime; (2.5) SM = X 1⩽q⩽P ∆ Z |z|⩽P −4+∆pq(z)S(q, z) dz. (2.5) If we take the major arcs to be narrow enough, then we may replace the function pq(z) inside the integral by 1, with an admissible error. For this, it will be enough to assume that If we take the major arcs to be narrow enough, then we may replace the function pq(z) inside the integral by 1, with an admissible error. For this, it will be enough to assume that ϕ + ∆ 2 ⩽2 5. (2.6) ϕ + ∆ 2 ⩽2 5. (2.6) Indeed, then |z| ⩽P −4+∆implies |z| ⩽Q−2 so that pq(z) = 1 + ON((q/Q)−N) for any N by (1.4). Thus we get Indeed, then |z| ⩽P −4+∆implies |z| ⩽Q−2 so that pq(z) = 1 + ON((q/Q)−N) for any N by (1.4). Thus we get (2.7) SM = S′ M + O(P n−N/2) = S′ M + O(P n−5), SM = S′ M + O(P n−N/2) = S′ M + O(P n−5), (2.7) say, where S′ M := X 1⩽q⩽P ∆ Z |z|⩽P −4+∆S(q, z) dz. Here and throughout the paper, we adopt the convention that when the quantity P −N appears in an estimate, that estimate is asserted for arbitrary positive integers N, and the implied constant is allowed to depend on N without mention. We may use the results from [2] to control this contribution. Here and throughout the paper, we adopt the convention that when the quantity P −N appears in an estimate, that estimate is asserted for arbitrary positive integers N, and the implied constant is allowed to depend on N without mention. We may use the results from [2] to control this contribution. ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 7 To this end, we recall the standard definition of the singular series To this end, we recall the standard definition of the singular series (2.8) S = lim R→∞S(R), where S(R) = X q⩽R 1 qn q X∗ a=1 X x (mod q) eq(aF(x)), (2.8) (2.9) I = lim R→∞I(R), where I(R) = Z R −R Z Rn ω(x)e(zF(x)) dx dz. (2.9) The contribution from the main term in (2.7) has been studied in [2, Lemma 23], which we restate here. (2.10) S(R) = S + Oψ(R−ψ) for some ψ > 0. Then I is absolutely convergent, and for any choice of ∆∈(0, 1/5) there exists δ > 0 such that 4 4 δ S′ M = SIP n−4 + Oψ(P n−4−δ). Furthermore, we have I > 0 provided that ρ is chosen small enough. The absolute convergence and positivity of S for n −σ ⩾26 is also established in [2]. Unfortunately, the power saving asymptotic formula (2.10), which is essential in employing Lemma 2.4, is established there only for n in the range n −σ ⩾42. We shall improve upon this range by refining arguments used there and proving Lemma 2.6 stated below. Before proceeding with a statement and proof of Lemma 2.6, we need a standard counting result, which is established in Lemma 2.5 below. To state this, we introduce the following notation, which will be adopted in the rest of the paper. For any q ∈N, and for any i ∈N, let (2.11) bi = Y pi||q pi, qi = Y pe||q e⩾i pe. bi = Y pi||q pi, qi = Y pe||q e⩾i pe. (2.11) Thus we have for example q = b1b2q3, q = b1b2q3, where the factor q3 is the cube-full part and b1 the square-free part of q. where the factor q3 is the cube-full part and b1 the square-free part of q. Lemma 2.5. For any positive real numbers R1, . . . , Rℓwe have the bound Lemma 2.5. For any positive real numbers R1, . . . , Rℓwe have the bound X b1∼R1,...,bℓ−1∼Rℓ−1 qℓ∼Rℓ 1 ≪ ℓY i=1 R1/i i . The proof is standard and similar to that of [2, Lemma 20], and we omit it here. Equipped with this result, we are now ready to state and prove Lemma 2.6. Lemma 2.6. If n −σ ⩾26, then the estimate S(R) = S + Oψ(R−ψ) Lemma 2.6. If n −σ ⩾26, then the estimate S(R) = S + O Lemma 2.6. If n −σ ⩾26, then the estimate S(R) = S + Oψ(R−ψ) holds for any ψ ∈(0, 1/24). holds for any ψ ∈(0, 1/24). holds for any ψ ∈(0, 1/24). Proof. The proof will follow along similar lines to that of [2, Theorem 2]. 2. Setup of the circle method The contribution from the main term in (2.7) has been studied in [2, Lemma 23], which we restate here. Lemma 2.4. Let n−σ ⩾26. Suppose that S is absolutely convergent, and satifies the estimate (2.10) S(R) = S + Oψ(R−ψ) (2.10) (2.10) The key idea here is a more refined treatment of the exponential sums arising in (2.8), when q is free of any 24-th power, as established by bounds in [2, Lemmas 7 and 25]. Fix a natural number ℓ⩾3 and O. MARMON AN b2 · · · bℓ−1qℓ. Using [2, Lemmas 7 an 1+ ( + +1)/2 O. MARMON AND P. VISHE 8 write q = b1b2 · · · bℓ−1qℓ. Using [2, Lemmas 7 and 25] along with bound [2, (6.12)], one then has write q = b1b2 · · · bℓ−1qℓ. Using [2, Lemmas 7 and 25] along with bound [2, (6.12)], one then has q X∗ a=1 X x (mod q) eq(aF(x)) ≪q1+ε(b1b2)(n+σ+1)/2b(2n+σ+1)/3 3 · · · b((ℓ−2)n+σ+1)/(ℓ−1) ℓ−1 q(23n+σ+1)/24 ℓ = qn+1+ε (b1b2)m/2bm/3 3 · · · bm/(ℓ−1) ℓ−1 qm/24 ℓ , = q (b1b2)m/2bm/3 3 · · · bm/(ℓ−1) ℓ−1 qm/24 ℓ , with m := n −σ −1. It follows that |S −S(R)| ⩽ X q⩾R q−n|Sq| ≪ X b1···bℓ−1qℓ⩾R (b1b2)−(m/2−1−ε)b−(m/3−1−ε) 3 · · · q−(m/24−1−ε) ℓ . We put ℓ= 24 and note that for any 3 ⩽k ⩽24 one has −( m k −1) ⩽−1 k We put ℓ= 24 and note that for any 3 ⩽k ⩽24 one has −( m k −1) ⩽−1 k as soon as m ⩾25. This gives as m ⩾25. This gives |S −S(R)| ≪R−1/24+2ε X b1···b23q24⩾R (b1b2)−1−εb−1/3−ε 3 · · · b−1/23−ε 23 q−1/24−ε 24 ≪R−1/24+2ε ∞ X b1,··· ,b23,q24=1 (b1b2)−1−εb−1/3−ε 3 · · · b−1/23−ε 23 q−1/24−ε 24 . Using a dyadic decomposition along with Lemma 2.5, one now concludes that the sum on the right hand side is convergent, so that the right hand side is Oε(R−1/24+ε), as required. This proves Lemma 2.6. □ Using a dyadic decomposition along with Lemma 2.5, one now concludes that the sum on the right hand side is convergent, so that the right hand side is Oε(R−1/24+ε), as required. This proves Lemma 2.6. □ Combining Lemma 2.4 with (2.7), we thus have Combining Lemma 2.4 with (2.7), we thus have (2.12) SM = cF P n−4 + O(P n−4−δ), (2.12) SM = cF P n−4 + O(P n−4−δ), SM = cF P n−4 + O(P n−4−δ), where cF depends on the parameter ρ, and cF > 0 if ρ is chosen to be small enough, assuming that Xns(AQ) ̸= ∅. (2.14) ∥F∥= the maximum modulus of the coefficients of F. (2.10) This settles our analysis of the contribution from the major arcs. 2.2. Minor arcs contribution. The main part of the paper will be devoted to showing that the remaining ranges for q, z give a negligible contribution to (1.5). More specifically, we consider the minor arcs contribution to Proposition 1.3: Sm = X 1⩽q⩽P ∆ Z P −4+∆⩽|z|⩽(qQ)−1+θ |pq(z)S(q, z)|dz θ |pq(z)S(q, z)|dz (2.13) + X P ∆⩽q⩽Q Z |z|⩽(qQ)−1+θ |pq(z)S(q, z)|dz. It should be noted that the choice of minor arcs depend on the parameter θ > 0, which arises in (1.5), as well as the choice of ∆> 0. We will sometimes write Sm = Sm,θ,∆to emphasize this dependence. Our main bound for the minor arc contribution will take the following form. It should be noted that the choice of minor arcs depend on the parameter θ > 0, which arises in (1.5), as well as the choice of ∆> 0. We will sometimes write Sm = Sm,θ,∆to emphasize this dependence. Our main bound for the minor arc contribution will take the following form. Proposition 2.7. Let F be a quartic form satisfying n −σ ⩾31. Then there is a choice of ρ ∈(0, 1] and, for any 0 < ∆< 1/5, there exists ϕ0 > 0, such that for any 0 < ϕ < ϕ0, one has Sm,θ,∆= On,∆,θ,ϕ,∥F∥(P n−4−δ). ∥ ∥ for some δ = δ(∆, n, ϕ) > 0, provided that θ ≪n,∆1. Here for some δ = δ(∆, n, ϕ) > 0, provided that θ ≪n,∆1. Here (2.14) (2.14) ∥F∥= the maximum modulus of the coefficients of F. ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 9 Here and in the following propositions, ρ is as in (2.3), and ϕ is as in (2.4) and (2.6). Fixing ∆and taking θ small enough that the conclusion of Proposition 2.7 is valid, we put together the bounds in (1.5), (2.12) and Proposition 2.7 to obtain Theorem 2.3. Thus, from now on, it is enough to concentrate on proving Proposition 2.7. 2.3. A more general minor arcs bound. The arguments used to bound the minor arcs con- tribution will not depend on the fact that F is homogeneous. We shall thus deduce the bound in Proposition 2.7 in a less restrictive setting. For a general polynomial f ∈Z[x1, . . . , xn], not necessarily homogeneous, we introduce the alternative “height function” ∥f∥P := ∥P −deg(f)f(Px1, . . (2.10) . , Pxn)∥. Suppose now that F ∈Z[x1, . . . , xn] is a quartic polynomial, not necessarily homogeneous. Let F0 be its leading form, defining a quartic hypersurface X0 ⊆Pn−1 Q . Rather than fixing the weight function in (2.3), we shall obtain a uniform estimate for an entire class Wn of weight functions. Given a positive real number c and a sequence (cj)∞ j=0 of positive real numbers, we let Wn = Wn(c), where c = (c, (cj)j) for short, be the set of infinitely differentiable functions W : Rn →R⩾0 with support inside [−c, c]n that satisfy max ( ∂j1+···+jn ∂xj1 1 · · · ∂xjn n W(x) | x ∈Rn, j1 + · · · + jn = j ) ⩽cj for all j ⩾0. In the sequel, we shall often suppress the dependence on c in our estimates. We shall assume that there is a constant M > 0 such that the following properties hold: for all j ⩾0. In the sequel, we shall often suppress the dependence on c in our estimates. We shall assume that there is a constant M > 0 such that the following properties hold: min x∈P supp(W) |∂x1F(x)| ⩾MP 3. (2.15) min x∈P supp(W) |∂x1F(x)| ⩾MP 3. (2.15) max i max x1,x2∈P supp(W) |∂xiF(x2) −∂xiF(x1)| ⩽ M 8n√ n! P 3. (2.16) (2.16) Proposition 2.8. Let F be a quartic polynomial and suppose that n −dim Sing(X0) ⩾31, where X0 ⊆Pn−1 Q is the hypersurface defined by the leading quartic form F0. Let W ∈Wn and assume further that (2.15)–(2.16) hold. Then, for any 0 < ∆< 1/5, there exists ϕ0 > 0 such that for any 0 < ϕ ⩽ϕ0, Proposition 2.8. Let F be a quartic polynomial and suppose that n −dim Sing(X0) ⩾31, where X0 ⊆Pn−1 Q is the hypersurface defined by the leading quartic form F0. Let W ∈Wn and assume further that (2.15)–(2.16) hold. Then, for any 0 < ∆< 1/5, there exists ϕ0 > 0 such that for any 0 < ϕ ⩽ϕ0, f y ϕ ⩽ϕ0, Sm = On,∆,θ,ϕ,∥F∥P ,c(P n−4−ψ) for some ψ = ψ(n, ∆, ϕ) > 0, provided that θ ≪n,∆,ϕ 1. Sm = On,∆,θ,ϕ,∥F∥P ,c(P n−4−ψ) provided that θ ≪n ∆ϕ 1. Sm = On,∆,θ,ϕ,∥F∥P ,c(P n−4−ψ) Let us verify that Proposition 2.7 follows from Proposition 2.8. (2.10) For any fixed choice of t, R appearing in the minor arcs (2.13), our final goal is to establish the bound (2.18) X R⩽q⩽2R I(q, t) ≪P n−4−ε, (2.18) which will be finally achieved in Section 10 using various estimates we derive in the following sections. which will be finally achieved in Section 10 using various estimates we derive in the following sections. (2.10) In the former, F is assumed to be homogeneous, which implies that ∥F∥P = ∥F∥. Thus it only remains to verify the conditions (2.15)–(2.16). By assumption, we have M0 = |∂x1F(x0)| > 0. By choosing ρ sufficiently small, we may ensure that none of the ∂xiF vary more than M0/(2 · 8n√ n!) on supp(ω). Since F is now a homogenenous polynomial by assumption, so are its derivatives ∂xiF. Therefore, for arbitrary y1, y2 ∈supp(ω) we have |∂xiF(Py1) −∂xiF(Py2)| = P 3|∂xiF(y1) −∂xiF(y2)| ⩽ M0 2 · 8n√ n! P 3. Furthermore, we have |∂x1F(Py1)| ⩾|∂x1F(Px0)| −P 3|∂x1F(x0) −∂x1F(y1)| ⩾M0P 3 − M0 2 · 8n√ n! P 3 ⩾M0 2 P 3, so we have verified (2.15)–(2.16) with M = M0/2, as required. 10 O MARMON AND P VISHE 10 O MARMON AND 10 O. MARMON AND P. VISHE We shall prove Proposition 2.8 by induction on dim Sing(X0), the base step being the following result for the non-singular case. Proposition 2.9. Suppose that n ⩾30 and that the leading quartic form F0 is non-singular. Let W ∈Wn and assume that (2.15)–(2.16) hold. Then, for any 0 < ∆< 1/5, there exists ϕ0 > 0 such that for any 0 < ϕ ⩽ϕ0, Sm = On,∆,ϕ,∥F∥P ,c(P n−4−ψ) for some ψ = ψ(n, ∆, ϕ) > 0, provided that θ ≪n,∆,ϕ 1. Sm = On,∆,ϕ,∥F∥P ,c(P n−4−ψ) > 0, provided that θ ≪n,∆,ϕ 1. Our efforts in the Sections 3 through 8 will culminate in the verification of Proposition 2.9 in Section 10. The inductive argument leading to Proposition 2.8 will be postponed to Section 11, as it is similar to another slicing argument that we shall need to apply in the non-singular case as well. In studying the minor arcs contribution, it will be convenient to split the integrals in (2.13) into suitable dyadic intervals. This allows us to consider the integrals I(q, t) = Z t⩽|z|⩽2t |S(q, z)|dz, (2.17) (2.17) where 1 ⩽q ⩽Q, 0 ⩽t ⩽(qQ)−1+θ. Note that we can trivially establish |I(q, t)| ≪qtP n. However, with some more work akin to Proposition 7.3, using cancellations due to an average over a in the definition (1.6) of S(q, z), for a large enough value of P, it is also possible to establish the bound b1/2 1 q2tP n, where q = b1q2, with b1 being the square-free part of q. 3. Van der Corput differencing Van der Corput differencing provides a key tool in our analysis of bounding (2.17). We will use both pointwise van der Corput differencing method in the spirit of [2] and averaged van der Corput differencing employed by Heath-Brown [12] and Hanselmann [9], the latter being more advantageous when t is not too small. A key advantage in using averaged van der Corput differencing is in the introduction of smoothed Gaussian averages in (3.7), which readily helps us establish Lemma 3.2. We begin with a brief survey of van der Corput differencing. Let f be a function on Rn supported in the set |x| ≪P and let H be a subset of Zn ∩{|x| ≪P}. The starting point of van der Corput differencing is the following identity: #H X x∈Zn f(x) = X h∈H X x∈Zn f(x + h) = X x∈Zn X h∈H f(x + h). A quick application of the Cauchy-Schwarz inequality implies #H2 X x∈Zn f(x) 2 ≪P n X h N(h) X y∈Zn f(y + h)f(y), (3.1) (3.1) where where where N(h) := #{h1, h2 ∈H : h = h1 −h2}, N(h) := #{h1, h2 ∈H : h = h1 −h2}, ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 11 and the sum over h is over h ∈Zn such that N(h) ̸= 0. (3.1) will denote a starting point for van der Corput differencing process for us. We shall apply it with f(x) being replaced by suitable exponential sums at various places in this section. al is to utilise this process to estimate the sum Our main goal is to utilise this process to estimate the sum S(q, z) = q X∗ a=1 X x∈Zn W(x/P)e((a/q + z)F(x)) introduced above, where W ∈Wn and where F is a quartic polynomial, not necessarily homogeneous. introduced above, where W ∈Wn and where F is a quartic polynomial, not necessarily homogeneous. 3.1. Pointwise van der Corput differencing. In the notation of (2.11), we put q = b1q2, where b1 is square-free and q2 square-full. We plan to benefit from an extra averaging over the sum over a (mod q) occurring in S(q, z), achieved for a fixed value of z. However, this saving is essential only from the square-free part of q. 3. Van der Corput differencing This significantly simplifies our work. Unfortunately, this treatment will not be enough to achieve n = 29 variables, and some early calculations show that a small extra saving in the sum over a in (3.2) will be vital to save one more variable. Remark. A key advantage of step (3.2) is that after an application of Poisson summation, an overall factor of size O(b1/2 1 ) can be saved via considerations of the resulting quartic exponential sums modulo the square-free part b1 of q. In the square-full case however, our sums coincide with the cubic sums in [2], allowing us to reuse those bounds. See Lemmas 4.2 and 4.4 in the following section for a more explicit version. This comment also applies to our bounds in Section 3.2 below. This significantly simplifies our work. Unfortunately, this treatment will not be enough to achieve n = 29 variables, and some early calculations show that a small extra saving in the sum over a in (3.2) will be vital to save one more variable. 3.2. Averaged van der Corput differencing. We next incorporate an averaging over z to estimate the integrals I(q, t) defined in (2.17). At this stage, we shall impose the conditions (2.15)–(2.16) occuring in Proposition 2.8. Choose an arbitrary point x ∈P supp(W). By (2.15), |∂x1F(x)| ⩾MP 3. By (3.2), we have I(q, t) ≪ q2 X∗ a=1 Z t⩽|z|⩽2t |Sa(q, z)|dz. I(q, t) ≪ q2 X∗ a=1 Z t⩽|z|⩽2t |S Let H = H(q, t) be an integer with 1 ⩽H ⩽P. We may choose a set T of cardinality O(1 + tHP 3) such that Let H = H(q, t) be an integer with 1 ⩽H ⩽P. We may choose a set T of cardinality O(1 + tHP 3) such that [t, 2t] ∪[−2t, −t] ⊆ [ τ∈T  τ −(HP 3)−1, τ + (HP 3)−1 . Then we can write I(q, t) ≪ q2 X∗ a=1 X τ∈T Z |τ−z|<(HP 3)−1 |Sa(q, z)|dz. An application of the Cauchy-Schwarz inequality next implies Z |τ−z|<(HP 3)−1 |Sa(q, z)|dz ≪(HP 3)−1/2 Z |τ−z|<(HP 3)−1 |Sa(q, z)|2 dz !1/2 . Inserting this into the above bounds, we may write I(q, t) ≪(HP 3)−1/2 q2 X∗ a=1 X τ∈T Ma(q, τ)1/2, where where (3.7) Ma(q, τ) := Z |τ−z|<(HP 3)−1 |Sa(q, z)|2dz ≪ Z ∞ −∞ exp(−H2P 6(τ −z)2)|Sa(q, z)|2 dz. 3. Van der Corput differencing To this end, we shall sum trivially over the square-full part of q2 of q: |S(q, z)| ⩽ q2 X∗ a=1 |Sa(q, z)|, (3.2) (3.2) (3.3) Sa(q, z) := b1 X∗ s=1 X x∈Zn W(x/P)e((s/b1 + a/q2 + z)F(x)). (3.3) We now apply van der Corput differencing (3.1) to the function f(x) = b1 X∗ s=1 W(x/P)e((s/b1 + a/q2 + z)F(x)), to bound |Sa(q, z)| for any fixed a: |Sa(q, z)|2 ≪#H−2P n X h N(h) X y∈Zn f(y + h)f(y) = #H−2P n X h N(h)Ta,h(q, z), (3.4) where o bound |Sa(q, z)| for any fixed a: |Sa(q, z)|2 ≪#H−2P n X h N(h) X y∈Zn f(y + h)f(y) = #H−2P n X h N(h)Ta,h(q, z) 3.4) where where (3.5) Ta,h(q, z) := X x∈Zn Wh(x/P) b1 X∗ s1,s2=1 eb1 s1Fh(x) + (s1 −s2)F(x)  eq2 aFh(x)  e(zFh(x)), with Wh(y) = W(y + P −1h)W(y) and Fh(x) = F(x + h) −F(x). Wh(y) = W(y + P −1h)W(y) and Fh(x) = F(x + h) −F(x). Wh(y) = W(y + P −1h)W(y) and Fh(x) = F(x + h) −F(x). Note that we may trivially bound |Ta,h(q, z)| ≪P nb2. We now let H = H(q, z) be an integer with 1 ⩽H ⩽P and put Note that we may trivially bound |Ta,h(q, z)| ≪P nb2. We now let H = H(q, z) be an integer with 1 ⩽H ⩽P and put (3.6) H := {h ∈Nn | 1 ⩽h1, ..., hn ⩽H}. (3.6) Then the bounds (3.2) and (3.4), along with the trivial bound N(h) ≪#H, imply the following result, which we refer to as the pointwise van der Corput bound with respect to z. Lemma 3.1. We have the bound Lemma 3.1. We have the bound |S(q, z)| ≪P n/2H−n/2 q2 X∗ a=1  X |h|⩽H |Ta,h(q, z)|   1/2 . O. MARMON AND P. VISHE 12 Remark. A key advantage of step (3.2) is that after an application of Poisson summation, an overall factor of size O(b1/2 1 ) can be saved via considerations of the resulting quartic exponential sums modulo the square-free part b1 of q. In the square-full case however, our sums coincide with the cubic sums in [2], allowing us to reuse those bounds. See Lemmas 4.2 and 4.4 in the following section for a more explicit version. This comment also applies to our bounds in Section 3.2 below. 3. Van der Corput differencing We now employ van der Corput differencing (3.4) with a modified differencing set. Let 1 ⩾c1 > 0 be a constant to be determined later, and put (3.8) H1 := {h ∈Nn | 1 ⩽h1 ⩽c1P, 1 ⩽h2, ..., hn ⩽H}. (3.8) H1 := {h ∈Nn | 1 ⩽h1 ⩽c1P, 1 ⩽h2, ..., hn ⩽H}. (3.8) to obtain to obtain Ma(q, τ) ≪#H−1 1 P n X h∈H1 Z ∞ −∞ exp(−H2P 6(τ −z)2)Ta,h(q, z) dz, (3.9) (3.9) with Ta,h as in (3.5). We have again used the trivial bound N(h) ≪#H1 here. We now set L := log P and use the lower bound |∂x1(F(x))| ⩾MP 3 for all x ∈P supp(W), to obtain that with Ta,h as in (3.5). We have again used the trivial bound N(h) ≪#H1 here. We now set L := log P and use the lower bound |∂x1(F(x))| ⩾MP 3 for all x ∈P supp(W), to obtain that ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 1 ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 13 the contribution in the above sum from the terms corresponding to h satisfying |h1| ≫HL is negligible, as derived in the following lemma. Lemma 3.2. Suppose that (2.15) holds. Then there is a constant c > 0 such that for any h ∈H1 such that |h1| ⩾cHL, we have Z ∞ −∞ exp(−H2P 6(τ −z)2)Ta,h(q, z) dz ≪N P −N. Proof. We follow the strategy in [12, Section 4] and [9, Section 5]. The parameter a is consid- ered fixed for now. Using (3.5) we have Proof. We follow the strategy in [12, Section 4] and [9, Section 5]. The parameter a is consid- ered fixed for now. Using (3.5) we have (3.10) Z ∞ −∞ exp(−H2P 6(τ −z)2)Ta,h(q, z) dz = X x∈Zn Wh(x/P)I(h, x), (3.10) where where I(h, x) = b1 X∗ s1,s2=1 eb1 s1Fh(x) + (s1 −s2)F(x)  eq2 aFh(x)  I(h, x) = b1 X∗ s1,s2=1 eb1 s1Fh(x) + (s1 −s2)F(x)  eq2 aFh(x)  × Z ∞ −∞ exp(−H2P 6(τ −z)2)e(zFh(x)) dz. × Z ∞ −∞ exp(−H2P 6(τ −z)2)e(zFh(x)) dz. × Z ∞ −∞ exp(−H2P 6(τ −z)2)e(zFh(x)) dz Calculating the inner integral explicitly, we may write I(h, x) = √π HP 3 exp  − π2 H2P 6 |Fh(x)|2  e(−τFh(x)) × b1 X∗ s1,s2=1 eb s1Fh(x) + (s1 −s2)F(x)  eq2 aFh(x)  . 3. Van der Corput differencing I(h, x) = √π HP 3 exp  − π2 H2P 6 |Fh(x)|2  e(−τFh(x)) × b1 X∗ s1,s2=1 eb s1Fh(x) + (s1 −s2)F(x)  eq2 aFh(x)  . s1,s2=1 We now claim that c can be chosen in such a way that if h ∈H1 satisfies |h1| ⩾cHL, then we have We now claim that c can be chosen in such a way that if h ∈H1 satisfies |h1| ⩾cHL, then we have (3.11) |Fh(x)| ≫HP 3L |Fh(x)| ≫HP 3L (3.11) for any x occurring in (3.10) with Wh(x/P) ̸= 0. If this is true, then for such values of h and x we have 2 |I(h, x)| ≪ q2 HP 3 exp(−c′(log P)2) the quantity in (3.10) is ≪P n+1 exp(−c′(log P)2) ≪P −N, |I(h, x)| ≪ q2 HP 3 exp(−c′(log P)2) for some constant c′ > 0, so the quantity in (3.10) is ≪P n+1 exp(−c′(log P)2) ≪P −N, q It remains to verify the above claim. For this we write Fh(x) = ∂F ∂x1 (x) · h1 + O(HP 3) + O(h2 1P 2), where the implied constants depend on ∥F∥P only (but not on ∥F∥). By (2.15), we have |∂x1F(x)| ⩾MP 3, yielding |Fh(x)| ⩾|h1|P 2 (MP −O(|h1|)) −O(HP 3). We may thus choose c1 in the definition (3.8) so that |Fh(x)| ⩾|h1|MP 3 2 −O(HP 3), We may thus choose c1 in the definition (3.8) so that |Fh(x)| ⩾|h1|MP 3 2 −O(HP 3), O. MARMON AND P. VISHE 14 say, for relevant values of x. This clearly gives the desired bound (3.11) as soon as |h1| ≫ HL. □ Using Lemma 3.2, and noting that the contribution from the range |τ −z| ⩾L(HP 3)−1 is also O(P −N), we get the following bound for (3.7): Ma(q, τ) ≪P −N + P n−1 Hn−1 Z |τ−z|<L(HP 3)−1  X |h|⩽HL Ta,h(q, z)  dz ≪P −N + L HP 3 P n−1 Hn−1 max |τ−z|<L(HP 3)−1 X |h|⩽HL |Ta,h(q, z)|. Thus we have proved the following result. Thus we have proved the following result. Lemma 3.3. For any 1 ⩽H ⩽P, we have mma 3.3. For any 1 ⩽H ⩽P, we have Lemma 3.3. 3. Van der Corput differencing For any 1 ⩽H ⩽P, we have I(q, t) ≪P −N + P ε  t + 1 HP 3   P H (n−1)/2 q2 X∗ a=1 max z (1)    X |h|⩽HP ε |Ta,h(q, z)|    1/2 , where max(1) is taken over t ⩽|z| ⩽max  2t, t + 1 HP 3−ε . (4.1) and that max{∥f∥P , ∥g∥P } ⩽H max{∥f∥P , ∥g∥P } ⩽H for some H ⩾1. Given a weight function w ∈Wn and a fixed integer a relatively prime to q we define for some H ⩾1. Given a weight function w ∈Wn and a fixed integer a relatively prime to q, we define (4.2) T (q, z) = Tn(a, q, z; f, g, w, P) := X x∈Zn w(x/P) b1 X∗ s1,s2=1 eb1 s1g(x) + (s1 −s2)f(x)  eq2 ag(x)  e(zg(x)). (4.2) T (q, z) = Tn(a, q, z; f, g, w, P) (4.2) T (q, z) = Tn(a, q, z; f, g, w, P) := X x∈Zn w(x/P) b1 X∗ s1,s2=1 eb1 s1g(x) + (s1 −s2)f(x)  eq2 ag(x)  e(zg(x)). For the result in this paper, we would only need to this result with d1 = 4, d2 = 3. However, we consider general degrees d1 and d2 since these bounds will be useful in bounding the contribution from the square-free part of q in subsequent applications to higher degree forms. We can also obtain them here without too much extra work. From Section 6 onwards however, we will specialise to the case d1 = 4, d2 = 3. 4.1. Hyperplane intersection: main lemma. Here, we will deviate slightly from our need for bounding (4.2), to consider a question of handling singularities of an arbitrary finite set of varieties, via hyperplane intersections. This is achieved by Lemma 4.1 below. In this paper, it will only be necessary to simultaneously bound singular locus of a system of a cubic and a quartic form and of their complete intersection. However, a general result can be obtained with not much more extra work. This lemma will be instrumental in establishing an important bound obtained in Proposition 8.1. Moreover, the extra condition that e1 can be chosen in a certain way, mentioned at the end of Lemma 4.1 will be useful in Section 11. To this end, we introduce some notation, which will be used throughout this paper. We use the symbol v to denote a place of Q, that is, v = ∞or v = p for a prime p. Given the forms F1, . . . , Fm ∈Z[x1, . . . , xn], we then denote by Vv(F1, . . . where max(1) is taken over t ⩽|z| ⩽max  2t, t + 1 HP 3−ε . where max(1) is taken over t ⩽|z| ⩽max  2t, t + 1 HP 3−ε . We note here that as a standard feature in applications of van der Corput differencing, H will eventually be chosen in order to obtain the bound (2.18). A minimum value of H is necessary to make the contribution from h = 0 work. However, choosing larger values of H increases the contribution from generic values of h’s. Lemma 3.3 will be crucial in dealing with the case when q, t are both large. We shall also state a corresponding result that is derived in the vein of the “classical” circle method used in [2]. Here, a typical minor arcs contribution takes the shape I(q, t) ⩽ Z t⩽|z|⩽2t q X∗ a=1 |S(a/q + z)| dz. An easy modification of the arguments above give us the following estimate, which in essence is contained in Hanselmann’s treatment [9, (5.11)]: Lemma 3.4. For any 1 ⩽H ⩽P, we have Lemma 3.4. For any 1 ⩽H ⩽P, we have Lemma 3.4. For any 1 ⩽H ⩽P, we have I(q, t) ≪P −N + P ε  t + 1 HP 3   P H (n−1)/2 q X∗ a=1 max z (1)    X |h|⩽HP ε |Sh(a/q + z)|    1/2 , where max(1) is defined as above and (3.12) Sh(α) := X x∈Zn w(x/P)e(αFh(x)). (3.12) A main difference here from Lemma 3.3 is that the sum (3.12) is a purely cubic exponential sum only containing the form Fh, as opposed to a mixture of cubic and quartic exponential sums (3.5), appearing in Lemma 3.3. In Section 9, we will apply Weyl bound directly to estimate Sh(α), via [3, Lemma 3.3]. This bound will turn out to be important for us. It will be used to supplement bounds from Lemma 3.3 and those from pure Weyl differencing. ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 15 4. Evaluation of quartic exponential sums; initial considerations 4. Evaluation of quartic exponential sums; initial considerations Our calculations above led us to the consideration of the exponential sums Ta,h(q, z). Here, we shall investigate a more general version of that sum. Let f, g ∈Z[x1, . . . , xn] be polynomials and suppose that deg(f) = d1, deg(g) = d2, where d1 ⩾d2 ⩾2, deg(f) = d1, deg(g) = d2, where d1 ⩾d2 ⩾2, (4.1) Moreover, the basis vectors ei may be chosen so that (4.3) L/2 ⩽|ei| ⩽L dim Sing (XI ∩Λη)v  = max{−1, dim Sing (XI)v  −η}. dim Sing (XI ∩Λη)v  = max{−1, dim Sing (XI)v  −η}. Moreover, the basis vectors ei may be chosen so that dim Sing (XI ∩Λη)v  = max{−1, dim Sing (XI)v  −η}. Moreover, the basis vectors ei may be chosen so that (4.3) L/2 ⩽|ei| ⩽L g ( I η)v  { , g ( I)v  η} Moreover, the basis vectors ei may be chosen so that (4.3) for all i = 1, . . . , n and Ln ≪det(e1, . . . , en) ≪Ln Ln ≪det(e1, . . . , en) ≪Ln for a constant L = On,d1,...,dm(r + 1). , 1, , m In addition, given any vector f ∈Rn, vector e1 in the basis above can be chosen such that it makes an angle of at most π/3 with f. Proof. First we give a short outline of the proof. We shall represent the collection of vectors ei by the matrix E having the ei as columns. There are now two main points to our argument. First we argue that each condition (⋆)I,η,v is implied by the non-vanishing of some polynomial, over Fv, in the entries ei,j. On the other hand, a positive proportion of all matrices E correspond to bases with the desired properties (4.3)–(4.4). We begin by noting that the case r = 0 follows from the case r = 1, so we may assume that r ⩾1, so that Π is non-empty. For fixed I, η and v, it is a consequence of Bertini’s theorem, as observed by Ghorpade and Lachaud [8, Prop. 1.3], that the set Uη,I of subspaces Λ satisfying the condition (⋆)I,η,v contains a Zariski-open subset of the Grassmannian Gn−η := G(n −1 − η, n −1). (Note that for those places v where the forms Fi, i ∈I, do not intersect completely, the condition is void.) Recall that Gn−η is a closed subvariety of PN Fv for a certain N = N(n, η) by virtue of the Pl¨ucker embedding. By an argument very similar to the proof of [18, Lemma 2.8], one sees that one may in fact find a hypersurface Zη,I ⊂PN of degree On,d1,...,dr(1) independent of v, such that Uη,I contains the complement of Zη,I in Gn−η. (4.1) , Fm) the closed subvariety of Pn−1 Fv defined by the image of the ideal ⟨F1, . . . , Fm⟩in Fv[x1, . . . , xn], and let sv(F1, . . . , Fm) := dim(Sing(Vv(F1, . . . , Fm))), δv(F1, . . . , Fm) := dim(Vv(F1, . . . , Fm)). We will consider the aim of simultaneously reducing the dimension of the singular locus of a system of forms F1, ..., Fm, their complete intersection, and the complete intersection of any sub-collection {Fi : i ∈I} for any subset I ⊆{1, ..., m}. Given a collection of primes Π, Lemma 4.1 presents us with a nice lattice basis, which in turn give us an entire chain of subspaces which successively achieve this aim. Lemma 4.1. Let F1, . . . , Fm be forms of degree d1, . . . , dm defining a complete intersection X = V (F1, . . . , Fm) ⊂Pn−1 Q of dimension n −1 −m. Let Π be a collection of primes, with #Π = r ⩾0, and write Πa := {p ∈Π | p > a} for each a ∈N. There is a constant c = c(n, d1, . . . , dm) and a collection of primitive linearly independent integer vectors e1, . . . , en ∈Zn e1, . . . , en ∈Zn O. MARMON AND P. VISHE 16 satisfying the following property for any integer 0 ⩽η ⩽n−1, any subset ∅̸= I ⊆{1, . . . , m} and any v ∈{∞} ∪Πcr: satisfying the following property for any integer 0 ⩽η ⩽n−1, any subset ∅̸= I ⊆{1, . . . , m} and any v ∈{∞} ∪Πcr: (⋆)I,η,v Provided that the closed subscheme XI ⊂Pn−1 Z defined by Fi = 0 for all i ∈I satisfies dim(XI)v = n −1 −|I|, the subspace Λη ⊂Pn−1 Fv spanned by the images of e1, . . . , en−η is such that (⋆)I,η,v Provided that the closed subscheme XI ⊂Pn−1 Z defined by Fi = 0 for all i ∈I satisfies dim(XI)v = n −1 −|I|, the subspace Λη ⊂Pn−1 Fv spanned by the images of e1, . . . , en−η is such that dim(XI ∩Λη)v = max{−1, dim(XI)v −η} and and dim Sing (XI ∩Λη)v  = max{−1, dim Sing (XI)v  −η}. (4.1) We conclude that there is a constant C1 = C1(n) such that We conclude that there is a constant C1 = C1(n) such that #M∗(B) ⩾C1Bn2. reover, it is evident that each matrix in M∗(B) satisfies (4.3) and (4.4). Moreover, it is evident that each matrix in M∗(B) satisfies (4.3) and (4.4). Let W be the set of matrices E ∈Mn×n(Z) with entries in [−B, B] for which the column vectors fail the condition (⋆)I,η,v for some choice of I, η and v ∈{∞} ∪Πa, where a is yet to be chosen. Our aim will be to show that W has cardinality strictly less than C1Bn2, implying the existence of a basis with the desired property. 2 Fix a place v, and let us identify n × n-matrices with points in An2. Let S be the closed subset of An2 defined by det(E) = 0. For each η, an appropriate Pl¨ucker map Φη : An2 \ S → Gn−η ×Gη maps An2 \S onto an affine open subset of Gn−η ×Gη, and we may define W = Wv to be the union of the closures in An2 of the inverse images Φ−1 η (Zη) of the subvarieties Zη introduced above. Then a matrix E lies in W only if either E ∈W∞or [E]p ∈Wp(Fp) for some p ∈Πa, where [E]p denotes the matrix where each entry is the reduction (mod p) of the corresponding entry of E. By [2, Lemma 4], the number of matrices E such that |ei,j| ⩽B for all i, j, and such that either E ∈W∞or [E]p ∈Wp(Fp) for a prime p is ⩽C2(Bn2p−1 + Bn2−1) for some constant C2 = C2(n, d1, . . . , dm). The number of matrices E satisfying this condition for at least one prime p ∈Πa is thus for some constant C2 = C2(n, d1, . . . , dm). The number of matrices E satisfying this condition for at least one prime p ∈Πa is thus ⩽C2  Bn X p∈Πa p−1 + rBn−1  ⩽C2r a Bn2 + C2rBn2−1. Choosing c = 4C2/C1 and a = cr = 4C2r/C1, this is ⩽1 2C1Bn2 Choosing c = 4C2/C1 and a = cr = 4C2r/C1, this is Choosing c = 4C2/C1 and a = cr = 4C2r/C1, this is ⩽1 2C1Bn2 as soon as B ⩽a, which is what we wanted to prove. (4.1) Consequently, the complement of the hypersurface Zη = S I Zη,I is contained in Uη = T I Uη,I. The hypersurface Zη embeds as a hypersurface in Gn−η × Gη under the natural map Gn−η ,→Gn−η × Gη. η η η η η η Define M(B), for any B > 0, to be the set of matrices E = (ei,j) ∈Mn×n(R) such that each column vector ej = (e1,j, . . . , en,j) satisfies B/2 ⩽∥ej∥⩽B. (Here, we use ∥· ∥to denote the usual Euclidean norm on Rn.) For any ψ ∈(0, π/2], let Mψ(B) be the set of E ∈M(B) such that each column vector ej makes an angle of at least ψ with the hyperplane spanned by the remaining vectors ek, k ̸= j. It is clear that the set Mψ(B) has a well-defined and positive volume, that vol(M(B)) = Bn2 vol(M) and vol(Mψ(B)) = Bn2 vol(Mψ), where M := M(1) and Mψ := Mψ(1), and that vol(Mψ) ⩾vol(M) −n vol(Nψ), vol(Mψ) ⩾vol(M) −n vol(Nψ), say, where Nψ is the set of E ∈M where the vector en is either the zero vector or makes an angle of less than ψ with the hyperplane spanned by the first n −1 column vectors. Now one has say, where Nψ is the set of E ∈M where the vector en is either the zero vector or makes an angle of less than ψ with the hyperplane spanned by the first n −1 column vectors. Now one vol(Nψ) ≪n ψ vol(M), ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 17 so choosing ψ small enough, we may conclude that there exists a constant c1 = c1(n) such that so choosing ψ small enough, we may conclude that there exists a constant c1 = c1(n) such that vol(Mψ) ⩾c1 vol(M). vol(Mψ) ⩾c1 vol(M). vol(Mψ) ⩾c1 vol(M). vol(Mψ) ⩾c1 vol(M). If we now put M(B) = Mψ(B) ∩Mn×n(Z), then it follows from the Lipschitz principle [6] that #M(B) = Bn2 vol(Mψ) + On(Bn2−1). (4.5) #M(B) = Bn2 vol(Mψ) + On(Bn2−1). (4.5) Letting M∗(B) denote the set of matrices E ∈M(B) for which gcd(e1,j, . . . , en,j) = 1 for j = 1, . . . , n, one may deduce from (4.5), using standard M¨obius inversion arguments, that #M∗(B) = Bn2 vol(Mψ) ζ(n)n + On(Bn2−1). (4.1) We have thus established the assertion in the first part of the lemma, where we may take L ≈cr. as soon as B ⩽a, which is what we wanted to prove. We have thus established the assertion in the first part of the lemma, where we may take L ≈cr. Now to prove the last condition, we now want to choose the vectors e1, . . . , en so that the vector e1 makes an angle of at most π/3, say, to the fixed vector f. Recall the sets M(B), Mψ(B) considered before, and define Mθ(B), Mθ ψ(B) to be the set of matrices E in M(B) or Mψ(B), respectively, such that the first column vector e1 makes an angle of at most θ with the vector f. Let Mθ = Mθ(1) and Mθ ψ = Mθ ψ(1). Since Mψ is invariant under O. MARMON AND P. VISHE 18 rotations, one sees that for any 0 < θ ⩽π/2, the set Mθ ψ has a well-defined and positive volume and that rotations, one sees that for any 0 < θ ⩽π/2, the set Mθ ψ has a well-defined and positive volume and that vol(Mθ ψ) ⩾vol(Mθ) −n vol(Nψ). One may now calculate that vol(Mθ) ≫n θ2 vol(M), so upon replacing the constant c1(n) in the proof of Lemma 4.1 by a smaller yet positive constant, one may replace Mψ(B) by Mπ/3 ψ (B) in the definition of the sets M(B) and M∗(B), and still conclude that there exists a matrix E ∈M∗(B) with the desired properties, for B = L large enough. □ Let us compare Lemma 4.1 with [2, Lemma 5], which specialises to the case of one form G. An argument akin to Bertini’s theorem was used in [2, Lemma 5] to find a primitive integer vector m such that intersecting with the hyperplane m.x = 0 lowers the dimension of the singular locus of Vv(G) for v ∈Π∪∞. In addition, at each stage, m could be chosen in a nice way. Lemma 4.1 can be seen a a generalisation of [2, Lemma 5]. Being able to deal with an arbitrary finite collection of forms of various degrees will be crucial in dealing with systems of forms of higher degree. We end this analysis by explaining how Lemma 4.1 will be used to bound the sums T (q, z). (4.1) For f, g as defined at the beginning of this section, above we let F(x0, . . . , xn) := xdeg(f) 0 f( x1 x0 , . . . , xn x0 ) and G(x0, . . . , xn) := xdeg(g) 0 g( x1 x0 , . . . , xn x0 ) be their homogenisations and be their homogenisations and F0(x1, . . . , xn) := F(0, x1, . . . , xn), G0(x1, . . . , xn) := G(0, x1, . . . , xn) the leading forms of f and g. Now, if n ⩾2, we define (4.6) s′ v = s′ v(f, g) := max {sv(F0), sv(G0), sv(F0, G0)} , F0(x1, . . . , xn) := F(0, x1, . . . , xn), G0(x1, . . . , xn) := G(0, x1, . . . , xn) the leading forms of f and g. Now, if n ⩾2, we define (4.6) s′ v = s′ v(f, g) := max {sv(F0), sv(G0), sv(F0, G0)} , (4.6) s′ v = s′ v(f, g) := max {sv(F0), sv(G0), sv(F0, G0)} , (4.6) provided that δv(F0) = δv(G0) = δv(F0, G0) + 1 = n −2, and s′ v = n−1 otherwise. Note that in this definition, Vv(G0) and Vv(F0, G0) are considered as subvarieties of Pn−1. Let us give an example of how Lemma 4.1 will be used: The exponential sum T (q, z) can be most efficiently estimated in the non-singular case, that is, when s′ ∞= −1. Therefore, when s′ ∞̸= −1, we will employ Lemma 4.1. 4.2. Bounding T (q, z). The starting point for our investigation of T (q, z) is an application of the Poisson summation formula, a standard technique. The proof follows from a minor modification of [2, Lemma 8] and we will skip it here. Lemma 4.2. We have T (q, z) = q−n X v∈Zn S(q, v)I(z, q−1v), where where S(q, v) := b1 X∗ s1,s2=1 X a (mod q) eb1 s1g(a) + (s1 −s2)f(a)  eq2(ag(a))eq(v.a) and I(z, v) = Z w(x/P)e(zg(x) −v.x)dx. ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 19 A key feature in our approach is that, when applying Lemma 4.2 in the case where f is a quartic polynomial and g a cubic polynomial, only the cubic polynomial g occurs in the exponential integral, allowing us to use [2, Lemma 9]. (4.1) Upon an application of [2, Lemma 9] we get the following estimate. Proposition 4.3. Suppose that d1 = 4 and d2 = 3. Then we have the estimate Proposition 4.3. Suppose that d1 = 4 and d2 = 3. Then we have the estimate T (q, z) ≪P −N + q−nP n max v0 X |v−v0|⩽P εV |S(q, v)| Proposition 4.3. Suppose that d1 = 4 and d2 = 3. Then we have the estimate T (q, z) ≪P −N + q−nP n max v0 X |v−v0|⩽P εV |S(q, v)| 3. Suppose that d1 = 4 and d2 = 3. Then we have the estimate T (q, z) ≪P −N + q−nP n max v0 X |v−v0|⩽P εV |S(q, v)| T (q, z) ≪P −N + q−nP n max v0 X |v−v0|⩽P εV |S(q, v)| where V = qP −1 max{1, (HP 3|z|)1/2}. (4.7) V = qP −1 max{1, (HP 3|z|)1/2}. (4.7) The following observation from [2, Lemma 10] will be useful for establishing multiplicativity relations for our exponential sums. Lemma 4.4. Let r, s be integers with (r, s) = 1, and let r, s be integers satisfying rr + ss = 1. Then, for any polynomial h ∈Z[x1, . . . , xn], and any x, y ∈Zn, we have h(rrx + ssy) ≡rrh(x) + ssh(y) (mod rs). Moreover, for any rational function R(x) = h1(x) h2(x), where h1, h2 ∈Z[x1, . . . , xn], we have R(rrx + ssy) ≡rrR(x) + ssR(y) (mod rs), provided that h2(x), h2(y) and h2(rrx + ssy) are invertible in Z/rsZ. provided that h2(x), h2(y) and h2(rrx + ssy) are invertible in Z/rsZ. Proof. The above statements are consequences of the fact that the map ϕ : Z/rsZ × Z/rsZ →Z/rsZ, (x, y) 7→rrx + ssy ϕ : Z/rsZ × Z/rsZ →Z/rsZ, (x, y) 7→rrx + ssy ϕ : Z/rsZ × Z/rsZ →Z/rsZ, (x, y) 7→rrx + ssy is a ring homomorphism, which is easily verifiable. □ □ Now we may prove the following multiplicativity property for the exponential sums: Now we may prove the following multiplicativity property for the exponential sums: Lemma 4.5. Let b1, q2 be integers such that b1b1 + q2q2 = 1. (4.1) Now, if we put Uv(s1, s2, x) = s1g(x) + (s1 −s2)f(x) + v.x, then Lemma 4.4 gives eq(Uv(s1, s2, x)) = er(sUv(s′ 1, s′ 2, x))es(rUv(s′′ 1, s′′ 2, x)), which establishes (4.11). eq(Uv(s1, s2, x)) = er(sUv(s′ 1, s′ 2, x))es(rUv(s′′ 1, s′′ 2, x)), eq(Uv(s1, s2, x)) = er(sUv(s′ 1, s′ 2, x))es(rUv(s′′ 1, s′′ 2, x)), hi h bli h (4 11) which establishes (4.11). ( ) The multiplicativity relation (4.12), finally, is precisely the one given in [2, Lemma 10]. p y ( ), y, p y g [ , ] We end this section by introducing a quantity that will appear in our estimates for the exponential sums T (q, z). For q = b1q2, define (4.14) D(q) = Df,g(q) = n Y i=1 Y p|b1 s′ p(f,g)=i−1 pi/2 Y p|q2 s′ p(f,g)=i−1 pi, (4.14) provided that n ⩾2. If n = 1, we instead define D(q) to be the product of all primes p such that G0 vanishes identically (mod p), that is D(q) = Y p|(q,cont(G0)) p. provided that n ⩾2. If n = 1, we instead define D(q) to be the product of all primes p such that G0 vanishes identically (mod p), that is D(q) = Y p|(q,cont(G0)) p. D(q) = Y p|(q,cont(G0)) p. (4.15) cont(G0) = gcd of all the coefficients of G0. cont(G0) = gcd of all the coefficients of G0. (4.15) (4.1) Then we have (4.8) S(q, v) = T(b1, q2v)T ∗(q2, b1v), T(q, v) = T(q, v; f, g) = X x (mod q) q X∗ s1,s2=1 eq(s1g(x) + (s1 −s2)f(x) + v.x) (4.9) (4.10) T ∗(q, v) = T ∗ a (q, v; g) := X x (mod q) eq ag(x) + v.x  for any q ∈N. Moreover, if r, s are coprime integers and r, s are integers such that rr+ss = 1, then for any q ∈N. Moreover, if r, s are coprime integers and r, s are integers such that rr+ss = 1, then for any q ∈N. Moreover, if r, s are coprime integers and r, s are integers such that rr+s then (4.11) T(rs, v; f, g) = T(r, sv; sf, sg)T(s, rv; rf, rg) and (4.11) T(rs, v; f, g) = T(r, sv; sf, sg)T(s, rv; rf, rg) T ∗ a (rs, v) = T ∗ sa(r, sv)T ∗ ra(s, rv). (4.12) O. MARMON AND P. VISHE 20 Proof. Substituting a = q2q2x1 + b1b1x2 in the definition of S(q, v), where the vectors x1 and x2 run through all residue classes (mod b1) and (mod q2), respectively, Lemma 4.4 gives Proof. Substituting a = q2q2x1 + b1b1x2 in the definition of S(q, v), where the vectors x1 and x2 run through all residue classes (mod b1) and (mod q2), respectively, Lemma 4.4 gives Proof. Substituting a = q2q2x1 + b1b1x2 in the definition of S(q, v), where the vectors x1 and x2 run through all residue classes (mod b1) and (mod q2), respectively, Lemma 4.4 gives s1g(a) + (s1 −s2)f(a) ≡s1g(x1) + (s1 −s2)f(x1) (mod b1), ag(a) ≡ag(x2) (mod q2), and eq(v.a) = eb1(q2v.x1)eq2(b1v.x2). This gives us the relation (4.8). This gives us the relation (4.8). To prove (4.11), we set 4.13) (s1, s2, x) = ss(s′ 1, s′ 2, x′) + rr(s′′ 1, s′′ 2, x′′) (s1, s2, x) = ss(s′ 1, s′ 2, x′) + rr(s′′ 1, s′′ 2, x′′) (4.13) in (4.9), where the s′ i run through (Z/rZ)∗, the s′′ i run through (Z/sZ)∗, and x′, x′′ run through (Z/rZ)n and (Z/sZ)n, respectively. Now, if we put in (4.9), where the s′ i run through (Z/rZ)∗, the s′′ i run through (Z/sZ)∗, and x′, x′′ run through (Z/rZ)n and (Z/sZ)n, respectively. 5. Exponential sums to square-free moduli In this section, we shall provide bounds for the exponential sums T(b1, v) defined in (4.9) for square-free integers b1. We would like to emphasize here that after a sufficient number of hyperplane intersections, we may end up with having to estimate exponential sums involving any number of variables ⩽n. Since we did not want to introduce an extra notation for the number of variables defining the polynomials f, g, in this section, we will derive bounds which are valid for all n including n = 1, 2. Here, we consider a more general version of the sum T(b1, v) where eb1(·) is replaced by an arbitrary primitive additive character ψ : Z/b1Z →C; put T(ψ, v) := X x (mod b1) b1 X∗ s1,s2=1 ψ s1g(x) + (s1 −s2)f(x) + v.x  . This is crucial, since after using our multiplicativity relation (4.11), we end up with exponential sums involving different characters, and we need a uniform way for bounding them. We shall sometimes attach the subscript b1 to the character ψ for clarity. ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 21 We should also note here that all the implied constants in this section are allowed to depend only on n, d1 and d2. Our main bound for the exponential sums to prime moduli is contained in the following result. Lemma 5.1. Let n ⩾2, d1 > d2 and assume that s′ ∞= −1. There exists a non-zero homogeneous polynomial Φ = Φf,g ∈Z[v1, . . . , vn] such that the estimate |T(ψp, v)| ≪n,d1,d2 p(n+3+s′ p)/2p, Φ(v))1/2 holds for any prime p such that the leading form F0 does not vanish identically modulo p (i.e. δp(F0) = n −2), any non-trivial additive character ψp on Fp and any v ∈Zn. The polynomial may be chosen so that (1) coefficients of Φ are co-prime, i.e., cont(Φ) = 1; (1) coefficients of Φ are co-prime, i.e., cont(Φ) = 1; (1) coefficients of Φ are co-prime, i.e., cont(Φ) = 1; (2) we have the bounds (2) we have the bounds deg Φ ≪n,d1,d2 1, log ∥Φ∥≪n,d1,d2 log ∥f∥+ log ∥g∥. deg Φ ≪n,d1,d2 1, log ∥Φ∥≪n,d1,d2 log ∥f∥+ log ∥g∥. Lemma 5.1 states, in other words, that we may find a polynomial Φ such that the optimal bound |T(ψp, v)| ≪p(n+3+s′ p)/2 is attained for all v such that p ∤Φ(v). 5. Exponential sums to square-free moduli Before giving the proof of Lemma 5.1, we state and prove a result that will supply the polynomial Φ in the statement. For any v ∈Zn, we let δ(v) be the dimension of the singular locus of the intersection of Vp(F0, G0) with the hyperplane Lv in Pn−1 Fp , i.e. δ(v) = sp(F0, G0, Lv). It turns out that the quantity δ(v) will govern the strength of our bound for T(ψp, v). Proposition 5.2. There exists a non-zero homogeneous polynomial Φ = ΦF0,G0 ∈Z[v1, . . . , vn] with the following properties: • we have δ(v) ⩽s′ p(f, g) as soon as p ∤Φ(v); • we have δ(v) ⩽s′ p(f, g) as soon as p ∤Φ(v); • cont(Φ) = 1; • cont(Φ) = 1; ( ) • we have the bounds • we have the bounds deg Φ ≪n,d1,d2 1, log ∥Φ∥≪n,d1,d2 log ∥f∥+ log ∥g∥. Proof. A necessary condition for the inequality sv(F0, G0, Lv) > sv(F0, G0) to hold is that Lv be tangent to the variety Vv(F0, G0) at one of its non-singular points, or in other words, that Lv belong to the dual variety Vv(F0, G0)∗in (Pn−1 Fv )∨. We shall obtain universal equations defining this dual variety. to hold is that Lv be tangent to the variety Vv(F0, G0) at one of its non-singular points, or in other words, that Lv belong to the dual variety Vv(F0, G0)∗in (Pn−1 Fv )∨. We shall obtain universal equations defining this dual variety. ( ) We parametrise homogeneous polynomials Fi of degree di by coefficient vectors c(i), viewed as points in projective spaces PNi. By Chevalley’s constructibility theorem, the set W = {(F1, F2, v) | Lv is tangent to V (F1, F2) at a non-singular point} W = {(F1, F2, v) | Lv is tangent to V (F1, F2) at a non-singular point} is a constructible subset of PN1 × PN2 × (Pn−1)∨Thus we may write is a constructible subset of PN1 Z × PN2 Z × (Pn−1 Z )∨. Thus we may write W = k[ i=1 Ui ∩Si, W = k[ i=1 Ui ∩Si, where each Ui is open and each Si is closed. Clearly we may assume that the Ui are non-empty. We may now fix, once and for all, a collection of non-zero homogeneous polynomials where each Ui is open and each Si is closed. 5. Exponential sums to square-free moduli Clearly we may assume that the Ui are non-empty. We may now fix, once and for all, a collection of non-zero homogeneous polynomials Ψ1, . . . , ΨR in the multigraded ring Z[c(1), c(2), v] such that generators for the vanishing ideals of all the closed sets Si may be found among the Ψj. 22 O MARMON AND P VISHE O. MARMON AND P. VISHE 22 For a fixed tuple (F1, F2) = (F0, G0) ∈Z[x]2, denote by Ui and Si the fiber over (F1, F2) of Ui and Si, respectively, and put W = k[ i=1 Ui ∩Si, Z = k[ i=1 Si. By discarding some of the indices if necessary, we may again assume that the Ui are all non- empty. As subsets of (Pn−1 Z )∨, W is constructible and Z is closed. Furthermore, generators for the vanishing ideals I(Si) of the sets Si in Z[v] may be found among the specialisations Θj of the polynomials Ψj above at (F1, F2). j j By definition, the dual variety V ∗ v ⊂(Pn−1 Fv )∨of the variety Vv = Vv(F1, F2) is the Zariski closure of WFv = W ⊗Spec Fv in (Pn−1 Fv )∨. If Vv is irreducible, then V ∗ v is irreducible of dimension at most n −2. In general, if Vv has a decomposition into irreducible components Vv = C1 ∪· · · ∪Cm, then it is easy to see that V ∗ v = C∗ 1 ∪· · · ∪C∗ m. For each 1 ⩽j ⩽R, put ˜Θj = Θj cont(Θj) ∈Z[v]. Φ := R Y j=1 ˜Θk ˜Θj = Θj cont(Θj) ∈Z[v]. Then we claim that Φ := R Y j=1 ˜Θk is a polynomial satisfying the desired properties. To see this, let p be any prime. If Vp is irreducible, then for some closed subscheme Si we have V ∗ p ⊆(Si)Fp ̸= Pn−1 Fp . Thus there is a polynomial Θj, with (p, cont(Θj)) = 1, such that δ(v) ⩽s′ p(F1, F2) as soon as p ∤Θj(v). But then the polynomial ˜Θj satifies the same property. On the other hand, if Vp is not irreducible, then by definition we have s′ p(F1, F2) ⩾n −4, so we trivially have δ(v) ⩽s′ p(F1, F2) for all v. □ Remark. 5. Exponential sums to square-free moduli 4], we have X x (mod p) p|g(x), p|f(x) ψ(v.x) ≪p(n−1+δ(v))/2, X ψ(v.x) ≪p(n−1+δ(v))/2, where δ(v) = sp(F0, G0, Lv). By a result of Zak and Fulton and Lazarsfeld, as explained in [15, p. 897], we have δ(v) ⩽s′ p + 1. Furthermore, by Proposition 5.2, we have δ(v) ⩽s′ p as soon as p ∤Φ(v). Thus we get Σ1 ≪p(n+3+s′ p)/2(p, Φ(v))1/2 if F0 and G0 intersect properly (mod p). On the other hand, if F0 and G0 do not intersect properly (mod p), then we have s′ p = n −1 by definition. Then we certainly still have the estimate |Σ1| ⩽p2 X x (mod p) p|g(x), p|f(x) 1 ≪pn+1 = p(n+3+s′ p)/2, by our assumption that F0 does not vanish entirely (mod p). Furthermore, we may use [2, Lemma 7] to obtain by our assumption that F0 does not vanish entirely (mod p). Furthermore, we may use [2, Lemma 7] to obtain by our assumption that F0 does not vanish entirely (mod p). Furthermore, we may use [2, Lemma 7] to obtain |Σ3| ⩽ X b (mod p) X x (mod p) ψ(bf(x) + v.x) ≪p(n+3+s′ p)/2, and the sum Σ2 satisfies the same bound, since the polynomial g(x) + f(x) has F0(x) as its leading form. Thus, we see that Σ2 and Σ3 both give negligible contributions to T0,g(p, v). (We could equally well have proved this by the arguments used to estimate Σ1 above.) The term Σ4 vanishes in this case. and the sum Σ2 satisfies the same bound, since the polynomial g(x) + f(x) has F0(x) as its leading form. Thus, we see that Σ2 and Σ3 both give negligible contributions to T0,g(p, v). (We could equally well have proved this by the arguments used to estimate Σ1 above.) The term Σ4 vanishes in this case. Finally, we treat the case where n = 2. Here, we need not discern whether p | v or not. The bounds for Σ2 and Σ3 from the previous case remain valid for any n ⩾1 and any v. Also, we trivially have |Σ4| ⩽p2 ⩽p(n+3+s′ p)/2 for n = 2. To estimate Σ1, note that we have s′ p = 1 if the binary forms F0 and G0 have a common projective zero, and s′ p = −1 otherwise. 5. Exponential sums to square-free moduli We would like to note that Proposition 5.2 holds for an arbitrary choice of d1 and d2, i.e., it does not require the assumption d1 > d2, as required by a future application. In fact, the differing degrees of f and g are only used in bounding the term Σ2 in the proof of Lemma 5.1 below. Proof of Lemma 5.1. We may write T(ψp, v) = Σ1 −Σ2 −Σ3 + Σ4, where where Σ1 = p X a,b=1 X x (mod p) ψ(ag(x) + bf(x) + v.x), Σ3 = p X b=1 X x (mod p) ψ(bf(x) + v.x) Σ2 = p X a=1 X x (mod p) ψ(a(g(x) + f(x)) + v.x), Σ4 = X x (mod p) ψ(v.x). Let us first treat the case where p | v. By a theorem of Hooley [13] (see [18, Lemma 3.2] for its affine reformulation), we then have Σ1 = p2#{x (mod p) | f(x) ≡g(x) ≡0 (mod p)} = pn + O p(n+4+s′ p)/2 . Σ1 = p2#{x (mod p) | f(x) ≡g(x) ≡0 (mod p)} = pn + O p(n+4+s′ p)/2 . By the same argument we have Σ2 = p  pn−1 + O p(n+1+s′ p)/2 = pn + O p(n+3+s′ p)/2) = Σ3, ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 23 23 since F0(x) is the leading form of f(x) + g(x). Since Σ4 = pn, we conclude that T(ψp, v) ≪p(n+4+s′ p)/2 = p(n+3+s′ p)/2(p, v)1/2. since F0(x) is the leading form of f(x) + g(x). Since Σ4 = pn, we conclude that T(ψp, v) ≪p(n+4+s′ p)/2 = p(n+3+s′ p)/2(p, v)1/2. since F0(x) is the leading form of f(x) + g(x). Since Σ4 = pn, we conclude that T(ψp, v) ≪p(n+4+s′ p)/2 = p(n+3+s′ p)/2(p, v)1/2. This agrees with our claim, since the polynomial Φ is homogeneous. This agrees with our claim, since the polynomial Φ is homogeneous. Now we turn to the case where p ∤v. We observe that (5.1) Σ1 = p2 X x (mod p) p|g(x), p|f(x) ψ(v.x). (5.1) Suppose first that n ⩾3. If F0 and G0 intersect properly (mod p), i.e. if the corresponding projective varieties intersect properly (mod p), then the exponential sum over the variety defined by the equations g = f = 0 in An Fp may be treated by means of a result of Katz [16]. Indeed, by [16, Thm. 5. Exponential sums to square-free moduli In the former case, we have |Σ1| ⩽p3 + p−1 X a=1 p X b=1 X x (mod p) ψ(af(x) + bg(x) + v.x) ≪p3 = p(n+3+s′ p)/2 O. MARMON AND P. VISHE 24 by [2, Lemma 7], since each polynomial af(x)+bg(x)+v.x has a multiple of F0 as its leading form. In the latter case, there are only O(1) solutions to f(x) ≡g(x) ≡0 (mod p), so by [2, Lemma 7], since each polynomial af(x)+bg(x)+v.x has a multiple of F0 as its leading form. In the latter case, there are only O(1) solutions to f(x) ≡g(x) ≡0 (mod p), so Σ1 = p2 X x (mod p) f(x)≡g(x)≡0 (mod p) ψ(v.x) ≪p2 = p(n+3+s′ p)/2. ψ(v.x) ≪p2 = p(n+3+s′ p)/2. x (mod p) ( p) f(x)≡g(x)≡0 (mod p) ( p) f(x)≡g(x)≡0 (mod p) In both cases, we used the fact that F0 was supposed not to vanish entirely (mod p). This concludes the proof of Lemma 5.1. We observe that we could have disposed of the factor (p, Φ(v))1/2 in the case n = 2, but this stronger bound is not needed in the sequel. □ In both cases, we used the fact that F0 was supposed not to vanish entirely (mod p). This concludes the proof of Lemma 5.1. We observe that we could have disposed of the factor (p, Φ(v))1/2 in the case n = 2, but this stronger bound is not needed in the sequel. □ In the case n = 1, the quantity s′ v is not meaningful. Instead, we have the following alternative bounds. Here Res(f, g) denotes the usual resultant of two univariate polynomials. Lemma 5.3. Let n = 1. Then we have Lemma 5.3. Let n = 1. Then we have |T(ψp, v)| ≪p(p, Res(f, g)) for any v ∈Z, provided that p does not divide the leading coefficient of f. for any v ∈Z, provided that p does not divide the leading coefficient of f. Proof We write Proof. We write Proof. We write T(ψ, v) = Σ1 −Σ2 −Σ3 + Σ4, as in the proof of Lemma 5.1. Here we have |Σ4| ⩽p, and |Σ2| and |Σ3| are bounded from above by p times the number of zeroes of f + g and f, respectively, in Z/pZ. Under our assumption, we therefore get Σi ≪p for i = 2, 3, 4. 5. Exponential sums to square-free moduli Finally, |Σ1| = p X a,b=1 p X x=1 ψ(af(x) + bg(x) + vx) ⩽p2#{x ∈Z/pZ | f(x) ≡g(x) ≡0 (mod p)}, □ Lemma 5.5. Let n = 1. Then the estimate Lemma 5.5. Let n = 1. Then the estimate |T(ψb1, v)| ≪b3/2 1 b1, Res(f, g) 1/2 |T(ψb1, v)| ≪b3/2 1 b1, Res(f, g) 1/2 holds for any square-free b1 such that (b1, cont(F0)) = 1, any primitive additive character ψb1 modulo b1 and any v ∈Z. 6. Exponential sums to square-full moduli Let g ∈Z[x1, . . . , xn] be a cubic polynomial. Let q2 be an arbitrary square-full integer. The exponential sums T ∗(q2, v) in (4.10) that we consider coincide precisely with those investigated in [2]. We again begin by writing q2 = b2q3, where q3 is cube-full part of q2. We begin by estimating the exponential sum T ∗(b2, v) for an arbitrary integer b2, which is purely a product of squares of distinct primes. The bound from [2, Lemma 7] implies: Lemma 6.1. Let n ⩾1. Then for any v ∈Zn, we have T ∗(b2, v) ≪bn/2+ε 2 D(b2) Lemma 6.1. Let n ⩾1. Then for any v ∈Zn, we have T ∗(b2, v) ≪bn/2+ε 2 D(b2) T ∗(b2, v) ≪bn/2+ε 2 D(b2) Rest of this section is dedicated to estimating exponential sums modulo cube-full integers q3 = c2d, where d is square-free. The following bound is proven in [2, Lemma 11]: Lemma 6.2. For any r ∈Z with (r, q3) = 1, we have the bound (6.1) |T ∗ ra(q3, rv)| ≪qn/2 3 X s (mod c) c|a∇g(s)+v Md(s)1/2, (6.1) where Md(s) :=  t ∈(Z/dZ)n | ∇2g(s)t ≡0 (mod d) . We shall need bounds for two kinds of averages of the exponential sums T ∗(q3, v). First, for any v0 ∈Rn and any V ⩾1, we shall evaluate the sum We shall need bounds for two kinds of averages of the ex for any v0 ∈Rn and any V ⩾1, we shall evaluate the sum (6.2) X |v−v0|⩽V T ∗(c2d, v). X |v−v0|⩽V T ∗(c2d, v). (6.2) In view of Lemma 6.2, we then need to estimate the quantity In view of Lemma 6.2, we then need to estimate the quantity X |v−v0|⩽V P(c2d, v), where P(c2d, v) := X s (mod c) c|a∇g(s)+v Md(s)1/2. (6.3) c|a∇g(s)+v We shall assume that the leading form G0 is non-singular, in other words that s∞(G0) = −1 in the notation of Section 4. Furthermore, we shall assume that ∥g∥P ⩽H (6.5) ∥g∥P ⩽H (6.5) for some H ⩾1. In [2], two alternative bounds for the quantity in (6.3) are presented. In the present situation we shall only need the latter of these, given by Lemma 16. Together with the discussion concluding [2, §5], this implies the following estimate. Lemma 6.3. If the cubic polynomial g ∈Z[x1, . . . o Σ1 vanishes unless f and g have a common zero (mod p), in which case Σ4 ≪p2. so Σ1 vanishes unless f and g have a common zero (mod p), in which case Σ4 ≪p2. Now let q = b1 be an arbitrary squarefree integer. The estimates above assumed that F0 does not vanish identically (mod p). To say that this should hold for all primes p | b1 amounts to the condition (b1, cont(F0)) = 1, where cont(F0) is defined by (4.15). Now we observe that (an easy extension of) the mul- tiplicativity property (4.11) may be reformulated to state that if ψb1 is a primitive additive character modulo b1 = rs, where (r, s) = 1, then there exist primitive additive characters ψr modulo r and ψs modulo s such that T(ψb1, v; f, g) = T(ψr, v; f, g)T(ψs, v; f, g). Decomposing b1 into prime factors and multiplying together the bounds obtained for each factor by Lemma 5.1 or 5.3, we obtain a bound where the implied constant C = C(n, d1, d2), say, is replaced by a factor which is at most Cω(b1) ≪bε 1. We thus arrive at the following results. Lemma 5.4. Let n ⩾2 and let b1 be a square-free integer. For the polynomial Φ from Lemma 5.1, the estimate |T(ψb1, v)| ≪b(n+2)/2+ε 1 D(b1)(b1, Φ(v))1/2 |T(ψb1, v)| ≪b(n+2)/2+ε 1 D(b1)(b1, Φ(v))1/2 holds for any square-free number b1 such that (b1, cont(F0)) = 1, any primitive additive char- acter ψb1 modulo b1 and any v ∈Zn. ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 25 6. Exponential sums to square-full moduli , xn] satisfies (6.4) and (6.5), then we have the bound / X |v−v0|⩽V P(q3, v) ≪qε 3D(d) V n + (q3H)n/3 , where D(·) is the quantity defined in (4.14). where D(·) is the quantity defined in (4.14). O. MARMON AND P. VISHE 26 We also need to consider versions of the sum (6.2) where the vectors v are restricted to the ones satisfying the equation Φ(v) = 0 for a certain polynomial Φ ∈Z[v1, . . . , vn], or a congruence Φ(v) ≡0 (mod m) for some integer m. Lemma 6.4. Let Φ ∈Z[v1, . . . , vn], where n ⩾1, be a non-zero polynomial. Then we have the bound (6.6) X |v−v0|⩽V Φ(v)=0 P(q3, v) ≪n,deg(Φ) qε 3cnD(d)1/2  1 + V c n−1 . (6.6) Furthermore, for any squarefree integer m with (m, c) = 1 = (m, cont(Φ)), we ha Furthermore, for any squarefree integer m with (m, c) = 1 = (m, cont(Φ)), we have (6.7) X |v−v0|⩽V Φ(v)≡0 (mod m) P(q3, v) ≪n,deg(Φ) (mq)εcnD(d)1/2 1 + V c n−1 + V c n m−1 ! . (6.7) X |v−v0|⩽V Φ(v)≡0 (mod m) P(q3, v) ≪n,deg(Φ) (mq)εcnD(d)1/2 1 + V c n−1 + V c n m−1 (6.7) Proof. We may write X |v−v0|⩽V Φ(v)=0 P(q3, v) ⩽ X s (mod c) Md(s)1/2 max r (mod c) Ur(V ), where Ur(V ) := #{v ∈Zn | |v −v0| ⩽V, Φ(v) = 0, v ≡r (mod c)}. X |v−v0|⩽V Φ(v)≡0 (mod m) P(q3, v) ⩽ X s (mod c) Md(s)1/2 max r (mod c) Ur,m(V ), where Ur,m(V ) := #{v ∈Zn | |v −v0| ⩽V, Φ(v) ≡0 (mod m), v ≡r (mod c)}. If c ⩾V , then we obviously have Ur(V ) ⩽1 and Ur,m(V ) ⩽1. If c < V , then we may write Ur(V ) ⩽#  u ∈Zn; |u| ⩽2V c , Ψ(u) = 0  and and Ur,m(V ) ⩽#  u ∈Zn; |u| ⩽2V c , Ψ(u) ≡0 (mod m)  , where Ψ(u) = Φ(v1 + cu) for some v1 ∈Zn. The polynomial Ψ ∈Z[x1, . . . , xn] is not the zero polynomial, and by our assumption that (m, c) = 1 = (m, cont(Φ)), its image in Fp[x1, . . . , xn] is also non-vanishing for all p | m. 6. Exponential sums to square-full moduli It then follows from [2, Lemma 4] that Ur(V ) ≪ V c n−1 and Ur,m(V ) ≪mε V c n−1 + V c n m−1 ! . In general, we therefore have the bounds In general, we therefore have the bounds Ur(V ) ≪1 + V c n−1 and Ur,m(V ) ≪mε 1 + V c n−1 + V c n m−1 ! . ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 27 By [2, Lemma 14] we have X s (mod c) Md(s) ≪  c d n X s (mod d) Md(s) ≪qε 3cnD(d). An application of the Cauchy-Schwarz inequality gives An application of the Cauchy-Schwarz inequality gives X s (mod c) Md(s)1/2 ≪cn/2    X s (mod c) Md(s)    1/2 ≪qε 3cnD(d)1/2. We conclude that X |v−v0|⩽V Φ(v)=0 P(q, v) ≪  1 + V c n−1 X s (mod c) Md(s)1/2 ≪qεcnD(d)1/2  1 + V c n−1 and similarly and similarly ) P(q3, v) ≪(mq3)εcnD(d)1/2 1 + V c n−1 + V c n m−1 ! , as claimed. as claimed. □ Note that if n ⩾2 and the polynomial Φ is absolutely irreducible of degree at least 2, then using a bound by Serre [23, Chapter 13], the exponent in the bound (6.6) can be improved to n −3/2. However the current bound suffices to establish Theorem 1.1. 7. Evaluation of quartic exponential sums; further considerations We shall use the bounds from the previous sections to evaluate the exponential sum T (q, z). From now on, we again restrict the degrees of the polynomials to be deg(f) = 4 and deg(g) = 3 (unless g vanishes entirely). Thus, the implied constants in our estimates depend only on n and ε. In the same vein as Section 5, we will obtain bounds for all n including n = 1, 2, since f and g will correspond to the forms obtained from applying various stages of hyperplane intersections to F and Fh respectively. We write q = bq3 = b1b2q3 and q3 = c2d as above. Here b1 is the square-free and q3 is the cube-full part of q, as per our notation (2.11). Moreover, d is square-free as chosen in Section 6. We shall assume that (b1, cont(F0)) = 1 (7.1) (b1, cont(F0)) = 1 (7.1) and furthermore that ∥g∥P ⩽H ⩽P A (7.2) ∥g∥P ⩽H ⩽P A (7.2) for some A > 0. To begin with, let us in addition assume that (7.3) s′ ∞= −1. s′ ∞= −1. (7.3) O. MARMON AND P. VISHE 28 Recall the bound in Proposition 4.3 for T (q, z) and the factorisation (4.8) for the sums S(q, v) occurring there. Assume that n ⩾2. For the first factor in (4.8) we note that Recall the bound in Proposition 4.3 for T (q, z) and the factorisation (4.8) for the sums S(q, v) occurring there. Assume that n ⩾2. For the first factor in (4.8) we note that T(b1, q2v) = X x (mod b1) b1 X∗ s1,s2=1 eb1(s1g(x) + (s1 −s2)f(x) + q2v.x) = X x (mod b1) b1 X∗ s1,s2=1 eb1  q2 s′ 1g(x) + (s′ 1 −s′ 2)f(x) + v.x  = T(ψ′, v), T(b1, q2v) = X x (mod b1) b1 X∗ s1,s2=1 eb1(s1g(x) + (s1 −s2)f(x) + q2v.x) = X x (mod b1) b1 X∗ s1,s2=1 eb1  q2 s′ 1g(x) + (s′ 1 −s′ 2)f(x) + v.x  = T(ψ′, v), where we have put s′ i = q2si for i = 1, 2 in the second step, and where ψ′(·) = eb1(q2 ·) is a primitive additive character modulo b1. 7. Evaluation of quartic exponential sums; further considerations Thus we have the bound where we have put s′ i = q2si for i = 1, 2 in the second step, and where ψ′(·) = eb1(q2 ·) is a primitive additive character modulo b1. Thus we have the bound |T(b1, q2v)| ≪b(n+2)/2+ε 1 D(b1)(b1, Φ(v))1/2, by Lemma 5.4, where the polynomial Φ = Φf,g satisfies the properties listed there. We split the second factor in (4.8) further into a cubefree and a cubefull part, thus writing T ∗ a (q2, b1v) = T ∗ q3a(b2, q3b1v)T ∗ b2a(q3, b2b1v). For the first factor, Lemma 6.1 applies to give T ∗ q3a(b2, q3b1v) ≪bn/2+ε 2 D(b2). Inserting these bounds into Proposition 4.3, and assuming that q ⩽P 2, say, we get (7.4) T (q, z) ≪N P −N + q−nP n+εb1bn/2D(b) max v0 X |v v |⩽P εV |T ∗ b2a(q3, b2b1v)|(b1, Φ(v))1/2. Inserting these bounds into Proposition 4.3, and assuming that q ⩽P 2, say, we get (7 4) T (q z) ≪N P −N + q−nP n+εb1bn/2D(b) max X |T ∗(q3 b2b1v)|(b1 Φ(v))1/2 Inserting these bounds into Proposition 4.3, and assuming that q ⩽P 2, say, we get (7.4) T (q, z) ≪N P −N + q−nP n+εb1bn/2D(b) max v0 X |v−v0|⩽P εV |T ∗ b2a(q3, b2b1v)|(b1, Φ(v))1/2. (7.4) T (q, z) ≪N P −N + q−nP n+εb1bn/2D(b) max v0 X |v v0|⩽P εV |T ∗ b2a(q3, b2b1v)|(b1, Φ(v) |v−v0|⩽P εV Using Lemma 6.2, we then get (7.5) T (q, z) ≪b1P n+εq−n/2D(b) max v0 X |v−v0|⩽P εV P(q3, b1v)(b1, Φ(v))1/2. We estimate the sum over v using Lemma 6.4, writing We estimate the sum over v using Lemma 6.4, writing X |v−v0|⩽P εV P(q3, b1v)(b1, Φ(v))1/2 ⩽ X m|b1 m1/2 Φ X −v0|⩽P εV P(q3, b1v)(b1, Φ(v))1/2 ⩽ X m|b1 m1/2 X |v−v0|⩽P εV Φ(v)≡0 (mod m) P(q3, b1v) P(q3, b1v) X |v−v0|⩽P εV ≪D(d)P ε X m|b1 m1/2(cn + cV n−1 + V nm−1) ≪D(d)P ε b1/2 1 (cn + cV n−1) + V n . Inserting this bound into (7.5), we obtain Inserting this bound into (7.5), we obtain T (q, z) ≪b1P n+εq−n/2D(q)  b1/2 1 (cn + cV n−1) + V n . 7. Evaluation of quartic exponential sums; further considerations Alternatively, we may use the trivial bound (b1, Φ(v))1/2 ⩽b1/2 1 and apply Lemma obtain Alternatively, we may use the trivial bound (b1, Φ(v))1/2 ⩽b1/2 1 and apply Lemma 6.3 to obtain X |v−v0|⩽P εV P(q3, b1v)(b1, Φ(v))1/2 ⩽b1/2 1 X |v−v0|⩽P εV P(q3, b1v) ≪b1P εD(d)(V + (q3H)1/3)n, X |v−v0|⩽P εV P(q3, b1v)(b1, Φ(v))1/2 ⩽b1/2 1 X |v−v0|⩽P εV P(q3, b1v) ≪b1P εD(d)(V + (q3H)1/3 X |v−v0|⩽P εV P(q3, b1v)(b1, Φ(v))1/2 ⩽b1/2 1 X |v−v0|⩽P εV P(q3, b1v) ≪b1P εD(d)(V + (q3H)1/3)n, which in turn yields T (q, z) ≪b3/2 1 P n+εq−n/2D(q)  V n + (q3H)n/3 . Observing that min{cn, V n} ⩽cV n−1, we conclude as follows. ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 29 Proposition 7.1. Suppose that n ⩾2. Then, under the conditions (7.1)–(7.3), we have the bound Proposition 7.1. Suppose that n ⩾2. Then, under the conditions (7.1)–(7.3), we have the bound 1. Suppose that n ⩾2. Then, under the conditions (7.1)–(7.3), we have the p bound (7.6) T (q, z) ≪b1q−n/2D(q)P n+ε V n + b1/2 1 cV n−1 + (q3H)n/3 , where V = q P max{1, (HP 3|z|)1/2} as in (4.7). (7.6) In the case n = 1, we repeat the arguments above, replacing Lemma 5.4 by 5.5, to obtain the following bound. Proposition 7.2. If n = 1 and (7.1)–(7.3) hold, then we have T (q, z) ≪b1q−1/2D(q2)P 1+ε(b1, Res(f, g))1/2(V + (q3H)1/3). We shall also derive a ’trivial’ bound, which is useful also in the case when g vanishes identically. In that particular case, the condition (7.3) is automatically violated. Instead, we only impose a non-singularity condition on f. Proposition 7.3. Suppose that P ⩾q1/2+1/n, that (7.1) holds, and in addition that sp(F0) = −1 for all primes p | b1. Then we have the bound T (q, z) ≪b1P n+ε. Proposition 7.3 will be a consequence of the following observation. Lemma 7.4. Let f ∈Z[x1, . . . , xn] be a polynomial of degree d ⩾2 and let w ∈Wn. Let m be a squarefree number such that the leading form F0 of f is non-singular (mod p) for every prime p | m. Then we have the bound X x∈Zn m|f(x) w(x/P) ≪P n+εm−1 whenever P ⩾m1/2+1/n. whenever P ⩾m1/2+1/n. Proof. 7. Evaluation of quartic exponential sums; further considerations By Poisson summation we have X x∈Zn m|f(x) w(x/P) = X z (mod m) m|f(z) X y∈Zn w(P −1(z + my)) = P n mn X v∈Zn ˆw( P mv)Σm(v), where where Σm(v) = X x (mod m) m|f(x) em(v.x). where Σm(v) = X x (mod m) m|f(x) em(v.x). |f( ) By Lemma 4.4 we have the multiplicativity relation By Lemma 4.4 we have the multiplicativity relation Σm1m2(v) = Σm1(m2v)Σm2(m1v) if (m1, m2) = 1, where m1, m2 ∈Z are such that mm1 + m2m2 = 1. Suppose that p | m is a prime. If p ∤v, then we have Σp(v) = p−1 p−1 X a=1 X x (mod p) ep(af(x) + v.x) ≪pn/2 by [2, Lemma 7]. In the opposite case we still have the bound Σp(v) ≪pn−1. In particular it follows that one has (7.7) Σm(v) ≪mn−1+ε Σm(v) ≪mn−1+ε (7.7) O. MARMON AND P. VISHE 30 for all v ∈Zn and Σm(v) ≪mn/2+ε (7.8) Σm(v) ≪mn/2+ε (7.8) Σm(v) ≪mn/2+ε (7.8) if gcd(m, v) := gcd(m, v1, . . . , vn) = 1. The asserted bound follows if we can prove that (7.9) X v∈Zn ˆw( P mv)|Σm(v)| ≪mn−1+ε. if gcd(m, v) := gcd(m, v1, . . . , vn) = 1. The asserted bound follo (7.9) X v∈Zn ˆw( P mv)|Σm(v)| ≪mn−1+ε. (7.9) Using repeated integration by parts, one may show that ˆw(t) ≪k (1 + |t|)−k for any k ⩾1, from which it follows that for any k ⩾1, from which it follows that X v∈Zn ˆw( P mv) ≪  1 + m P n . In case m ⩽P, the bound (7.9) therefore follows directly from (7.7). We may thus suppose from now on that m ⩾P. In this case we prove (7.9) by induction on the number ω(m) of prime divisors of m. The case where ω(m) = 0, that is, m = 1, is trivial. For the induction step, we write X v∈Zn ˆw( P mv)|Σm(v)| = Σ1 + Σ2, where Σ1 denotes the sum ranging over v ∈Zn satisfying (m, v) = 1, and Σ2 denotes the sum over those v for which there exists a divisor m1 > 1 of m such that m1|v. There then exists one such divisor m1, for which Σ2 ≪mεΣ′ 2, say, where Σ′ 2 is the sum over v such that m1|v. 8. Finalisation of van der Corput differencing In this section, we use the results from Section 7 to estimate the quantity X |h|⩽H Ta,h(q, z) (8.1) X |h|⩽H Ta,h(q, z) (8.1) occurring in Section 3. q will be a fixed arbitrary number throughout this section. Our final aim is to prove the minor arcs bound in Proposition 2.9, so we recall that F is a quartic polynomial whose leading form F0 is non-singular. From now on, the implied constants in our estimates are allowed to depend on the height ∥F∥P and the quantity M introduced in (2.15)–(2.16). We shall prove the following result: Proposition 8.1. Provided that q ⩽P 2−4/(n+2), we have the bound Proposition 8.1. Provided that q ⩽P 2−4/(n+2), we have the bound (8.2) X |h|⩽H Ta,h(q, z) ≪b1P n+ε   1 + n−1 X η=0 Yη   , (8.2) where where where Yη := Hn−η q(n−η)/2 Xη for any 0 ⩽η ⩽n −1, with for any 0 ⩽η ⩽n −1, with Xη := V n−η + b1/2 1  q1/2 3 V n−η−1 + (q3H)(n−η)/3 for 0 ⩽η ⩽n −2, and for 0 ⩽η ⩽n 2, and Xn−1 := V + q3H1/3. Here V = qP −1 max{1, (HP 3|z|)1/2}, as per (4.7). The terms Yη appearing on the equation (8.2) roughly correspond to h’s for which the maximum dimension of the singular locus for a system of projective varieties corresponding to F, the leading form of Fh or their complete intersection is η −1. Given the nature of the bounds presented, the terms 1, Y0, Yn−1 and Yn−2 will be critical to our analysis. In this paper, the main contribution is presented by the terms 1 and Y0. Moreover, the term b1/2 1 q1/2 3 V n−1 turns out to give the dominating contribution in X0, which is partly why we are unable to establish the result for n = 29. Given q = b1q2 and h ∈Zn, we write b1 = b1,0(h) · · · b1,n(h) and q2 = q2,0(h) · · · q2,n(h), where b1,i(h) = Y p∥b1 s′ p(h)=i−1 p and q2,i(h) = Y pe|q2 s′ p(h)=i−1 pe. Here s′ p(h) = s′ p(F, Fh). Firstly, since q is fixed, there are then only O(P ε) possible different choices for 2(n + 1)-tuples (b1,0(h), . . . , b1,n(h), q2,0(h), . . . , q2,n(h)) ∈Nn+1 × Nn+1. (b1,0(h), . . . , b1,n(h), q2,0(h), . . . 7. Evaluation of quartic exponential sums; further considerations Writing m = m1m2 and v = m1w, we have where Σ1 denotes the sum ranging over v ∈Zn satisfying (m, v) = 1, and Σ2 denotes the sum over those v for which there exists a divisor m1 > 1 of m such that m1|v. There then exists one such divisor m1, for which Σ2 ≪mεΣ′ 2, say, where Σ′ 2 is the sum over v such that m1|v. Writing m = m1m2 and v = m1w, we have Σ′ 2 = X v∈Zn m1|v ˆw( P mv)|Σm1(m2v)||Σm2(m1v)| ≪mn−1+ε 1 X w∈Zn ˆw( P m2 w)|Σm2(w)| ≪mn−1+ε, by (7.7) and the induction hypothesis. by (7.7) and the induction hypothesis. Furthermore, by (7.8) one has Σ1 ≪mn/2+ε X v∈Zn ˆw( P mv) ≪mn/2+ε mn P n ⩽mn−1+ε, □ so we have proved the bound (7.9). so we have proved the bound (7.9). Proof of Proposition 7.3. We may write |T (q, z)| ⩽ X x∈Zn w(x/P) Z b1, f(x) + g(x), f(x)  , where Z(r, x, y) = r X∗ s1,s2=1 er s1x −s2y  . It is easy to see that Z(r, x, y) is a multiplicative function of r for fixed x and y, and that for prime p one has Z(p, x, y) = 1 if x and y are both non-zero (mod p), Z(p, x, y) = 1 −p if precisely one of x and y vanish (mod p) and Z(p, x, y) = (p −1)2 if x ≡y ≡0 (mod p). It follows that |Z(r, x, y)| ⩽(r, x)(r, y). Thus we have |T (q, z)| ⩽ X x∈Zn w(x/P)(b1, f(x) + g(x))(b1, f(x)) ⩽b1 X x∈Zn w(x/P)(b1, f(x)). |T (q, z)| ⩽ X x∈Zn w(x/P)(b1, f(x) + g(x))(b1, f(x)) ⩽b1 X x∈Zn w(x/P)(b1, f(x)). But But X ∈Zn w(x/P)(b1, f(x)) ⩽ X m|b1 m X x∈Zn m|f(x) w(x/P) ≪P nd(b1) ≪P n+ε X x∈Zn w □ by Lemma 7.4. ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 31 31 8. Finalisation of van der Corput differencing , q2,n(h)) ∈Nn+1 × Nn+1. h t l (b b ) f hi h Thus there is one such tuple (b1,0, . . . , b1,n, q2,0, . . . , q2,n), for which X |h|⩽H Ta,h(q, z) ≪P ε X h∈H′ Ta,h(q, z) = P ε n−1 X s=−1 X h∈H′s Ta,h(q, z), O. MARMON AND P. VISHE 32 say, where say, where H′ := {h ∈Zn | |h| ⩽H, b1,i(h) = b1,i and q2,i(h) = q2,i for all i = 0, . . . , n} set of H = {|h| ⩽H} and H′ := {h ∈Zn | |h| ⩽H, b1,i(h) = b1,i and q2,i(h) = q2,i for all i = 0, . . . , n} is a subset of H = {|h| ⩽H} and is a subset of H = {|h| ⩽H} and H′ s := {h ∈H′ | s′ ∞(h) = s} for each s = −1, . . . , n −1. Furthermore, put for each s = −1, . . . , n −1. Furthermore, put ˜q2,i = Y p|q2,i p. for any i. We note that since s′ p(h) ⩾s′ ∞(h), we have H′ s = ∅unless b1,i = q2,i = 1 for i ⩽s. To estimate the cardinality of the set H′ s, we need the following result. Lemma 8.2. The set Vv,i = {h ∈An Fv | s′ v(F, Fh) ⩾i −1} is a closed subvariety in An Fv of degree O(1), and there is a constant C, with log C ≪n log ∥F0∥, such that dim(Vv,i) ⩽n −i as soon as v = ∞or v = p > C. More precisely, Vv,i is the affine cone over a closed subvariety of Pn−1 Fv of dimension n −i −1. as soon as v = ∞or v = p > C. More precisely, Vv,i is the affine cone over a closed subvariety of Pn−1 Fv of dimension n −i −1. Remark. If F is a form of degree d1, then the homogeneous part of Fh of degree d1 −1 is the form F h(x) := h.∇F(x). For any i, the set Vv,i under consideration may be written as a union of the three sets V (j) v,i , say, for j = 1, 2, 3, defined by the conditions sv(F) ⩾i −1, sv(F h) ⩾i −1, and sv(F, F h) ⩾i −1, respectively. 8. Finalisation of van der Corput differencing It is clear that V (1) v,i = An or ∅, and the latter holds as soon as i ⩾1 and v ≫F 1. The fact that V (2) v,i satisfies the conclusions in the lemma was proven in [2]. Thus it suffices to prove the assertion with Vv,i replaced by V (3) v,i . Such a statement was first proven by Salberger in an unpublished note [22]. The same arguments were used to prove a similar result in a paper by the first author [19, Lemma 2.2], the proof of which may be used with only minor modifications to obtain a proof of Lemma 8.2. We omit the proof here, but it is worth pointing out that it uses a version of Bertini’s theorem that is only valid in characteristic zero, hence producing a condition on p that was not present in the corresponding result in [2]. respectively. It is clear that V (1) v,i = An or ∅, and the latter holds as soon as i ⩾1 and v ≫F 1. The fact that V (2) v,i satisfies the conclusions in the lemma was proven in [2]. Thus it suffices to prove the assertion with Vv,i replaced by V (3) v,i . Such a statement was first proven by Salberger in an unpublished note [22]. The same arguments were used to prove a similar result in a paper by the first author [19, Lemma 2.2], the proof of which may be used with only minor modifications to obtain a proof of Lemma 8.2. We omit the proof here, but it is worth pointing out that it uses a version of Bertini’s theorem that is only valid in characteristic zero, hence producing a condition on p that was not present in the corresponding result in [2]. To estimate #H′ s, we follow the argument in [2, §7], with a difference that the absolute constant c in [2] is replaced by a constant C = O∥F0∥(1). Since H′ s ⊂{h ∈V∞,s+1 ∩[−H, H]n | [h]p ∈Vp,i for all p | b1,iq2,i} , we may use [2, Lemma 4] to show that we may use [2, Lemma 4] to show that (8.3) #H′ s ≪qε max s+1⩽η⩽n Hn−η n Y i=η+1 (b1,i˜q2,i)i−η . (8.3) Proof of Proposition 8.1. Let −1 ⩽s ⩽n −1 be fixed and put Us = X h∈Hs Ta,h(q, z). Us = X h∈Hs Ta,h(q, z). 8. Finalisation of van der Corput differencing ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 33 Each exponential sum Ta,h(q, z) is an instance Each exponential sum Ta,h(q, z) is an instance T (q, z) = Tn(a, q, z; F, Fh, Wh, P) of the general sum introduced in Section 4. We may assume that the condition (7.1) holds. Indeed, if b′ 1 = (b1, cont(F0)), then b′ 1 ⩽∥F0∥⩽∥F∥P , so all exponential sums T(b′, v) satisfy a trivial upper bound O∥F∥P (1). By our convention on the implied constant, it suffices to bound Ta,h(q/b′ 1, z). Moreover, one sees that ∥Fh∥P ≪H, so that the condition (7.2) is also verified, at least upon replacing H with CH for some C ≪1. Choose η such that the maximum in (8.3) is attained. Let us first treat the case where η ⩽n −2. In this case, for each h ∈H′ s, we apply Lemma 4.1, with Π being the set of primes p | q, so that r = ω(q), and with {F1, F2} = {F0, F h 0 }. We obtain a lattice Λη of rank n −η and a basis e1, . . . , en−η for Λη, with the property that for any t ∈Zn, the polynomials ˜ft(y1, . . . , yn−η) := F(t + P yiei) and ˜gt(y1, . . . , yn−η) := Fh(t + P yiei) satisfy s′ v( ˜ft, ˜gt) = max{−1, s′ v(F, Fh) −η} (8.4) s′ v( ˜ft, ˜gt) = max{−1, s′ v(F, Fh) −η} (8.4) for all v ∈{∞} ∪Πcr. Indeed, denote by ˜F0 the leading form of ˜f0, which is simultaneously the leading form of ˜ft for all t. Similarly, let ˜G0 be the common leading form of the ˜gt. Then we see that for each v ∈{∞} ∪Πcr, we have for all v ∈{∞} ∪Πcr. Indeed, denote by ˜F0 the leading form of ˜f0, which is simultaneously the leading form of ˜ft for all t. Similarly, let ˜G0 be the common leading form of the ˜gt. Then we see that for each v ∈{∞} ∪Πcr, we have Vv( ˜F0) ∼= Vv(F0) ∩Λη, Vv( ˜G0) ∼= Vv(G0) ∩Λη and Vv( ˜F0, ˜G0) ∼= Vv(F0, G0) ∩Λη, Vv(F0) Vv(F0) ∩Λη, Vv(G0) Vv(G0) ∩Λη and Vv(F0, G0) Vv(F0, G0) ∩Λη, so (8.4) is precisely the condition that is asserted in Lemma 4.1. 8. Finalisation of van der Corput differencing VISHE 34 From this we conclude that we may choose Tη to be the set of vectors of the form Pn i=n−η+1 λiei, with λi ≪P. fi h f From this we conclude that we may choose Tη to be the set of vectors of the form Pn i=n−η+1 λiei, with λi ≪P. Defining new weight functions ˜wt(y1, . . . , yn−η) := Wh(P −1t + L−1 X yiei), we then have (8.6) Ta,h(q, z) ⩽ X t∈Tη Ta,h,t(q, z), where Ta,h,t(q, z) = Tn−η(q, z; ˜ft, ˜gt, ˜wt, P/L) (8.6) in the notation of (4.2). The verification that ˜wt ∈Wn−η for t ≪P follows as in the proof of [2, Prop. 2]. Indeed, the deciding property of the basis e1, . . . , en−η is the bound (8.5) derived above. One also sees, following [2], that ∥˜gt∥P/L ≪L3∥Fh∥P ≪P εH∥F∥P ≪P εH. Thus the conditions (7.1) and (7.2) are verified, at least upon replacing H by ˜H for some ˜H ≪ P εH. By (8.4) and our assumption that η ⩾s+1, we also have (7.3), so that Ta,h,t(q, z) indeed qualifies as an instance of the exponential sum treated in Proposition 7.1, with dimension n replaced by n−η. At this point it is worth noting that this is the main reason behind obtaining bounds for exponential sums defined over an arbitrary number of variables including 1 and 2 in Sections 5 through 7. Using (8.3), we may now write (8.7) Us ≪ P η+εHn−η n Y i=η+1 (b1,i˜q2,i)i−η · max′ h∈H′s max t∈Tη Ta,h,t(q, z). (8.7) By (8.4) it follows, again arguing as in the proof of [2, Prop. 2], that for each t occurring in (8.7) one has By (8.4) it follows, again arguing as in the proof of [2, Prop. 2], that for each t occurring in (8 7) one has D ˜ft,˜gt(q) = n−η Y i=1 Y p|b1 s′ p( ˜ft,˜gt)=i−1 pi/2 Y p|q2 s′ p( ˜ft,˜gt)=i−1 pi ≪qε n Y i=η+1 Y p|b1 s′ p(F,Fh)=i−1 p(i−η)/2 Y p|q2 s′ p(F,Fh)=i−1 pi−η ≪qε n Y i=η+1 (b1/2 1,i ˜q2,i)i−η. Inserting the bound from Proposition 7.1 into (8.7) thus yields Us ≪b1P n+ε Hn−η q(n−η)/2 Xη, where Xη is the quantity defined in the statement of the proposition. where Xη is the quantity defined in the statement of the proposition. It remains to deal with the possibility that η ∈{n −1, n}. 8. Finalisation of van der Corput differencing It also follows that deg ˜F0 = 4 and deg( ˜G0) = 3, and that is precisely the condition that is asserted in Lemma 4.1. It also follows that deg ˜F0 = 4 ( ˜G0) = 3, and that (b1, cont( ˜F0)) = 1. As there are at most O(qε) = O(P ε) choices for a basis satisfying (4.3), there is in fact one such choice for which we may write Us ≪P ε X′ h∈H′s |Ta,h(q, z)|, where the superscript ′ denotes that the sum is taken over those vectors h occurring in the original sum for which the condition (8.4) holds for the designated basis e1, . . . , en. For such an element h, we may partition the sum over x ∈Zn defining Ta,h(q, z) into cosets Λη + t of the lattice Λη, where t runs over some subset Tη ⊂(Z ∩[−P, P])n. We claim that the set Tη may be chosen of cardinality O(P η). To see this, consider a general linear combination where the superscript ′ denotes that the sum is taken over those vectors h occurring in the original sum for which the condition (8.4) holds for the designated basis e1, . . . , en. For such an element h, we may partition the sum over x ∈Zn defining Ta,h(q, z) into cosets Λη + t of the lattice Λη, where t runs over some subset Tη ⊂(Z ∩[−P, P])n. We claim that the set Tη may be chosen of cardinality O(P η). To see this, consider a general linear combination x = n X i=1 yiei. Denoting by πi, for each 1 ⩽i ⩽n, the projection onto the orthogonal complement of the subspace spanned by the vectors ej, j ̸= i, one then has ∥x∥⩾∥πix∥= |yi|∥πiei∥= |yi| | det(Λ)| | det(Λi)|, where Λ ⊆Zn denotes the full-dimensional lattice spanned by e1, . . . , en and Λi the lattice spanned by all ej, j ̸= i. Now it follows from the conditions (4.3) and (4.4) that where Λ ⊆Zn denotes the full-dimensional lattice spanned by e1, . . . , en and Λi the lattice spanned by all ej, j ̸= i. Now it follows from the conditions (4.3) and (4.4) that |yi| ≪∥x∥ L . (8.5) |yi| ≪∥x∥ L . (8.5) O. MARMON AND P. 8. Finalisation of van der Corput differencing Suppose first that η = n −1. Since s ⩽η −1 = n −2, the variety defined by F = Fh = 0 in An Q has dimension n −2 for all h occurring in the sum. We apply Lemma 4.1 again, although the non-singularity condition in the lemma is automatic in this case, since the resulting varieties all have dimension at most zero. Using similar arguments as in the previous case, we may then write (8.8) Us ≪P εHn−1 b1,n˜q2,n · max′ h∈Hs X t∈Tn−1 Ta,h,t(q, z). (8.8) For each t occurring in the sum, we may apply Proposition 7.2 to obtain Ta,h,t(q, z) ≪b1q−1/2D(q2)P 1+ε(b1, Res( ˜ft, ˜gt))1/2(V + (q3H)1/3). ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 35 ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 35 In order to capitalise the average over t, we use the explicit description Tn−1 = ( t = n X i=2 ziei; zi ∈Z, |zi| ≪P ) . The resultant Res( ˜ft, ˜gt) is then a polynomial R(z) in the variables z2, . . . , zn with integer coefficients. The fact that F and F h intersect completely implies that R(t) does not vanish identically in t. Similarly, the congruence R(t) ≡0 (mod p) defines a hypersurface in An−1 Fp unless either p | b1,n or p ≪P ε. Thus we write The resultant Res( ˜ft, ˜gt) is then a polynomial R(z) in the variables z2, . . . , zn with integer coefficients. The fact that F and F h intersect completely implies that R(t) does not vanish identically in t. Similarly, the congruence R(t) ≡0 (mod p) defines a hypersurface in An−1 Fp unless either p | b1,n or p ≪P ε. Thus we write X t∈Tn−1  b1, Res( ˜ft, ˜gt) 1/2 ≪P εb1/2 1,n X z∈Zn−1 z≪P 1+ε (b∗ 1, R(z))1/2 = P εb1/2 1,n X ℓ|b∗ 1 ℓ1/2#{z ∈Zn−1 | z ≪P 1+ε, ℓ| R(z)}, where we have put b∗ 1 = b−1 1,nb1 = b1,0 · · · b1,n−1. By [2, Lemma 4] we have #{z ∈Zn−1 | z ≪P 1+ε, ℓ| R(z)} ≪P ε(P n−2 + P n−1ℓ−1), which in turn gives the bound which in turn gives the bound X t∈Tn−1  b1, Res( ˜ft, ˜gt) 1/2 ≪b1/2 1 P n−2+ε + b1/2 1,nP n−1+ε. 8. Finalisation of van der Corput differencing Inserting this into (8.8), and arguing as above that D ˜ft,˜gt(q) ≪qε˜q2,n, gives Us ≪b1q−1/2 H b1,n˜q2,n P n+ε b1/2 1 ˜q2,n V + (q3H)1/3 P + b1/2 1,n ˜q2,n(V + (q3H)1/3) ! ≪b1 H q1/2 P n+ε(V + (q3H)1/3) 1 + q1/2 P ! ≪b1 H q1/2 P n+ε(V + (q3H)1/3) as claimed. as claimed. Finally, suppose that η = n. Invoking the hypothesis that q ⩽P 2−4/(n+2), we may use Proposition 7.3 for arbitrary h, yielding the bound Us ≪b1P n+ε. Summing the contribution from all possible values of s and η, we arrive at the bound stated in the lemma. □ 9. Weyl differencing We now describe an approach parallel to our main argument. To bound the exponential sum in (3.12), we shall then use a result of Browning and Prendiville-[3, Lemma 3.3], with d = 3. This can be summarised as applying three consecutive Weyl differencing steps to the cubic exponential sum Sh(α). Whenever h ̸= 0, this produces the bound Sh(a/q + z) ≪P n+ε P −2 + q|z|H + qP −3 + (q|z|P 3)−1n/8 . (9.1) (It follows from the nonsingularity of F that the cubic part of Fh does not vanish, for example as a special case of Lemma 8.2, so it is indeed a cubic polynomial.) Inserting this bound into (It follows from the nonsingularity of F that the cubic part of Fh does not vanish, for example as a special case of Lemma 8.2, so it is indeed a cubic polynomial.) Inserting this bound into O. MARMON AND P. VISHE 36 Lemma 3.4 and adding the contribution from h = 0, yields Lemma 3.4 and adding the contribution from h = 0, yields (9.2) I(q, t) ≪P n−1/2+εq(t + 1 HP 3 )H−(n−1)/2 × n 1 + Hn/2 P −n/8 + (qtH)n/16 + (qP −3)n/16 + (qtP 3)−n/16o . Alternatively, we may apply Weyl differencing four times to the original quartic exponential sum. We then obtain a bound Alternatively, we may apply Weyl differencing four times to the original quartic exponential sum. We then obtain a bound S(a/q + z) ≪P n+ε q|z| + q−1|z|−1P −4n/24 , or, if one will, I(q, t) ≪P n+εqt  qt + q−1t−1P −4n/24 . (9.3) I(q, t) ≪P n+εqt  qt + q−1t−1P −4n/24 . (9.3) 10. Bounds for the minor arc contribution, nonsingular case 10. Bounds for the minor arc contribution, nonsingular case In this section we shall combine several different approaches to achieve the bound Sm = O(P n−4−ψ) in Proposition 2.9. First we observe that when estimating the integrals comprising Sm, we may replace the integrand |pq(z)S(q, z)| by |S(q, z)|. Indeed, by (1.3), this happens at the cost of introducing a dependence on the parameter θ in the implied constant. We divide the ranges for q and its factors b1, b2, q3 into O(Qε) dyadic intervals (10.1) R < q ⩽2R, R1 < b1 ⩽2R1, R2 < b2 ⩽2R2, R3 < q3 ⩽2R3, (10.1) ( ) q , 1 1 1, 2 2 2, 3 q3 (Note that our notation differs from that in [2, (9.4)]). We then put (Note that our notation differs from that in [2, (9.4)]). We then put (10.2) K(t, R, R) := X q,(10.1) Z t⩽|z|⩽2t |S(q, z)| dz = X q,(10.1) I(q, t) (10.2) for any t, where R := (R1, R2, R3). The quantity K(t, R, R) is relevant for the minor arcs estimate only if for any t, where R := (R1, R2, R3). The quantity K(t, R, R) is relevant for the minor arcs estimate only if (10.3) R ⩽Q, R1R2R3 ≍R, t ⩽(RQ)−1+θ, where A ≍B stands for the usual notation that A ≪B ≪A. In addition, either t ⩾P −4+∆ or R ⩾P ∆. (10.4) t ⩾P −4+∆ or R ⩾P ∆. (10.4) Clearly we then have ) Sm ≪P ε max t,R,R satisfying (10.3)+(10.4) K(t, R, R) + O(P n−5), where the error term arises from very small values of t, say |t| ⩽P −10. What follows in this section is a delicate comparison of various estimates that we have obtained so far to establish the bound K(t, R, R) ≪P n−4−ε, for every choice t, R, R relevant to the minor arcs contribution. We will start by comparing three main bounds. The first bound is obtained by an application of van der Corput dif- ferencing followed by Weyl differencing in (9.2), which gives rise to Proposition 10.1 below. This bound is sufficient, unless at least one of (10.7), (10.8) or (10.9) is violated, which will be assumed henceforth. 10. Bounds for the minor arc contribution, nonsingular case When t is not very small, we further compare the Weyl differencing bound in Proposition 10.2 (obtained from (9.3)) and our main bound in Proposition 10.3 (a ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 37 consequence of Proposition 8.1). This delicate optimisation is carried in Propositions 10.4 and 10.5. Proposition 10.4 provides a satisfactory bound for all the contribution from all terms in (8.2), except from a part of bounds coming from terms Yn−2 and Y0. The comparison of these bounds with those from Proposition 10.2 is rather challenging. To this end, we introduce variables Z and α via (10.24), and view the problem of comparing these contributions as a rational linear optimisation problem in Z and α. We are thus able to calculate an explicit minimum value for each of these, using Mathematica. Finally, when t is very small, our averaged van der Corput bound in Proposition 10.3 is rather wasteful, and we instead need to use an alternate point-wise van der Corput bound in Lemma 3.1 and compare it with Proposition 10.2 to achieve a satisfactory bound. p y Let us put B1 = R1R2 for short, and introduce the quantity B2 := B1R1/3 3 = R1R2R1/3 3 . The significance of B2 is explained by the bound The significance of B2 is explained by the bound (10.6) X q,(10.1) 1 ≪R1R1/2 2 R1/3 3 ⩽B2, (10.6) following from Lemma 2.5. In other words, B2 denotes a suitable upper bound for the number of q’s satisfying (10.1). We seemingly give up a factor of R1/2 2 here. However, this is firstly due to the fact that this saving is not necessary for us, and secondly, since this simplifies our linear optimisation process later by allowing us to work with only two variables Z and α (see (10.24)). We next put T := Rt and note that (10.3) implies following from Lemma 2.5. In other words, B2 denotes a suitable upper bound for the number of q’s satisfying (10.1). We seemingly give up a factor of R1/2 2 here. However, this is firstly due to the fact that this saving is not necessary for us, and secondly, since this simplifies our linear optimisation process later by allowing us to work with only two variables Z and α (see (10.24)). 10. Bounds for the minor arc contribution, nonsingular case We next put T := Rt and note that (10.3) implies T ⩽Q−1+2θ ⩽P −8/5−ϕ/2 if only ϕ ⩾8θ, say, which we may assume from now on. if only ϕ ⩾8θ, say, which we may assume from now on. We are now ready to record the bounds for K(t, R, R) obtained by the Weyl differencing procedure in Section 9. The first alternative (one van der Corput differencing step followed by three Weyl differencing steps) provides the following bound: Proposition 10.1. We have the bound K(t, R, R) ≪P n−4−ϕ/4 provided that n ⩾21 and the following three conditions are satisfied: B2 ⩽P 4n 45 −179 90 , B2 ⩽P 4n 45 −179 90 , (10.7) T ⩾B 16 n−17 2 P −3n−59 n−17 +4ϕ, (10.8) T ⩾B−2/(n+1) 2 R1−2/(n+1)P −3−1/(n+1). (10.9) B2 ⩽P 4n 45 −179 90 , (10.7) (10.7) T ⩾B 16 n−17 2 P −3n−59 n−17 +4ϕ, (10.8) T ⩾B−2/(n+1) 2 R1−2/(n+1)P −3−1/(n+1). (10.9) T ⩾B 16 n−17 2 P −3n−59 n−17 +4ϕ, T ⩾B−2/(n+1) 2 R1−2/(n+1)P −3−1/(n+1). (10.9) Proof. By (9.2) we get K(t, R, R) ⩽ X q,(10.1) I(q, t) ≪B2R(t + 1 HP 3 )P n−1/2+εH−(n−1)/2 × n 1 + Hn/2 P −n/8 + (RtH)n/16 + (RP −3)n/16 + (RtP 3)−n/16o . (10.10) K(t, R, R) ⩽ X q,(10.1) I(q, t) ≪B2R(t + 1 HP 3 )P n−1/2+εH−(n−1)/2 × n 1 + Hn/2 P −n/8 + (RtH)n/16 + (RP − (10.10) We put H := B2/(n−1) 2 T 2/(n−1)P 7/(n−1)+cϕ = (B2TP 7/2)2/(n−1)P cϕ, H := B2/(n−1) 2 T 2/(n−1)P 7/(n−1)+cϕ = (B2TP 7/2)2/(n−1)P cϕ, O. MARMON AND P. VISHE 38 for some suitable constant c. The upper bound T ≪P −8/5−ϕ/2 implies that 1 ⩽H ⩽P as required, for n in the required range. Furthermore, the lower bound (10.9) implies that we always have t ⩾(HP 3)−1, allowing us to simplify (10.10) to for some suitable constant c. The upper bound T ≪P −8/5−ϕ/2 implies that 1 ⩽H ⩽P as required, for n in the required range. 10. Bounds for the minor arc contribution, nonsingular case Furthermore, the lower bound (10.9) implies that we always have t ⩾(HP 3)−1, allowing us to simplify (10.10) to 10.11) P −n+4K(t, R, R) ≪B2TP 7/2+ε H(n−1)/2 n 1 + Hn/2 P −n/8 + (TH)n/16 + (TP 3)−n/16o Our choice of H implies that the contribution from the term ’1’ inside the brackets is ≪P −ϕ/2, ≪P −ϕ/2, say, provided only that c ⩾2/(n −1) and ϕ ⩾2ε. Note that the hypothesis (10.7) implies that y ( ) (10.12) B2TP 7/2 ⩽P 4(n−1)/45−ϕ/2. The contribution from the term P −n/8 is then ≪(B2TP 7/2)n/(n−1)P −n/8+cϕ/2+ε ≪P −13n/360+(c−1)ϕ/2+ε, ( ) (10.12) B2TP 7/2 ⩽P 4(n−1)/45−ϕ/2. The contribution from the term P −n/8 is then (10.12) B2TP 7/2 ⩽P 4(n−1)/45−ϕ/2. (10.12) which is clearly admissible for any n, provided c ⩽1 and ε is chosen small enough. The hypothesis (10.7) also ensures that the term (TH)n/16 gives an admissible contribution Indeed, by (10.12), this contribution is ≪B2TP 7/2+εH(n+8)/16T n/16 ≪(B2TP 7/2)9n/(8(n−1))P −n/10−nϕ(1/2−c)/16+cϕ/2+ε ≪B2TP 7/2+εH(n+8)/16T n/16 ≪ ≪P −nϕ(1/2−c)/16+ε ≪P −ϕ/4 ≪P −nϕ(1/2−c)/16+ε ≪P −ϕ/4 provided that n ⩾16, c ⩽1/8 and ϕ ⩾8ε. provided that n ⩾16, c ⩽1/8 and ϕ ⩾8ε. Finally, under the hypothesis (10.8), the contribution from the term (TP 3)−n/16 p , / ϕ Finally, under the hypothesis (10.8), the contribution from the term (TP 3)−n/16 is ≪(B2TP 7/2)n/(n−1)(TP 3)−n/16P ε+cϕ/2 ≪(B2TP 7/2)n/(n−1)(TP 3)−n/16P ε+cϕ/2 ≪Bn/(n−1) 2 P −n(3n−59)/(16(n−1))T −n(n−17)/(16(n−1))P ε+cϕ/2 ≪P −ϕ+ε+cϕ/2 ≪P −ϕ/4, provided that n ⩾21, c ⩽1 and ϕ ⩾4ε. It is thus possible to choose c and ε such that a these bounds are satisfied. □ ≪(B2TP 7/2)n/(n−1)(TP 3)−n/16P ε+cϕ/2 ≪Bn/(n−1) 2 P −n(3n−59)/(16(n−1))T −n(n−17)/(16(n−1))P ε+cϕ/2 ≪P −ϕ+ε+cϕ/2 ≪P −ϕ/ ≪Bn/(n−1) 2 P −n(3n−59)/(16(n−1))T −n(n−17)/(16(n−1))P ε+cϕ/2 ≪P −ϕ+ε+cϕ/2 ≪P −ϕ/4, ≪ 2 ≪ ≪ , provided that n ⩾21, c ⩽1 and ϕ ⩾4ε. It is thus possible to choose c and ε such that all these bounds are satisfied. □ Remark. In the case n = 30, the conditions in Proposition 10.1 read B2 ⩽P 61/90, T ⩾B16/13 2 P −31/13+4ϕ and T ⩾B−2/31 2 R29/31P −94/31. The approach featuring four consecutive Weyl differencing steps produces the following bound, where δ > 0 is a parameter to be chosen at a later stage. Alternatively, if T ⩾P −2, say, then T −1P −4 ⩽T, so we get K(t, R, R) ≪B2T 1+n/24P n+ε. Alternatively, if T ⩾P −2, say, then T −1P −4 ⩽T, so we get K(t, R, R) ≪B2T 1+n/24P n+ε. □ Next we give the estimate for K(t, R, R) coming from our main approach, where only one van der Corput differencing step was carried out. To this end we define the quantity B3 := B1/2 1 R1/3 3 , assuming the role of B2 in the previous two results. The saving here by of a factor of R1/2 1 over B2 is a major achievement of our van der Corput differencing method devised in Section 3. osition 10.3. Provided that R ⩽P 2−4/(n+2), one has K(t, R, R) ≪B3R(t + 1 HP 3 )P n−1/2+εH−(n−1)/2 (1 + Y0 + · · · + Yn−1)1/2 , where Yη := Hn−η R(n−η)/2 Xη for any 0 ⩽η ⩽n −1, with Xη := V n−η + R1/2 1  R1/2 3 V n−η−1 + (R3H)(n−η)/3 for 0 ⩽η ⩽n −2, and for 0 ⩽η ⩽n −2, and Xn−1 := V + (R3H)1/3, where V = R P max{1, √ tHP 3}. Note that there is a slight abuse of notation in that we have reused the same letters for the quantities V , Xη and Yη although replacing q, b1, q3 and |z| by R, R1, R3 and t, respectively, in their definition. Proof. Suppose that q satisfies (10.1), and recall the bound for I(q, t) from Lemma 3.3. We employ Proposition 8.1 for each z in the range t ⩽|z| ⩽max  2t, t + 1 HP 3−ε , yielding I(q, t) ≪R1/2 1 R2R3  t + 1 HP 3  P n−1/2+εH−(n−1)/2 (1 + Y0 + · · · + Yn−1)1/2 . Indeed, the shape of V and the presence of a factor P ε in the bound allows us to replace |z| by t in the final estimate, although slightly larger values of |z| are also considered. The asserted bound now follows from (10.6). □ We shall later choose the parameter ϕ to be fairly small, so we may certainly assume that Q ⩽P 2−4/(n+2), as required for Proposition 10.3. Let K′(t, R, R) := B3R(t + 1 HP 3 )P n−1/2+εH−(n−1)/2, corresponding to the contribution to K(t, R, R) from the term ’1’ in the expression in brackets in Proposition 10.3. Proposition 10.2. Suppose that Proposition 10.2. Suppose that T ⩾min{B24/(n−24) 2 P −4+δ, P −2}. Then we have the estimate K(t, R, R) ≪B2T 1+n/24P n+ε + P n−4−δ(n/24−1)+ε. Proof. By (9.3) we get K(t, R, R) ⩽ X q,(10.1) I(q, t) ≪B2TP n+ε T + T −1P −4n/24 . Let us assume that T ⩾B24/(n−24) 2 P −4+δ. Then we have K(t, R, R) ≪B2T 1+n/24P n+ε + B2T 1−n/24P n−n/6+ε ≪B2T 1+n/24P n+ε + P n−4−(n−24)δ/24+ε. ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 39 Alternatively, if T ⩾P −2, say, then T −1P −4 ⩽T, so we get K(t, R, R) ≪B2T 1+n/24P n+ε. Alternatively, if T ⩾P −2, say, then T −1P −4 ⩽T, so we get K(t, R, R) ≪B2T 1+n/24P n+ε. We put H = max{H1, H2}, where where H1 = B2/(n−1) 3 T 2/(n−1)P 36/(5(n−1)), H2 = B2/(n+1) 3 R2/(n+1)P 6/(5(n+1)), (10.14) (10.14) 40 O MARMON AND P VISHE O. MARMON AND P. VISHE 40 a choice that produces the bound K′(t, R, R) ≪P n−4−1/10+ε. H1 and H2 are chosen such that when H1 ⩾H2, then HP 3t ⩾1 and vice versa. This can be easily checked by setting H1 = H2, which in turn implies that H1 and H2 are chosen such that when H1 ⩾H2, then HP 3t ⩾1 and vice versa. This can be easily checked by setting H1 = H2, which in turn implies that T = Rt = B−2/(n+1) 3 R1−2/(n−1)P −18/5+3/5−6/(5(n+1)) = R(H2P 3)−1. Let us now treat the other terms. Our choice of H guarantees that for the other terms to be admissible, it is enough to check that the the remaining terms satisfy the following bound: Y0, · · · , Yn−1 ≪P 1/5−4ε. For 0 ⩽η ⩽n −2, we more precisely write For 0 ⩽η ⩽n −2, we more precisely write Xη ≪X ′ η + X ′′ η , where, for 0 ⩽η ⩽n −2, X ′ η = V n−η + B1/2 1 R1/2 3 V n−η−1, X ′′ η = B1/2 1 (R3H)(n−η)/3, 1/3 where, for 0 ⩽η ⩽n −2, X ′ η = V n−η + B1/2 1 R1/2 3 V n−η−1, X ′′ η = B1/2 1 (R3H)(n−η)/3, X ′ n−1 = V, X ′′ n−1 = (R3H)1/3. Note that V ≍R/P + H1/2R1/2P 1/2T 1/2 = R1/2V0, say, where V0 = R1/2/P + H1/2P 1/2T 1/2. X ′ η = V n−η + B1/2 1 R1/2 3 V n−η−1, X ′′ η = B1/2 1 (R3H)(n−η)/3, n 1 n 1 ( ) Note that V ≍R/P + H1/2R1/2P 1/2T 1/2 = R1/2V0, say, where V0 = R1/2/P + H1/2P 1/2T 1/2. Note that V ≍R/P + H1/2R1/2P 1/2T 1/2 = R1/2V0, say, where We may then in turn write Yη ≪Y′ η + Y′′ η , where Y′ η = Hn−η R(n−η)/2 X ′ η, Y′′ η = Hn−η R(n−η)/2 X ′′ η . Upon inspecting the shape of these expressions, one easily sees that 1 + Y0 + · · · + Yn−1 ≪1 + Y′ 0 + Y′′ 0 + Y′ n−2 + Y′′ n−2 + Y′′ n−1. Alternatively, if T ⩾P −2, say, then T −1P −4 ⩽T, so we get K(t, R, R) ≪B2T 1+n/24P n+ε. (10.16) We begin by inspecting the contribution from the Y′ η terms. First we simplify the expression to obtain Y′ η ≪Hn−ηV n−η−1 0 (V0 + 1), (10.17) Y′ η ≪Hn−ηV n−η−1 0 (V0 + 1), Y′ η ≪Hn−ηV n−η−1 0 (V0 + 1), (10.17) the key observation here being that B1R3 ≪R. For η = 0, this gives Y′ 0 ≪HnV n−1 0 (V0 + 1) . the key observation here being that B1R3 ≪R. For η = 0, this gives Y′ 0 ≪HnV n−1 0 (V0 + 1) . Y′ 0 ≪HnV n−1 0 (V0 + 1) . Note that the expressions for H and V0 contain non-negative powers of R, B3 and T. Therefore, the maximum value of these expressions is achieved when (10.18) B3 ≍R1/2 ≍P 4/5+ϕ/2 and T ≍P −8/5−ϕ/2. B3 ≍R1/2 ≍P 4/5+ϕ/2 and T ≍P −8/5−ϕ/2. Inserting these bounds, we get Inserting these bounds, we get H1 = P 8/5(n−1)+ϕ/(n−1)P −16/5(n−1)−ϕ/(n−1)P 36/(5(n−1)) = P 28/5(n−1), (10.19) H2 = P 8/5(n+1)P 16/5(n+1)+2ϕ/(n+1)P 6/5(n+1) = P 6/(n+1)+3ϕ/(n+1). (10.20) (10.20) Note that H2 ⩾H1, when n ⩾29. Thus, V0 ≍R1/2/P ≍P ϕ/2−1/5. For a small enough value of ϕ, we may assume that V0 ≪1. Note that H2 ⩾H1, when n ⩾29. Thus, V0 ≍R1/2/P ≍P ϕ/2−1/5. For a small enough value of ϕ, we may assume that V0 ≪1. ϕ y Upon using this value of V0, we get that ϕ y Upon using this value of V0, we get that ϕ Upon using this value of V0, we get that Y′ 0 ≪Hn 2 V n−1 0 ≪P 6n/(n+1)+3ϕ−(n−1)/5+(n−1)ϕ/2 ≪P 1/5−6/31+nϕ, Y′ 0 ≪Hn 2 V n−1 0 ≪P 6n/(n+1)+3ϕ−(n−1)/5+(n−1)ϕ/2 ≪P 1/5−6/31+nϕ, for n ⩾30, which is an admissible contribution, for a small enough value of ϕ. for n ⩾30, which is an admissible contribution, for a small enough value of ϕ. ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 41 Next, we consider η = n −2, where we have Y′ n−2 ≪H2V0(V0 + 1). Again, the right hand side is non-decreasing in B3, R, T, and therefore the maximum value here is reached when (10.18) holds. Replacing the corresponding values of H2 and V0, we get that Y′ n−2 ≪P 12/(n+1)+6ϕ/(n+1)P −1/5+ϕ/2 ≪P 1/5−2/155+ϕ for n ⩾30, which is again an admissible contribution for a small enough value of ϕ. (10.26) Let us first examine this range. In the case η = 0, (10.22) implies that Let us first examine this range. In the case η = 0, (10.22) implies that P −n+4K′′ 0 (t, R, R)P −n+4 ≍K′′ 0,2(t, R, R) ≪B 4n 3(n+1) 3 P 4n 5(n+1) −1 10 +εR1/4 1 Rn/6 3 R 4n 3(n+1) −n 4 ≪P h2(Z,α)+ε, say, where say, where h2(Z, α) = 4n 3(n + 1) Z 3 + α 6  + n 6 (Z −α) + α 4 +  4n 3(n + 1) −n 4  Z + 4n 5(n + 1) −1 10. One may check that h2(Z, α) is decreasing as a function of n for admissible values of Z and α. This feature will repeat in all the bounds that we will derive in this section. Therefore we may assume that n = 30 and thus h2(Z, α) = −145 186Z −1687 372 α + 209 310. (10.27) h2(Z, α) = −145 186Z −1687 372 α + 209 310. Alternatively, if T ⩾P −2, say, then T −1P −4 ⩽T, so we get K(t, R, R) ≪B2T 1+n/24P n+ε. VISHE 42 so that R3 ≍P Z−α and so that R3 ≍P Z−α and B2 ≍P Z 3 + 2α 3 , B3 ≍P Z 3 + α 6 . B2 ≍P Z 3 + 2α 3 , B3 ≍P Z 3 + α 6 . Our strategy will be to express our bounds for the quantities Y′′ η,j as functions of Z and α, and check that the desired bounds hold throughout the region Ωdefined by Our strategy will be to express our bounds for the quantities Y′′ η,j as functions of Z and α, and check that the desired bounds hold throughout the region Ωdefined by 0 ⩽α ⩽Z ⩽8 5. (10.25) 0 ⩽α ⩽Z ⩽8 5. (10.25) (10.25) Note that the Weyl bound contribution roughly grows as R increases, and the contributions in (10.21) and (10.22) increase as R decreases. Thus, it is natural to expect the minimum value is obtained when Z is in the intermediate range between 1 and 8/5. This is asserted by our findings in Appendix A. g pp Recall that H = H2 if and only if Recall that H = H2 if and only if T ⩽R(H2P 3)−1 = B−2/(n+1) 3 R1−2/(n+1)P −6/(5(n+1))−3. Alternatively, if T ⩾P −2, say, then T −1P −4 ⩽T, so we get K(t, R, R) ≪B2T 1+n/24P n+ε. Finally, Y′′ n−1 ≪H4/3R−1/6 = max{R4/3(n−1)P 16/3(n−1), R4/(n+1)P 8/5(n+1)}R−1/6 ≪P 16/3(n−1) ≪P 1/5−0.01, ≪P 16/3(n−1) ≪P 1/5−0.01, for n ⩾30. We summarise our findings in the following result. Proposition 10.4. Suppose that n ⩾30. There exist absolute constants ϕ0, ε0 > 0, depending only on n, such that the estimate K(t, R, R) ≪ε P n−4−ε + P n−4−1/10+ε(Y′′ 0 + Y′′ n−2)1/2 holds for any ε ⩽ε0, provided that ϕ ⩽ϕ0. Remark. With some more work, appealing to the previously mentioned bound of Serre [23, Chapter 13] to improve the exponent in (6.6), the conclusion of Proposition 10.4 could also have been obtained for n = 29. However, satisfactorily handling contribution from the remaining terms Y′′ 0 , Y′′ n−2 in the case n = 29 requires substantially new arguments. We defer the necessary refinements of our method to a forthcoming paper. It remains to treat the contributions from Y′′ η for η = 0, n −2. We begin by writing Y′′ η ≪Y′′ η,1 + Y′′ η,2, where Y′′ η,j denotes the contribution obtained by replacing H with Hj. Let K′′ j,η(t, R, R), for η ∈{0, n −2}, j ∈{0, 1}, denote the corresponding contribution to K(t, R, R) from the term Y′′ j,η. More explicitly, P −n+4K′′ η,1(t, R, R) ≪P −1/10+εH2(n−η)/3 1 R1/4 1 R(n−η)/6 3 R−(n−η)/4 = (B3T) 4(n−η) 3(n−1) P 24(n−η) 5(n−1) −1 10 +εR1/4 1 R(n−η)/6 3 R−(n−η)/4, (10.21) P −n+4K′′ η,2(t, R, R) ≪P −1/10+εH2(n−η)/3 2 R1/4 1 R(n−η)/6 3 R−(n−η)/4 = (B3R) 4(n−η) 3(n+1) P 4(n−η) 5(n+1) −1 10 +εR1/4 1 R(n−η)/6 3 R−(n−η)/4. (10.22) (10.21) (10.21) (10.22) We will compare these contributions to the relevant contribution coming from Proposition 10.2, which we denote by We will compare these contributions to the relevant contribution coming from Proposition 10.2, which we denote by KWeyl(t, R, R) := B2T 1+n/24P n+ε. Thus we shall assume until further notice that (10.23) T ⩾min{B24/(n−24) 2 P −4+δ, P −2}, (10.23) so that Proposition 10.2 applies. The remaining range where T is very small will be treated at the end of this section by different means. W so that Proposition 10.2 applies. The remaining range where T is very small will be treated at the end of this section by different means. B1 = P α, R = P Z, (10.24) B1 = P α, R = P Z, O. MARMON AND P. (10.27) In this case we have P −n+4KWeyl(t, R, R) ≪B2  B−2/(n+1) 3 R1−2/(n+1)P −6/(5(n+1))−31+n/24 P 4+ε ≪B2B − 2 n+1 24+n 24 3 R n−1 n+1 24+n 24 P 4−15n+21 5(n+1) 24+n 24 +ε ≪P w2(Z,α)+ε, where w2(Z, α) = Z 3 + 2α 3 − 24 + n 12(n + 1) Z 3 + α 6  + (n −1)(24 + n) 24(n + 1) Z + 4 −(15n + 21)(24 + n) 120(n + 1) . Again, this is decreasing in n, and for n = 30 translates to Again, this is decreasing in n, and for n = 30 translates to w2(Z, α) = 889 372Z + 239 372α −1759 620 . w2(Z, α) = 889 372Z + 239 372α −1759 620 . (10.28) (10.28) One now reaches a simple linear optimization problem of checking whether One now reaches a simple linear optimization problem of checking whether max (Z,α)∈Ωmin{h2(Z, α), w2(Z, α)} < 0. max (Z,α)∈Ωmin{h2(Z, α), w2(Z, α)} < 0. Indeed, it is then obvious that min{K′′ η(t, R, R), KWeyl(t, R, R)} ≪P n−4−ϕ/4, say, provided that ϕ and ε are chosen small enough. These quantities may be messy to compute by hand, but could be easily computed using a simple code in Mathematica/sage. We will ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 43 attach these codes in the appendix, for the aid of the reader. Using this, it is easy to check that the required maximum is < −0.1, giving an admissible bound in the range (10.26). In the case of η = n −2, (10.22) implies that P −n+4K′′ n−2(t, R, R) ≍P −n+4K′′ n−2,2(t, R, R) ≪(B3R) 8 3(n+1) P 8 5(n+1) −1 10 +εB1/4 1 R1/3 3 R−1/2 ≪R 4 (n+1) −1 6 P 8 5(n+1) −1 10 +ε ≪P 8 5(n+1) −1 10 +ε ≪P 8/155−1/10+ε, which gives an admissible contribution. Here we have again used the fact that B1R3 ≍R. We now consider the complementary range which gives an admissible contribution. Here we have again used the fact that B1R3 ≍R. We now consider the complementary range (10.29) T > B−2/(n+1) 3 R1−2/(n+1)P −6/(5(n+1))−3. (10.29) where we always have H = H1. Observe that we only need to study the range where Propo- sition 10.1 fails. Let us split that range up into three parts as follows: where we always have H = H1. (10.27) Observe that we only need to study the range where Propo- sition 10.1 fails. Let us split that range up into three parts as follows: B−2/(n+1) 3 R1−2/(n+1)P −3−6/(5(n+1)) < T ⩽B 16 n−17 2 P −3n−59 n−17 +4ϕ, (10.30) B−2/(n+1) 3 R1−2/(n+1)P −3−6/(5(n+1)) < T ⩽B−2/(n+1) 2 R1−2/(n+1)P −3−1/(n+1), (10.31) B2 ⩾P 4n 45 −179 90 . (10.32) B2 ⩾P 4n 45 −179 90 . Suppose first that (10.30) applies. Then, inserting the upper bound for T, we have Suppose first that (10.30) applies. Then, inserting the upper bound for T, we have P −n+4K′′ η(t, R, R) ≍P −n+4K′′ η,1(t, R, R) ≪B 4(n−η) 3(n−1) 3 B 64(n−η) 3(n−1)(n−17) 2 P 24(n−η) 5(n−1) −3n−59 n−17 4(n−η) 3(n−1) +c1ϕ−1/10+εB1/4 1 R(n−η)/6 3 R−(n−η)/4 ≪B 4(n−η) 3(n−1) 3 B 64(n−η) 3(n−1)(n−17) 2 P 24(n−η) 5(n−1) −3n−59 n−17 4(n−η) 3(n−1) +c1ϕ−1/10+εB1/4 1 R(n−η)/6 3 R−(n−η)/4 ≪P h1(Z,α,η)+c1ϕ+ε ≪P h1(Z,α,η)+c1ϕ+ε, say. Here, and in the sequel, ci’s will denote absolute constants whose precise value is imma- terial to our arguments. These constants may have an implicit dependence on η and n only. The cases of interest for us are η = 0 and n −2. More explicitly, say. Here, and in the sequel, ci’s will denote absolute constants whose precise value is imma- terial to our arguments. These constants may have an implicit dependence on η and n only. The cases of interest for us are η = 0 and n −2. More explicitly, 0) = 4n 3(n −1) Z 3 + α 6  + 64n 3(n −1)(n −17) Z 3 + 2α 3  + 1 4α + n 6 (Z −α) −n 4 Z + 24n 5(n −1) −3n −59 n −17 4n 3(n −1) −1 10. For n = 30 this gives h1(Z, α, 0) = −115 78 Z −15329 4524 α + 5943 3770. Si il l 30 this gives h1(Z, α, 0) = −115 78 Z −15329 4524 α + 5943 3770. h1(Z, α, 0) = −115 78 Z −15329 4524 α + 5943 3770. Similarly, h1(Z, α, n −2) = 8 3(n −1) Z 3 + α 6  + 128 3(n −1)(n −17) Z 3 + 2α 3  + 1 4α + 1 3(Z −α) −1 2Z + 48 5(n −1) −3n −59 n −17 8 3(n −1) −1 10, which for n = 30 translates to slates to h1(Z, α, n −2) = −23 234Z + 101 13572α + 133 11310. If we instead feed the upper bound for T into the bound in Proposition 10.2, we get If we instead feed the upper bound for T into the bound in Proposition P −n+4KWeyl(t, R, R) = B2T 1+n/24P 4+ε ≪B 1+ 16 n−17 24+n 24 2 P 4−3n−59 n−17 24+n 24 +c2ϕ+ε ≪P w1(Z,α)+c2ϕ+ε, O. MARMON AND P. VISHE 44 where where w1(Z, α) =  1 + 16 n −17 24 + n 24  Z 3 + 2α 3  + 4 −3n −59 n −17 24 + n 24 .  Again for n = 30 one has w1(Z, α) = 49 39Z + 98 39α −71 52. 39 Using linear optimization again one checks that Using linear optimization again one checks that ax α)∈Ωmin{h1(Z, α, 0), w1(Z, α)} ⩽−0.01, max (Z,α)∈Ωmin{h1(Z, α, n −2), w1(Z, α)} ⩽−0.02. max (Z,α)∈Ωmin{h1(Z, α, 0), w1(Z, α)} ⩽−0.01, max (Z,α)∈Ωmin{h1(Z, α, n −2), w1(Z, α)} ⩽−0.02. Next we will treat the range (10.31). Again using H = H1 and the upper bound for T in (10.31), we have Next we will treat the range (10.31). Similarly, Again using H = H1 and the upper bound for T in (10.31), we have P −n+4K′′ η(t, R, R) ≍P −n+4K′′ η,1(t, R, R) ≪B 4(n−η) 3(n−1) 3 B − 8(n−η) 3(n+1)(n−1) 2 R 4(n−η) 3(n+1) −n−η 4 P −4(n−η) 3(n−1) 3n+4 n+1 + 24(n−η) 5(n−1) −1 10 +εB1/4 1 R(n−η)/6 3 ≪P h3(Z,α,η)+ε, P −n+4K′′ η(t, R, R) ≍P −n+4K′′ η,1(t, R, R) ≪B 4(n−η) 3(n−1) 3 B − 8(n−η) 3(n+1)(n−1) 2 R 4(n−η) 3(n+1) −n−η 4 P −4(n 3(n h (Z α η)+ε P −n+4K′′ η(t, R, R) ≍P −n+4K′′ η,1(t, R, R) 4(n−η) 8(n−η) 4(n η) n η 4(n where h3(Z, α, 0) = 4n 3(n −1) Z 3 + α 6  − 8n 3(n + 1)(n −1) Z 3 + 2α 3  +  4n 3(n + 1) −n 4  Z + 1 4α + n 6 (Z −α) − 4n 3(n −1) 3n + 4 n + 1 + 24n 5(n −1) −1 10, h3(Z, α, n −2) = 8 3(n −1) Z 3 + α 6  − 16 3(n + 1)(n −1) Z 3 + 2α 3  +  8 3(n + 1) −1 2  Z + 1 4α + 1 3(Z −α) − 8 3(n −1) 3n + 4 n + 1 + 48 5(n −1) −1 10. Specialising to n = 30, we get Specialising to n = 30, we get h3(Z, α, 0) = −145 186Z −49403 10788α + 6141 8990, h3(Z, α, n −2) = −29 558Z −2329 32364α −1289 26970. The Weyl differencing bound in this range for T yields h3(Z, α, 0) = −145 186Z −49403 10788α + 6141 8990, h3(Z, α, n −2) = −29 558Z −2329 32364α −1289 26970 The Weyl differencing bound in this range for T yields The Weyl differencing bound in this range for T yields P −n+4KWeyl(t, R, R) ≪B2  B−2/(n+1) 2 R1−2/(n+1)P −1/(n+1)−31+n/24 P 4+ε ≪P w3(Z,α)+ε, ≪P w3(Z,α)+ε, where w3(Z, α) = Z 3 + 2α 3   1 − 2 n + 1 24 + n 24  + (n −1)(24 + n) (n + 1)24 Z + 4 − 3n + 4 n + 1  24 + n 24  . For n = 30, this becomes w3(Z, α) = 889 372Z + 53 93α −175 62 . Similarly, Clearly, the exponent h3(Z, α, n −2) ⩽−1289 26970 For n = 30, this becomes w3(Z, α) = 889 372Z + 53 93α −175 62 . Clearly, the exponent h3(Z, α, n −2) ⩽−1289 26970 suffices. In the other case, one calculates suffices. In the other case, one calculates max (Z,α)∈Ωmin{h3(Z, α, 0), w3(Z, α)} ⩽−0.008, max (Z,α)∈Ωmin{h3(Z, α, 0), w3(Z, α)} ⩽−0.008, giving an admissible bound in the range (10.31). giving an admissible bound in the range (10.31). ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 45 Finally, suppose that (10.32) applies. In this range, in some cases, it will be more convenient to refer to an earlier bound in Proposition 10.3 directly: Finally, suppose that (10.32) applies. In this range, in some cases, it will be more convenient to refer to an earlier bound in Proposition 10.3 directly: P −n+4K′′ η(t, R, R) ≪B3R(t + 1 HP 3 )P 7/2+εH−(n−1)/2(Y′′ η )1/2 ≪B3R(t + 1 HP 3 )P 7/2+εB1/4 1 H(1−η)/2(R3H)(n−η)/6R−(n−η)/4. P −n+4K′′ η(t, R, R) ≪B3R(t + 1 HP 3 )P 7/2+εH−(n−1)/2(Y′′ η )1/2 ≪B3R(t + 1 HP 3 )P 7/2+εB1/4 1 H(1−η)/2(R3H)(n−η)/6R−(n−η)/4. Again, we start with the case η = 0 first. Comparing with the bounds (10.19) and (10.20), one also always has H ⩽P 1/5, so that H1/2(R3H)n/6R−n/4 ≍H(n+3)/6B−n/4 2 ⩽P n+3 30 + 179n 360 −n2 45 ⩽P −239/60 for n ⩾30. This yields P −n+4K′′ 0 (t, R, R) ≪B3B1/4 1 (T + RP −3)P 7/2+εH1/2(R3H)n/6R−n/4 ≪RP −7/5+ϕP 7/2−239/60+ε ≪P −17/60+2ϕ+ε a bound which is clearly admissible. y For the contribution K′′ n−2(t, R, R), we start by writing P −n+4K′′ n−2(t, R, R) ≪P −1/10(Y′′ n−2)1/2 ≪P −1/10+εR−1/2B1/4 1 R1/3 3 H4/3. As a consequence, P −n+4K′′ n−2,1(t, R, R) ≪P −1/10+εR−1/2B1/4 1 R1/3 3 (B3T) 8 3(n−1) P 48 5(n−1) . P −n+4K′′ n−2,1(t, R, R) ≪P −1/10+εR−1/2B1/4 1 R1/3 3 (B3T) 8 3(n−1) P 48 5(n−1) . We insert the trivial upper bound T ⩽P −8/5 to obtain We insert the trivial upper bound T ⩽P −8/5 to obtain P −n+4K′′ n−2,1(t, R, R) ≪B 8 3(n−1) 3 P 16 3(n−1) −1 10 +εB1/4 1 R1/3 3 R−1/2. (10.33) The inequality (10.16) is still valid, and therefore, it is enough to only look at the corre- sponding terms. Therefore, we shall now estimate the different contributions K′ η(t, R, R), K′′ η(t, R, R) for η ∈{0, n −2} and K′′ n−1(t, R, R), named according to the contribution from the corresponding terms in (10.16). We begin by investigating the term K′ 0(t, R, R) first: The inequality (10.16) is still valid, and therefore, it is enough to only look at the corre- sponding terms. Therefore, we shall now estimate the different contributions K′ η(t, R, R), K′′ η(t, R, R) for η ∈{0, n −2} and K′′ n−1(t, R, R), named according to the contribution from the corresponding terms in (10.16). We begin by investigating the term K′ 0(t, R, R) first: P −n+4K′ η(t, R, R) ≪B3TP 4+εR−(n−η)/4V (n−η)/2 1 + R1/4V −1/2 . We note that when inserting the chosen value of H, we have B3, B2, R and T occurring to non-negative exponents, as long as η ⩽n −2 so it suffices to estimate this contribution when B2 3 ≍B2 ≍R ≍P 8/5+ϕ and T ≍P −2. In that case one has tHP 3 ≍HPR−1 ≍PR−1(B2 2B3P 2)2/n ≍P 1+4/nR−1+5/n ≍P −3/5+12/n−(n−5)ϕ/n ≪1 as soon as n > 20 Thus we may assume that V ≍R/P Then we get tHP 3 ≍HPR−1 ≍PR−1(B2 2B3P 2)2/n ≍P 1+4/nR−1+5/n ≍P −3/5+12/n−(n−5)ϕ/n ≪1 as soon as n > 20. Thus we may assume that V ≍R/P. Then we get P −n+4K′ 0(t, R, R) ≪B3TP 4+εRn/4P −n/2 1 + R−1/4P 1/2 ≪B3P 2+ε−n/10+nϕ/4 1 + P 1/10 ≪P −(n−29)/10+(n+2)ϕ/4+ε, tHP 3 ≍HPR−1 ≍PR−1(B2 2B3P 2)2/n ≍P 1+4/nR−1+5/n ≍P −3/5+12/n−(n−5)ϕ/n ≪1 as soon as n > 20. Thus we may assume that V ≍R/P. Then we get P −n+4K′ 0(t, R, R) ≪B3TP 4+εRn/4P −n/2 1 + R−1/4P 1/2 ≪B3P 2+ε−n/10+nϕ/4 1 + P 1/10 ≪P −(n−29)/10+(n+2)ϕ/4+ε, ≪B3P 2+ε−n/10+nϕ/4 1 + P 1/10 ≪P −(n−29)/10+(n+2)ϕ/4+ε, so an admissible contribution as soon as n ⩾30. We next observe that in this range, we have V ≪R/P < R1/2, which means that so an admissible contribution as soon as n ⩾30. We next observe that in this range, we have V so an admissible contribution as soon as n ⩾30. Similarly, By (10.32), one now has B 8 3(n−1) 3 B1/4 1 R1/3 3 R−1/2 ≪B 4 3(n−1) −1 4 1 R 8 9(n−1) −1 6 3 ≪B 4 3(n−1) −1 4 2 ≪P ( 4 3(n−1) −1 4 )( 4n 45 −179 90 ) mplying that implying that P −n+4K′′ n−2,1(t, R, R) ≪P 16 3(n−1) −1 10 +( 4 3(n−1) −1 4 )( 4n 45 −179 90 )+ε ≪P −0.05+ε, for n ⩾30. ⩾ We have now obtained a bound which is valid everywhere except for very small values of T. We have now obtained a bound which is valid everywhere except for very small values of T. Proposition 10.5. Suppose that n ⩾30 and that T ⩾min{B24/(n−24) 2 P −4+δ, P −2}. Proposition 10.5. Suppose that n ⩾30 and that T ⩾min{B24/(n−24) 2 P −4+δ, P −2}. Then we have the bound K(t, R, R) ≪P n−4 P −5δ/24 + P −ϕ/4 provided only that ϕ ⩽ϕ0, where ϕ0 is an absolute constant. In the remaining range, we shall employ the pointwise van der Corput differencing bound in Lemma 3.1. Using this result in place of Lemma 3.3 in the above argument, one obtains the bound K(t, R, R) ≪B3TP n+εH−n/2 1 + Y0 + · · · + Yn−1 1/2 . Let us put H := 1 + B4/n 2 (B3TP 4)2/n, O. MARMON AND P. VISHE 46 yielding yielding B3TP n+εH−n/2 ≪P n−4+εB−2 2 . Recall that we are assuming that T ⩽min{B24/(n−24) 2 P −4+δ, P −2}. Since n ⩾30, we have in particular T ⩽B4 2P −4+δ. On the other hand, by (10.4) we have either B3TP n+εH−n/2 ≪P n−4+εB−2 2 . B3TP n+εH−n/2 ≪P n−4+εB−2 2 . Recall that we are assuming that T ⩽min{B24/(n−24) 2 P −4+δ, P −2}. Since n ⩾30, we have in particular T ⩽B4 2P −4+δ. On the other hand, by (10.4) we have either T ⩾RP −4+∆ or R ⩾P ∆. Since R1/3 ≪B2 ≪R, we may conclude in both these cases that Since R1/3 ≪B2 ≪R, we may conclude in both these cases that B2 ≫P (∆−δ)/3. In the range in the contention, H ⩽B(4(n−12)/(n(n−24)) 2 B2/n 3 P 2δ/n. (10.33) H ⩽B(4(n−12)/(n(n−24)) 2 B2/n 3 P 2δ/n. (10.33) We next observe that in this range, we have V ≪R/P < R1/2, which means that P −n+4K′ n−2(t, R, R) ≪B−2 2 P ε(HR−1/2V + HR−1/4V 1/2) ≪B−2 2 P εHR−1/4V 1/2 ≪B4/n 2 (B3P 2)2/nP ε−1/2B−2 2 R1/4 ≪P ε−1/2+4/nR5/n−7/4 ≪P ε−1/2+4/n, clearly, for n ⩾3, rendering us with an appropriate contribution. Similarly, P −n+4K′′ n−1(t, R, R) ≪P εB−2 2 H1/2R−1/4R1/6 3 H1/6 ≪P εB−3/2 2 H2/3R−1/4 ≪P ε+4δ/3nB−3/2 2 R−1/4B8(n−12)/(3n(n−24)) 2 B4/(3n) 3 ≪P ε+4δ/3nB8(n−12)/(3n(n−24))+4/(3n)−7/4 2 ≪P ε+4δ/75B−31/100 2 ≪P ε−(∆−2δ)/10, for n ⩾25, giving a reasonable contribution as long as δ < ∆/4, say. We have not tried to optimise the exponents here. ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 47 Next, we have 4 2 2 /3 /4 1/4 /6 Next, we have R) ≪P εB−2 2 H2n/3R−n/4B1/4 1 Rn/6 3 ≪P ε+4δ/3B8(n−12)/(3(n−24))−2 2 B4/3 3 R−n/4B1/4 1 Rn/6 3 ≪P ε+4δ/3B8(n−12)/(3(n−24))−13/12−n/4 1 R8(n−12)/(9(n−24))−n/12−2/9 3 ≪P ε+4δ/3B−1/2 1 R−0.05 3 ≪P ε+4δ/3B−0.15 2 ≪P ε−δ/6 P −n+4K′′ 0 (t, R, R) for n ⩾30, provided that δ ⩽∆/31. Finally, for n ⩾30 we have for n ⩾30, provided that δ ⩽∆/31. Finally, for n ⩾30 we have for n ⩾30, provided that δ ⩽∆/31. Finally, for n ⩾30 we have P −n+4K′′ n−2(t, R, R) ≪B−2 2 P ε H R1/2 B1/4 1 (R3H)1/3 ≪B−2 2 P εB1/4 1 R1/3 3 R−1/2(B3B2(n−12)/(n−24) 2 P δ)8/(3n) ≪B−2 2 P εB1/4 1 R1/3 3 R−1/2(B3B6 2P δ)4/45 = B−22/15 2 B4/45 3 B1/4 1 R1/3 3 R−1/2P 4δ/45+ε ≪B−22/45 2 P 4δ/45+ε ≪P −22(∆−δ)/135+4δ/45+ε ≪P −δ+ε, under the previous assumption on δ. Choosing δ = ∆/31, and ε small enough, we have now finally verified the assertion in Proposition 2.9, where we may take ψ = min{ϕ/4, ∆/186} for any ϕ ⩽ϕ0. under the previous assumption on δ. Choosing δ = ∆/31, and ε small enough, we have now finally verified the assertion in Proposition 2.9, where we may take ψ = min{ϕ/4, ∆/186} for any ϕ ⩽ϕ0. 11. Bounds for the minor arcs contribution, general case In this section we shall give the inductive argument to deduce the Proposition 2.8 from the Proposition 2.9. Assume that dim Sing(X0) ⩾0. We subject the exponential sum S(q, z) = S(q, z; F, W, P, n) = q X∗ a=1 X x∈Zn W(x/P)e((a/q + z)F(x)) to a similar slicing argument to that used in the proof of Proposition 8.1. We may apply Lemma 4.1 for the single form F0, with Π = ∅, and with f = (f1, . . . , fn) = ∇F(Px0), where x0 is the point in (2.3), to obtain a linearly independent set of vectors e1, . . . , en with the properties listed there and such that the angle of e1 with f is at most π/3. We then split the sum above into cosets of the lattice spanned by e1, . . . , en−1, as in the proof of Proposition 8.1, to obtain S(q, z) = X t∈T St(q, z) = X t∈T S(q, z; Ft, Wt, P/L, n −1), say, where say, where Ft(y) = F(t + X yiei) and Wt(y) = W(P −1t + L−1 X yiei) for some L = O(1), and where T is a set of cardinality O(P). The sum over t can be moved outside to obtain for some L = O(1), and where T is a set of cardinality O(P). The sum over t can be moved outside to obtain (11.1) Sm = Sm(F, W, P, n) = X t Sm,t, Sm = Sm(F, W, P, n) = X t Sm,t, (11.1) O. MARMON AND P. VISHE 48 where where Sm,t = Sm(Ft, Wt, P ′, n −1) = X 1⩽q⩽P ∆ Z P −4+∆⩽|z|⩽(qQ)−1+θ |pq(z)||S(q, z; Ft, Wt, P ′, n −1)| dz + X P ∆⩽q⩽Q Z |z|⩽(qQ)−1+θ |pq(z)||S(q, z; Ft, Wt, P ′, n −1)| dz. θ |pq(z)||S(q, z; Ft, Wt, P ′, n −1)| dz Here we have put P ′ := P/L. We must then investigate whether the polynomials Ft and the weight functions Wt fulfil the hypotheses in Proposition 2.8, in order to invoke our induction hypothesis. Here we have put P ′ := P/L. We must then investigate whether the polynomials Ft and the weight functions Wt fulfil the hypotheses in Proposition 2.8, in order to invoke our induction hypothesis. 11. Bounds for the minor arcs contribution, general case yp Much like in the proof of Proposition 8.1, one checks that Wt ∈Wn−1 and ∥Ft∥P ′ ≪L4∥F∥P ≪∥F∥P . We shall now find M′ > 0 such that the conditions (2.15)–(2.16) hold with F, W, P replaced by Ft, Wt, P ′ and M replaced by M′. If t ∈Zn is arbitrary and y ∈Zn is such that x = t + Pn−1 i=1 yiei occurs in the exponential sums St(q, z), then we have ∇Ft(Py) = ∇F(Px).E, where we denote by E the n × (n −1)-matrix with the basis vectors e1, . . . , en−1 as columns. By (2.15) we then have |f1| ⩾MP 3. |f1| ⩾MP . Now, let y1, y2 ∈P ′ supp(Wt) and put xj = t + P i yiei ∈P supp(W) for j = 1, 2. If fj := ∇F(xj) and gj := ∇Ft(yj) then we have gj = fj.E for j = 1, 2. Thus we see that |g1 1| = |f1.e1| ⩾|f.e1| −|(f1 −f).e1|. |f1| Now, let y1, y2 ∈P ′ supp(Wt) and put xj = t + P i yiei ∈P supp(W) for j = 1, 2. If fj := ∇F(xj) and gj := ∇Ft(yj) then we have gj = fj.E for j = 1, 2. Thus we see that |g1,1| = |f1.e1| ⩾|f.e1| −|(f1 −f).e1|. |g1,1| = |f1.e1| ⩾|f.e1| −|(f1 −f).e1|. Since e1 was chosen to have an angle of at most π/3 to the vector f, we get Since e1 was chosen to have an angle of at most π/3 to the vector f, we get |g1,1| ⩾1 2|f|2|e1|2 −|f1 −f|2|e1|2 ⩾L 2 1 2|f|∞−√n|f1 −f|∞  . Using (2.15)–(2.16) the expression in brackets satisfies 1 2|f|∞−√n|f1 −f|∞⩾MP 3 2 − MP 3 8np (n −1)! ⩾MP 3 4 , so the condition (2.15) holds with M replaced by M′ = L4M 8 ≪M. F th h 1 2|f|∞−√n|f1 −f|∞⩾MP 3 2 − MP 3 8np (n −1)! ⩾MP 3 4 , so the condition (2.15) holds with M replaced by M′ = L4M 8 ≪M. M replaced by M′ = L4M 8 ≪M. (2.15) holds with M replaced by M′ = L4M 8 ≪M. M′ = L4M 8 ≪M. Furthermore, we have Furthermore, we have , |(g1)i −(g2)i| = |ei.(f1 −f2)| ⩽|ei|2|f1 −f2|2 ⩽L√n · MP 3 8n√ n! = M′(P ′)3 8n−1p (n −1)! , so that (2.16) also holds for M′. Appendix A. Mathematica code Here, we give the Mathematica code verifying our linear optimisation bounds in Section 10. Maximize[Min[-145/ 186 X - 1687 / 372 a + 209/ 310, 889/ 372 X + 239/ 372 a - 1759 / 620], Here, we give the Mathematica code verifying our linear optimisation bounds in Section 10 Maximize[Min[-145/ 186 X - 1687 / 372 a + 209/ 310, 889/ 372 X + 239/ 372 a - 1759 / 620], 0 ≤a ≤X ≤8/ 5 , {X, a}] - 1117 5895 , X →2177 1965 , a →0 Maximize[Min[-115/ 78 X - 15 329 / 4524 a + 5943 / 3770, 49/ 39 X + 98/ 39 a - 71/ 52], Maximize[Min[-115/ 78 X - 15 329 / 4524 a + 5943 / 3770, 49/ 39 X + 98/ 39 a - 71/ 52], 0 ≤a ≤X ≤8/ 5 , {X, a}] - 1469 123 540 , X →22 181 20 590 , a →0 Maximize[Min[-23/ 234 X + 101/ 13 572 a + 133/ 11 310, 49/ 39 X + 98/ 39 a - 71/ 52], Maximize[Min[-23/ 234 X + 101/ 13 572 a + 133/ 11 310, 49/ 39 X + 98/ 39 a - 71/ 52], 0 ≤a ≤X ≤8/ 5 , {X, a}] Maximize[Min[-23/ 234 X + 101/ 13 572 a + 133/ 11 310, 49/ 39 X + 98/ 39 a - 71/ 52], 0 ≤a ≤X ≤8/ 5 , {X, a}] 0 ≤a ≤X ≤8/ 5 , {X, a}] - 7213 349 260 , X →31 151 87 315 , a →31 151 87 315  Maximize[Min[-145/ 186 X - 49 403 / 10 788 a + 6141 / 8990, 889/ 372 X + 53/ 93 a - 175/ 62], 0 ≤a ≤X ≤8/ 5 , {X, a}] - 61 271 341 910 , X →63 032 56 985 , a →0 Maximize[Min[-145/ 186 X - 49 403 / 10 788 a + 6141 / 8990, 889/ 372 X + 53/ 93 a - 175/ 62], 0 ≤a ≤X ≤8/ 5 , {X, a}] - 61 271 341 910 , X →63 032 56 985 , a →0 0 ≤a ≤X ≤8/ 5 , {X, a}] 11. Bounds for the minor arcs contribution, general case Si so that (2.16) also holds for M′. Since d S Since dim Sing(X0 ∩H) = dim Sing(X0) −1, dim Sing(X0 ∩H) = dim Sing(X0) −1, dim Sing(X0 ∩H) = dim Sing(X0) −1, where H is the projective hyperplane spanned by the vectors e1, . . . , en−1, we may now assume by induction that for each t occurring in (11.1), the bound where H is the projective hyperplane spanned by the vectors e1, . . . , en−1, we may now assume by induction that for each t occurring in (11.1), the bound Sm,t = O((P ′)n−5−ψ) = O(P n−5−ψ) holds for some ψ = ψ(∆), for an appropriate choice of ϕ and θ. The implied constant depends on the quantity ∥Ft∥P ′, but we easily see that∥Ft∥P ′ ≪∥F∥P for any t ∈[−P, P]n. Since ON THE HASSE PRINCIPLE FOR QUARTIC HYPERSURFACES 49 the sum in (11.1) includes at most O(P) choices of t ∈[−P, P]n, we immediately obtain the desired bound S O(P n 4 ψ) the sum in (11.1) includes at most O(P) choices of t ∈[−P, P]n, we immediately obtain the desired bound S O(P n−4−ψ) Sm = O(P n−4−ψ), Sm = O(P n−4−ψ), which is valid for the same choice of ϕ and θ. This completes the proof of Proposition 2.8 References [1] B. J. Birch. Forms in many variables. Proc. Roy. Soc. Ser. A, 265:245–263, 1961/1962. [2] T. D. Browning and D. R. Heath-Brown. Rational points on quartic hypersurfaces. J. Reine Angew. Math., 629:37–88, 2009. [3] T. D. Browning and S. M. Prendiville. Improvements in Birch’s theorem on forms in many variables. To appear in J. Reine Angew. Math., arXiv:1402.4489, 2014. [4] T. D. Browning and P. Vishe. Cubic hypersurfaces and a version of the circle method for number fi Duke Math. J., 163(10):1825–1883, 2014. , ( ) , [5] Tim Browning and Pankaj Vishe. 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Centre for Mathematical Sciences, Lund University, Box 118, 221 00 Lund, Sweden Email address: oscar.marmon@math.lu.se Department of Mathematical Sciences,, Durham University, Durham, DH1 3LE, UK Email address: pankaj.vishe@durham.ac.uk
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Marc‑André Ehrenfels1 · Caroline Fretter1 · Jennifer von Stein1 · Maria Isabel Körber1 · Hendrik Wienemann1 · Stephan Baldus1 · Roman Pfister1 · Christos Iliadis1 Received: 4 December 2023 / Accepted: 27 February 2024 © The Author(s) 2024 * Christos Iliadis christos.iliadis@uk-koeln.de Clinical Research in Cardiology https://doi.org/10.1007/s00392-024-02428-z Clinical Research in Cardiology https://doi.org/10.1007/s00392-024-02428-z ORIGINAL PAPER Echocardiographic assessment Nevertheless, the functional status of patients following transcatheter tricuspid valve repair (TTVr) regarding symp- tom burden and quality of life (QoL) remains poorly studied. Health-related QoL is a strong predictor of clinical outcome and all-cause mortality in patients with heart failure [8]. Hence, a major goal of TTVr treatment is to increase QoL in patients with severe symptoms [9]. It is fundamental to acknowledge TR as a heterogeneous valve disease with dis- tinguishable phenotypes [10, 11]. Chronic volume overload occurring in severe TR culminates in right ventricular (RV) dilation and dysfunction, which has been shown to nega- tively affect prognosis in patients with heart failure [10, 12]. In patients undergoing isolated tricuspid valve (TV) surgery, preoperative RV remodeling (RVR) can be identified as a predictor of poor outcomes and has been associated with decreased overall survival [13, 14]. However, a systematic analysis of the TR phenotype and disease stage, which are both associated with RVR, is lacking. As TTVr is gaining importance, it is essential to evaluate the effect of prepro- cedural RVR in the transcatheter context. Therefore, this study aimed to analyze the impact of TTVr on procedural success, various measures of functional capacity and QoL, and mid-term clinical outcomes in the context of the pres- ence or absence of preprocedural RVR. All patients underwent transthoracic echocardiographic assessment one day prior to the procedure. All echocardio- grams were evaluated by a trained cardiologist following the current guidelines for echocardiographic assessment of valve regurgitation [15, 16]. In order to capture the severity of TR and to study the reduction in TR properly, we used a five-class grading scheme to quantify TR grade, previ- ously proposed by Hahn and Zamorano [17]. The TR grade was classified as none or mild (I°), moderate (II°), severe (III°), massive (IV°), and torrential (V°) using qualitative (color flow jet), semiquantitative (vena contracta, systolic flow reversal in hepatic veins), and quantitative (effective regurgitant orifice area, regurgitant volume) parameters according to current guidelines. TR was secondary in all of the cases. Furthermore, right heart catheterization was per- formed to assess pulmonary hypertension (PH) prior TTVr. PH was classified in precapillary, isolated postcapillary, and combined pre- and postcapillary PH according to current guidelines [18]. Chronic thromboembolic pulmonary hyper- tension (CTEPH) was excluded via computed tomography or ventilation/perfusion scan in all patients with clinical suspi- cion and precapillary PH. Abstract Keywords  Tricuspid regurgitation · Ventricular dilation · Transcatheter tricuspid valve repair Abbreviations 6 MWD Six-minute walking distance CI Confidence interval EROA Effective regurgitant orifice area FAC Fractional area change HF Heart failure HR Hazard ratio IQR Interquartile range MLHFQ Minnesota Living with Heart Failure Questionnaire NYHA New York Heart Association QoL Quality of life RV Right ventricle/right ventricular RVR Right ventricular remodeling SF-36 36-Item Short Form Health Survey TAPSE Tricuspid annular plane systolic excursion TR Tricuspid regurgitation TTVr Transcatheter tricuspid valve repair TV Tricuspid valve VC Vena contracta 1 Department for Internal Medicine III, Faculty of Medicine and University Hospital Cologne, University of Cologne, Kerpener Strasse 62, 50937 Cologne, Germany :(0123456789) :(01234 3456789) Clinical Research in Cardiology 1975 Declaration of Helsinki and is in line with the estab- lished Ethical Guidelines for Epidemiological Research. Consecutive patients who underwent TTVr and signed an informed consent form at our high-volume referral center were included in the study. We retrospectively studied patients between January 2017 and October 2020 and prospectively enrolled patients between October 2020 and December 2022. Despite receiving optimal medical therapy according to current guidelines, all patients were classified as New York Heart Association (NYHA) func- tional class ≥ II. The entire study population underwent discussion in the interdisciplinary heart team conference with a concordant decision on interventional treatment with transcatheter edge-to-edge repair or transcatheter direct annuloplasty via Cardioband implantation. This study was approved by the local ethics committee of the University of Cologne. Echocardiographic assessment Patients with diagnosed CTEPH were treated with specific therapy and not with TTVr and were therefore excluded from this study. RV size was evaluated by measuring the RV cavity area and RV cavity diameter in two-dimensional RV-focused api- cal four-chamber view. RVR was defined as dilation of the RV at the mid-level (> 35 mm diameter, according to current guidelines) [15]. The RV basal diameter was not considered a distinct indicator of RVR, as it can be dilated along with the TV annulus in isolated RA enlargement [19]. RV dys- function was defined as a TAPSE < 17 mm or FAC < 35%. Introduction Tricuspid regurgitation (TR) is one of the most prevalent valvular diseases in the elderly population and significantly worsens clinical outcomes and survival in patients with heart failure [1]. With an aging population, the prevalence of TR is expected to increase in the future [2]. Therefore, there is intensive debate about the necessity of treatment options that might impact clinical outcomes and at the same time provide sufficient safety. As conventional surgery for isolated severe TR has a significant perioperative risk and has not been proven to improve survival compared to medical manage- ment alone, treatment of symptomatic severe isolated TR has shifted towards a conservative approach for the majority of patients [3, 4]. Over the last few years, several transcatheter technologies have been developed to address the need for alternative treatment options for high-risk populations that are not eligible for surgery [5, 6]. First steps have been taken by proving the feasibility and safety of the procedure [7]. Abstract Abstract Background  Severe tricuspid regurgitation (TR) is associated with chronic volume overload and right ventricular remod- eling (RVR). Transcatheter tricuspid valve repair (TTVr) reduces TR and can improve quality of life (QoL), but the role of preprocedural RVR on TTVr outcomes remains unclear. Aims  To investigate the role of RVR on outcomes after TTVr for severe TR. Methods  Consecutive patients undergoing TTVr (61% edge-to-edge vs. 39% direct annuloplasty) for severe TR were retro- spectively compared by preexisting RVR which was defined as dilation of RV mid-level diameter (> 35 mm) according to guidelines. QoL was evaluated using NYHA class, Minnesota Living with Heart Failure Questionnaire (MLHFQ), 36-Item Short Form Health Survey (SF-36), and 6-min walking distance (6MWD) 1-month after TTVr. Mid-term mortality and heart failure (HF) hospitalization were assessed through 1 year. Results  RVR was present in 137 of 223 patients (61%). Symptoms and QoL improved equally in both groups: ≥ 1 NYHA class (57% vs. 65% of patients with vs. without RVR, respectively), 6MWD (36% vs. 34%), MLHFQ (81% vs. 69%), and SF-36 (68% vs. 65%) improvement. One-year mortality and HF hospitalization were significantly higher in patients with RVR (24% and 30%, respectively) than in patients without (8% and 13%, both p < 0.05). In multivariable analysis, RVR was independently associated with mortality (HR 2.3, 95%CI (1.0–5.0), p = 0.04) and the combined endpoint of mortality or rehospitalization (HR 2.0, 95%CI (1.1–3.8), p = 0.03).i Conclusions  TTVr was associated with significant QoL improvement after 1 month, irrespective of RVR. Despite increased mortality and rehospitalization for heart failure, TTVr in the presence of RVR still provides substantial symptomatic benefit for patients with severe TR. Study population This study was conducted at the Heart Centre at the Uni- versity of Cologne. The study protocol conformed to the Clinical Research in Cardiology Echocardiographic evaluation was repeated at discharge and at follow-up, approximately 4–6 weeks after the procedure. as well as successful deployment and correct positioning of the device. Additionally, procedural success was confirmed if the postprocedural TR grade at discharge was moderate or less (≤ II°). Evaluation of the functional capacity and QoL We obtained a thorough functional status for all prospec- tive patients. Functional capacity and QoL assessments were performed by a trained medical student who was blinded to procedural and echocardiographic results. Evaluation was incorporated into the clinical routine prior to TTVr as part of the prospective analysis of our study. Six-min walk- ing distance (6 MWD) was measured [20]. Acknowledg- ing the occurrence of peripheral edema as a major cardinal symptom of right heart failure and TR, we quantitatively analyzed the occurrence of edema [21, 22]. The grading of peripheral edema used in this study is a modification of the classic clinical assessment described by Seidel et al. [23]. We classified three grades of edema according to severity, as measured by physical examination. Grade I was defined as pitting edema with up to 2 mm of depression and immediate rebound. Grade II was defined as pitting edema with 2–4 mm of depression and rebounding within 10–25 s. Grade III was defined as pitting edema with more than 4 mm of depression and rebound in more than 1 min. The Minnesota Living with Heart Failure Questionnaire (MLHFQ) was used to assess QoL [24]. The MLHFQ contains 21 questions (− 0–5 points for each question) representing physical, mental, and social components that can be affected by heart failure (HF). Addi- tionally, a short version of the Short Form-36 (SF-36) health survey questionnaire containing 12 items reflecting the origi- nal 8 subscales of the SF-36 was conducted [25]. Item scores were coded, totalized, and transformed on a scale from 0 (worst health) to 100 (best health), covering functional status and general well-being as well as physical and mental health. Assessment was conducted during hospitalization before the procedure and at follow-up 4–6 weeks after the procedure. Statistical analysis Characteristics are expressed as mean ± standard deviation (SD) for continuous variables if normally distributed and as median (IQR) if not normally distributed. Normal distribu- tion was tested for all variables using the Shapiro–Wilk test. Categorical data were presented as counts (percentages). Differences in baseline characteristics between patients with and without preprocedural RVR were determined by applying the independent t-test if normally distributed and the Mann–Whitney U test if not normally distributed for continuous variables. Differences in categorical variables were determined using Pearson’s χ2 test. Changes in NYHA functional class, TR grade, grade of peripheral edema, 6 MWD, and MLWFQ were studied using the Wilcoxon matched pairs signed rank test. Changes in the occurrence of peripheral edema were analyzed using the McNemar’s test. Changes in SF-36 scores were analyzed by applying a paired t-test. Kaplan–Meier plots were used to depict the event curves of the patients with and without RVR. Furthermore, we used Cox regression models to investigate the impact of preprocedural RVR on mid-term clinical outcomes, includ- ing all-cause mortality, HF hospitalization, and the com- bined endpoint. Risk was expressed as hazard ratio (HR), 95% confidence interval (CI), and p-value. Univariable Cox regression analysis was conducted for all baseline variables. Variables with a p < 0.05 in the univariable Cox regression analysis were selected for adjustment in a multivariable Cox regression model. For redundant variables, such as echocar- diographic severity (VC, EROA, and RV), only one was con- sidered for multivariable analysis. Natriuretic peptides were not included in the regression analysis because of multiple confounders (renal dysfunction and obesity). The multipara- metric scores (EuroSCORE II, TRI-SCORE) were also not considered for multivariable analysis because the individual Endpoints Next to TR grade, the presence and grade of peripheral edema, NYHA functional class, and QoL scores (for the prospective patient population) were assessed at 4–6 weeks following TTVr. Preprocedural RVR was assessed in all patients to analyze its impact on clinical and functional out- comes. We captured all-cause mortality, occurrence of first rehospitalization for decompensated HF, and occurrence of repeat TV intervention as mid-term clinical outcomes by contacting patients’ general physicians and using register queries. Procedural results of TTVr Cardioband implantation was performed in 87 patients (39%), and MitraClip in the tricuspid position was used in 11 patients (5%). TriClip devices were used in 49 patients (22%). PASCAL devices were used in 76 patients (34%). Technical success was achieved in 209 (94%) patients. In seven patients (3%), TTVr was unsuccessful due to unfa- vorable anatomical, technical, or procedural circumstances, and the device was removed without complications. Proce- dural success, defined as a moderate or less TR at hospital discharge, was achieved in 64% of the attempted procedures. None of the patients was lost to follow-up at 1 month regard- ing vital status. Seven patients (3%) died postprocedural or during the 1-month follow-up period.f A total of 137 patients (61%) had preprocedural RVR, and 86 patients (39%) had no RVR. Patients with RVR were significantly more male (p < 0.001). Both groups were highly symptomatic, with a similar distribution of NYHA func- tional class grading. Patients with RVR received a signifi- cantly (p < 0.001) higher dose of loop diuretics at baseline (60 mg/day, IQR, 20 mg/day to 100 mg/day with RVR, vs. 30 mg/day, IQR, 20 mg/day to 50 mg/day without RVR; p < 0.001) than patients without RVR. The occurrence of peripheral edema did not differ between the groups. Prior to TTVr, patients with RVR had more frequently a history of hospitalization for HF (67% vs. 50%, p = 0.01), diabetes mellitus (31% vs. 14% of patients, p = 0.003), prior myocar- dial infarction (7% vs. 1%, p = 0.039), higher NT-proBNP level (median 2,627 vs. 1,368 ng/l, p < 0.001), and higher TRI-SCORE (6.5 ± 1.9 vs. 6.0 ± 1.8, p = 0.026). A detailed comparison of the baseline characteristics of the patients with and without RVR is presented in Table 1. There was no difference regarding the presence of RVR at baseline between patients undergoing edge-to-edge repair and patients undergoing direct annuloplasty (59% vs. 65%, p = 0.316). The technical success rate was higher in patients without RVR (98% vs. 91% with RVR, p = 0.054). All-cause mortality rate within 1 month of TTVr was higher in patients with RVR (p = 0.033). Procedural success rate was higher in the absence of RVR (78% vs. 55%, p < 0.001). There were no differences in procedural aspects or complications between TTVr devices. TTVr procedures The procedures were performed using MitraClip, TriClip (Abbott Vascular), PASCAL P10, PASCAL Ace (Edwards Lifesciences) devices, or Cardioband device (Edwards Lifesciences), as previously described [6, 26, 27]. As direct annuloplasty is more complex than TEER, both regarding patient screening including computed tomography as well as longer and more complex procedure, TEER is the first treatment choice in daily clinical practice. In general, every patient which showed a tricuspid valve anatomy with dif- ficulty in leaflet grasping (e.g., extremely large coaptation gap, short leaflets, or multiple-scallop leaflets) was evaluated for direct annuloplasty. Technical success was defined as successful device implantation without conversion to emer- gent TV surgery or re-intervention and absence of mortality, Clinical Research in Cardiology did not differ among the groups (76% vs. 69%, p = 0.231). The mean pulmonary artery pressure measured in right heart catheterization was higher in patients with RVR (31 vs. 27 mmHg, p = 0.049). Additionally, the mean pulmonary artery wedge pressure was higher in patients with RVR (19 vs. 16 mmHg, p = 0.006). The etiology of pulmonary hyper- tension showed marked differences between the two groups, with patients with RVR having a distribution of 16% with precapillary pulmonary hypertension, 55% with isolated postcapillary hypertension, and 29% with combined precap- illary and postcapillary hypertension, whereas those with- out RVR had percentages of 30%, 33%, and 37%, respec- tively, for the aforementioned classifications (p = 0.036). Whereas the mean TAPSE was not statistically different among groups (17 mm vs. 18 mm, p = 0.126), reduced FAC (< 35%) was more frequent in patients with RVR (68% vs. 27%, p < 0.001). Hepatic systolic flow reversal was more frequent in patients with RVR (82% vs. 62%, p = 0.005). A detailed comparison of the baseline echocardiographic assessments of the patients with and without RVR is pre- sented in Table 1. components of the scores were already included. A two- tailed p value < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS Statistics version 28 (IBM, Armonk, NY, USA). Study population A total of 223 patients who underwent TTVr were included in the study. Eighty-two patients were retrospectively ana- lyzed, and 141 patients underwent prospective evaluation. The baseline clinical and echocardiographic characteristics are shown in Table 1. The median age was 80 years (IQR, 75–83), and 69% of the patients were female. The median European System for Cardiac Operative Risk Evaluation score II (EuroScore II) was 4.1% (IQR, 2.8–7.2%), and the mean TRI-SCORE was 6.3 ± 1.9 points. All patients had right heart failure and were symptomatic, with signs and symptoms of at least one of the following: chest discomfort, breathlessness, palpitations, and edema. Most patients were severely symptomatic at admission, with 85% presenting with NYHA functional class III or IV. Procedural results of TTVr Head-to-head comparison of procedural information and major complications associated with the two different TTVr strategies are shown in the supplemental file. A detailed comparison of procedural results is presented in Table 2. TR grade at discharge was significantly lower in patients without RVR (p = 0.004). In comparison with TR grade at baseline, the TR grade at 1-month follow-up was Echocardiographic assessment The TR grade was significantly higher in patients with RVR compared with those without RVR. Forty-seven percent vs. 33% of patients with vs. without RVR, respectively, presented a TR grade of massive or torrential (p = 0.008). Patients with RVR also showed higher values for all param- eters of quantitative TR grade measurement (EROA 0.62 vs. 0.46 ­cm2, p = 0.007; RV 50 vs. 39 ml, p < 0.001; VC 12 vs. 9 mm, p < 0.001). The frequency of pulmonary hypertension Clinical Research in Cardiology Table 1   Study population Total (N = 223) RV remodeling (N = 137) No RV remodeling (N = 86) P-value (RVR vs. Echocardiographic assessment No RVR) CpcPH 55 (25) 43 (31) 12 (14) Diabetes mellitus 37 (17) 26 (19) 11 (13) 0.003 COPD 19 (8) 15 (11) 4 (5) 0.227 Peripheral artery disease 6 (3) 5 (4) 1 (1) 0.101 Dependent on dialysis 205 (92) 124 (91) 81 (94) 0.264 Atrial fibrillation 11 (5) 10 (7) 1 (1) 0.327 Prior myocardial infarction 26 (12) 16 (12) 10 (12) 0.039 Prior stroke 48 (21) 30 (22) 18 (21) 0.991 Previous heart surgery 0.864 Previous valvular intervention 40 (18) 27 (20) 13 (15) Mitral valve repair/replacement 10 (4) 5 (4) 5 (6) 0.384 Tricuspid valve repair 9 (4) 5 (4) 4 (5) 0.447 Cardioband 1 (0.5) 0 (0) 1 (1) Edge-to-edge + Cardioband Baseline medical treatment Dose of loop diuretic agents, mg/d 40 (20–100) 5 60 (20–100) 30 (20–52)  < 0.001 Sequential nephron blockade 41 (18) 29 (21) 12 (14) 0.176 Aldosterone antagonists 110 (49) 71 (52) 39 (45) 0.346 ACE inhibitors/ARBs 91 (41) 57 (42) 34 (39) 0.759 Beta-blockers 191 (86) 117 (85) 74 (86) 0.894 Cardiac devices Pacemaker 49 (22) 35 (25) 14 (16) 0.104 ICD 4 (2) 4 (3) 0 (0) 0.110 CRT​ 7 (3) 6 (4) 1 (1) 0.180 Table 1   (continued) 6MWD, 6-min walk distance; ACE, angiotensin-converting enzyme; ARB, angiotensin receptor blocker; BMI, body mass index; CABG, coronary artery bypass grafting; COPD, chronic obstructive pulmonary disease; CpcPH, combined post- and precapillary pulmonary hypertension; CRT​ , cardiac resynchronization therapy; eGFR, estimated glomerular filtration rate; EROA, effective regurgitant orifice area; EuroSCORE, European System for Cardiac Operative Risk Evaluation; FAC, fractional area change; HF, heart failure; ICD, implantable cardioverter defibrillator; IpcH, isolated postcapillary pulmonary hypertension; NT-proBNP, N-terminal pro-B-type natriuretic peptide; NYHA, New York Heart Association; PA, pulmonary artery; PAmean, mean pulmonary artery pressure; PAPsys, systolic pulmonary artery pressure; PH, pulmonary hypertension; RV, regurgitant volume; TAPSE, tricuspid annular plane systolic excursion; TR, tricuspid regurgitation; VC, vena contracta p-values < 0.05 are shown in boldface 6MWD, 6-min walk distance; ACE, angiotensin-converting enzyme; ARB, angiotensin receptor blocker; BMI, body mass index; CABG, coronary artery bypass grafting; COPD, chronic obstructive pulmonary disease; CpcPH, combined post- and precapillary pulmonary hypertension; CRT​ , cardiac resynchronization therapy; eGFR, estimated glomerular filtration rate; EROA, effective regurgitant orifice area; EuroSCORE, European System for Cardiac Operative Risk Evaluation; FAC, fractional area change; HF, heart failure; ICD, implantable cardioverter defibrillator; IpcH, isolated postcapillary pulmonary hypertension; NT-proBNP, N-terminal pro-B-type natriuretic peptide; NYHA, New York Heart Association; PA, pulmonary artery; PAmean, mean pulmonary artery pressure; PAPsys, systolic pulmonary artery pressure; PH, pulmonary hypertension; RV, regurgitant volume; TAPSE, tricuspid annular plane systolic excursion; TR, tricuspid regurgitation; VC, vena contracta p-values < 0.05 are shown in boldface significantly reduced regardless of RVR. Echocardiographic assessment No RVR) CpcPH 55 (25) 43 (31) 12 (14) Diabetes mellitus 37 (17) 26 (19) 11 (13) 0.003 COPD 19 (8) 15 (11) 4 (5) 0.227 Peripheral artery disease 6 (3) 5 (4) 1 (1) 0.101 Dependent on dialysis 205 (92) 124 (91) 81 (94) 0.264 Atrial fibrillation 11 (5) 10 (7) 1 (1) 0.327 Prior myocardial infarction 26 (12) 16 (12) 10 (12) 0.039 Prior stroke 48 (21) 30 (22) 18 (21) 0.991 Previous heart surgery 0.864 Previous valvular intervention 40 (18) 27 (20) 13 (15) Mitral valve repair/replacement 10 (4) 5 (4) 5 (6) 0.384 Tricuspid valve repair 9 (4) 5 (4) 4 (5) 0.447 Cardioband 1 (0.5) 0 (0) 1 (1) Edge-to-edge + Cardioband Baseline medical treatment Dose of loop diuretic agents, mg/d 40 (20–100) 5 60 (20–100) 30 (20–52)  < 0.001 Sequential nephron blockade 41 (18) 29 (21) 12 (14) 0.176 Aldosterone antagonists 110 (49) 71 (52) 39 (45) 0.346 ACE inhibitors/ARBs 91 (41) 57 (42) 34 (39) 0.759 Beta-blockers 191 (86) 117 (85) 74 (86) 0.894 Cardiac devices Pacemaker 49 (22) 35 (25) 14 (16) 0.104 ICD 4 (2) 4 (3) 0 (0) 0.110 CRT​ 7 (3) 6 (4) 1 (1) 0.180 Clinical Research in Cardiology V l di (IQR) (%) SD P l h i d fi d l 20 H P Table 1   (continued) Total (N = 223) RV remodeling (N = 137) No RV remodeling (N = 86) P-value (RVR vs. Echocardiographic assessment In comparison with TR grade at discharge, the TR grade at the 1-month follow- up did not change in either group. Fifty-six percent vs. 78% of patients with vs. without RVR presented with a NYHA class of ≤ II at discharge (p = 0.004). the 1-month follow-up (p < 0.001). Grade III peripheral edema decreased from 10% at baseline to 4% at the 1-month follow-up (p = 0.003). The mean 6 MWD increased from baseline to 1-month follow-up (p < 0.001), with 35% of patients showing a clinically relevant improvement of 50 m or more. The median MLHFQ score decreased from baseline to 1-month follow-up (p < 0.001), with 76% of the patients presenting with a clinically relevant improvement of at least 5 points. Additionally, the mean SF-36 score increased from baseline 1-month follow-up (p < 0.001), with 67% of patients Values are median (IQR), n (%), or mean ± SD. Pulmonary hypertension was defined as mean pulmonary artery pressure > 20 mmHg. Pre- capillary pulmonary hypertension was defined as PAmean > 20  mmHg and PAWP ≤ 15  mmHg and PVR > 2 WU. IpcPH was defined as PAmean > 20 mmHg and PAWP > 15 mmHg and PVR ≤ 2 WU. CpcPH was defined as PAmean > 20 mmHg and PAWP > 15 mmHg and PVR > 2 WU. Right ventricular remodeling was defined as RV diameter > 35 mm at mid-level. RV dysfunction was defined as FAC < 35% or TAPSE < 17 mm 1 N = 219, 2N = 132, 3N = 114, 4N = 214, 5N = 222, 6N = 164, 7N = 215, 8N = 151, 9N = 210, 10N = 209, 11N = 211, 12N = 162, 13N = 163, 14N = 157, 15N = 146, 16N = 128 Echocardiographic assessment N RVR) Age, y 80 (75–83) 79 (74–83) 81 (76–82) 0.177 Female 153 (69) 82 (60) 71 (83)  < 0.001 BMI, kg/m2 25.4 (22.3–29.1) 25.4 (22.7–28.9) 25.4 (21.9–29.4) 0.530 EuroSCORE II, % 4.1 (2.8–7.2) 4.5 (2.9–7.3) 3.8 (2.7–7.2) 0.163 TRI-SCORE, points 6.3 ± 1.9 1 6.5 ± 1.9 6.0 ± 1.8 0.026 NYHA functional class I 4 (2) 2 (1) 2 (2) 0.677 II 30 (13) 18 (13) 12 (14) III 161 (72) 97 (71) 64 (74) IV 28 (13) 20 (15) 8 (9) MLHFQ, score 41 (27–52) 2 41 (30–54) 39 (24–49) 0.179 SF-36, % 50 ± 21 2 49 ± 20 51 ± 21 0.531 6MWD, m 270 ± 106 3 258 ± 111 285 ± 99 0.166 NT-proBNP, ng/L 2117 (1,344–4,129) 4 2627 (1,607–4,661) 1638 (1,081–2,716)  < 0.001 Kidney disease (GFR < 60 mL/min) 187 (84) 118 (86) 69 (80) 0.244 Increased bilirubin (> 1.2 mg/dL) 37 (17) 0.7 (0.5–1.1) 0.7 (0.5–1.0) 0.787 Clinical presentation Previous hospitalization due to HF 135 (60) 92 (67) 43 (50) 0.011 Prior peripheral edema 176 (79) 113 (82) 63 (73) 0.100 Peripheral edema at admission 129 (58) 83 (61) 46 (54) 0.296 Prior ascites 26 (12) 18 (13) 8 (9) 0.385 Echocardiographic assessment TR ­grade5 0.008 II 3 (1) 1 (1) 2 (2) III 126 (57) 70 (52) 56 (65) IV 54 (24) 32 (23) 22 (26) V 39 (18) 33 (24) 6 (7) EROA, ­cm2 0.53 (0.42–0.78) 6 0.62 (0.42–0.96) 0.46 (0.41–0.64) 0.007 VC, mm 11 (8–15) 7 12 (9–16) 9 (8–13)  < 0.001 RV, ml 46 (35–60) 8 50 (38–65) 39 (32–53)  < 0.001 TAPSE, mm 17 (14–20) 1 17 (14–20) 18 (15–20) 0.126 PAPsys, mmHg 43 (35–54) 9 43 (34–55) 43 (35–52) 0.784 RV/PA coupling, mm/mmHg 0.4 (0.3–0.5) 10 0.4 (0.3–0.5) 0.4 (0.3–0.5) 0.812 RV dysfunction 133 (62) 5 87 (66) 46 (55) 0.086 Reduced FAC (< 35%) 95 (45) 11 68 (53) 27 (32) 0.003 Hepatic systolic flow reversal 120 (74) 12 81 (82) 39 (62) 0.005 Reduced Ejection Fraction (< 50%) 42 (19) 5 30 (22) 12 (14) 0.133 Comorbidities Coronary artery disease 91 (41) 56 (41) 35 (41) 0.979 Previous CABG 20 (9) 16 (12) 4 (5) 0.074 Hypertension 184 (83) 113 (83) 71 (83) 0.988 Pulmonary hypertension 163 (73) 104 (76) 59 (69) 0.231 PAmean, mmHg 29 (24–35) 13 31 (24–36) 27 (24–32) 0.049 PAWP, mmHg 18 (14–22) 14 19 (16–23) 16 (14–20) 0.006 PVR, WU 3 (2–3) 15 2 (2–4) 3 (2–3) 0.693 Etiology of ­PH16 13 (16) 14 (30) 0.036 P ill PH 27 (21)60 (47) 45 (55) 15 (33) Clinical Research in Cardiology Clinical Research in Cardiology Clinical Research in Cardiology Values are median (IQR), n (%), or mean ± SD. Echocardiographic assessment Pulmonary hypertension was defined as mean pulmonary artery pressure > 20 mmHg. Pre- capillary pulmonary hypertension was defined as PAmean > 20  mmHg and PAWP ≤ 15  mmHg and PVR > 2 WU. IpcPH was defined as PAmean > 20 mmHg and PAWP > 15 mmHg and PVR ≤ 2 WU. CpcPH was defined as PAmean > 20 mmHg and PAWP > 15 mmHg and PVR > 2 WU. Right ventricular remodeling was defined as RV diameter > 35 mm at mid-level. RV dysfunction was defined as FAC < 35% or TAPSE < 17 mm 1 N = 219, 2N = 132, 3N = 114, 4N = 214, 5N = 222, 6N = 164, 7N = 215, 8N = 151, 9N = 210, 10N = 209, 11N = 211, 12N = 162, 13N = 163, 14N = 157, 15N = 146, 16N = 128 6MWD, 6-min walk distance; ACE, angiotensin-converting enzyme; ARB, angiotensin receptor blocker; BMI, body mass index; CABG, coronary artery bypass grafting; COPD, chronic obstructive pulmonary disease; CpcPH, combined post- and precapillary pulmonary hypertension; CRT​ , cardiac resynchronization therapy; eGFR, estimated glomerular filtration rate; EROA, effective regurgitant orifice area; EuroSCORE, European System for Cardiac Operative Risk Evaluation; FAC, fractional area change; HF, heart failure; ICD, implantable cardioverter defibrillator; IpcH, isolated postcapillary pulmonary hypertension; NT-proBNP, N-terminal pro-B-type natriuretic peptide; NYHA, New York Heart Association; PA, pulmonary artery; PAmean, mean pulmonary artery pressure; PAPsys, systolic pulmonary artery pressure; PH, pulmonary hypertension; RV, regurgitant volume; TAPSE, tricuspid annular plane systolic excursion; TR, tricuspid regurgitation; VC, vena contracta p-values<0 05 are shown in boldface Table 1   (continued) Total (N = 223) RV remodeling (N = 137) No RV remodeling (N = 86) P-value (RVR vs. Functional capacity and QoL Overall, at the 1-month follow-up, the rate of NYHA func- tional class III/IV reduced from 83% at baseline to 34% at Clinical Research in Cardiology showing a clinically relevant improvement of at least 2.5 i from 41 (IQR, 29–54) at baseline to 27 (IQR, 15–41) at the 1 h f ll ( 0 001) d 81% h d i Table 2   Procedural aspects of TTVr Values are n (%). Technical success was defined as successful device implantation without conversion to emergent TV surgery or re-intervention, absence of mortality, and successful deployment and correct posi- tioning of the device. Procedural success was defined as postprocedural TR grade at discharge was moder- ate or less (≤ II) p-values < 0.05 are shown in boldface Total (N = 223) RV remodeling (N = 137) No RV remodeling (N = 86) P-value (RVR vs. No RVR) Technical success 209 (94) 125 (91) 84 (98) 0.054 Procedural success 143 (64) 76 (55) 67 (78)  < 0.001 TR grade at discharge I 58 (26) 28 (20) 30 (35) 0.004 II 86 (39) 49 (36) 37 (43) III 62 (28) 44 (32) 18 (21) IV 12 (5) 11 (8) 1 (1) V 5 (2) 5 (4) 0 (0) Clinical Research in Cardiology showing a clinically relevant improvement of at least 2.5 points.i from 41 (IQR, 29–54) at baseline to 27 (IQR, 15–41) at the 1-month follow-up (p < 0.001), and 81% showed an improve- ment of at least 5 points. Additionally, the mean SF-36 score in patients without RVR improved from 51 ± 22% at base- line to 62 ± 19% at the 1-month follow-up (p < 0.001), with 65% of the patients showing an improvement of at least 2.5 points. The mean SF-36 score in patients with RVR increased from 50 ± 21% at baseline to 61 ± 21% at the 1-month follow-up (p < 0.001), with 68% of patients show- ing an improvement of at least 2.5 points. Functional capac- ity and QoL did not differ between devices. The changes in the MLHFQ and SF-36 scores are shown in Fig. 2. Significant improvement in NYHA functional class was observed in patients regardless of RVR. At the 1-month follow-up, NYHA functional class improved by at least one grade in 57% and 65% of the patients with and without RVR, respectively (p = 0.262). The rate of NYHA functional class III/IV decreased from 84 to 39% vs. Functional capacity and QoL from 82 to 27%, at the 1-month follow-up in patients with and without RVR, respectively (p < 0.001). The occurrence of peripheral edema decreased from 59 to 53% vs. from 55 to 37% at the 1-month follow-up in patients with and without RVR, respectively. A significant reduc- tion in the occurrence of peripheral edema was observed only in patients without RVR (p = 0.015). Patients with and without RVR showed a significant decrease in the grade of peripheral edema, with a decrease of at least one grade in 30% of patients with RVR and 31% of patients without RVR. Changes in NYHA class and the status of peripheral edema are illustrated in Fig. 1. QoL data were available for 35 patients at 12 months. NYHA functional class was available for 59 patients at 12 months. 6MWD was available for 30 patients at 12 months. Median MLHFQ scores at 12 months were 21 (IQR, 11–39) and 32 (IQR, 19–37) in patients with and with- out RVR, respectively. The mean SF-36 scores at 12 months were 65 ± 20% and 53 ± 19% in patients with and without RVR, respectively. In patients with RVR, the mean 6 MWD increased from 262 m ± 109 at baseline to 307 m ± 110 at the 1-month fol- low-up (p < 0.001), whereas in patients without RVR, the mean 6 MWD increased from 280 m ± 104 to 311 m ± 98 at the 1-month follow-up (p = 0.016). Thirty-six percent and 34% showed a clinically relevant increase in the 6 MWD of at least 50 m for patients with and without RVR, respec- tively, as shown in Fig. 2.i The rate of NYHA functional class III/IV at 12 months was 36% and 32% in patients with and without RVR, respec- tively. The incidence of peripheral edema at 12 months was 33% and 30% in patients with and without RVR, respec- tively. The mean 6MWD at 12 months was 324 ± 148 and 309 ± 108 in patients with and without RVR, respectively. Fig. 1   A Comparison of 1-month changes in NYHA functional class after TTVr in patients with and without RVR. B Comparison of 1-month changes in the occurrence of peripheral edema after TTVr in patients with and without RVR. C Comparison of 1-month changes in grade of peripheral edema after TTVr in patients with and without RVR Fig. 2   A Comparison of 1-month changes in 6MWD after TTVr in patients with and without RVR. B Comparison of 1-month changes in MLHFQ after TTVr in patients with and without RVR. C Comparison of 1-month changes in SF-36 after TTVr in patients with and without RVR Mid‑term clinical outcomes Both groups showed significant improvement in QoL at the 1-month follow-up compared with the preprocedural baseline. In patients without RVR, the median MLHFQ score decreased from 41 (IQR, 24–49) at baseline to 27 (IQR, 17–39) at the 1-month follow-up (p < 0.001), and 69% showed an improvement of at least 5 points. In comparison, the mean MLHFQ score in patients with RVR decreased Mid-term follow-up data were available after 463 ± 403 days (median, 374 days; IQR, 156–607). Six- month follow-up was available in 84% of patients, and 12-month follow-up was available in 68% of patients. Information about death after TTVr was available in all Clinical Research in Cardiology Clinical Research in Cardiology Clinical Research in Cardiology Fig. 3   A Kaplan–Meier curves for the endpoint of all-cause mortal- ity after TTVr. B Kaplan–Meier curves for the endpoint of rehospi- talization for heart failure after TTVr. C Kaplan–Meier Curves for the combined endpoint of all-cause mortality and rehospitalization for heart failure after TTVr ▸ Fig. 3   A Kaplan–Meier curves for the endpoint of all-cause mortal- ity after TTVr. B Kaplan–Meier curves for the endpoint of rehospi- talization for heart failure after TTVr. C Kaplan–Meier Curves for the combined endpoint of all-cause mortality and rehospitalization for heart failure after TTVr ▸ patients, and information about rehospitalization due to HF after TTVr was available in 76% of patients. One year after the procedure, 19% of the patients died, 25% were readmitted to the hospital due to decompensated heart failure, and one patient had to undergo repeat TTVr because of severe residual TR.i All-cause mortality 1 year after TTVr was significantly higher in patients with RVR (24% vs. 8%, p = 0.013), and RVR was associated with a HR of 2.6 (95% CI, 1.2–5.6) for all-cause mortality (p = 0.016). One-year heart failure (HF) rehospitalization after TTVr was significantly higher in patients with RVR (30% vs. 13%, p = 0.045), and RVR was associated with a HR of 2.4 (95% CI, 1.1–5.7) for rehospitalization due to HF after TTVr (p = 0.038). The combined endpoint of 1-year mortality or 1-year rehos- pitalization due to HF after TTVr was seen significantly more often in patients with RVR (44% vs. 21%, p = 0.012), and RVR was associated with a HR of 2.4 (95% CI, 1.3–4.5) for the combined endpoint of mortality or rehos- pitalization due to HF after TTVr (p = 0.005). Outcomes between devices were not different. The need for repeat TTVr was not associated with RVR. The Kaplan–Meier curves of all-cause mortality and rehospitalization due to HF are shown in Fig. 3. In the multivariable analysis, RVR remained to be inde- pendently associated with mid-term mortality (HR 2.3, 95%CI (1.0–5.0), p = 0.042) and the combined endpoint (HR 2.0, 95%CI (1.0–3.8), p = 0.027). Mid-term clinical outcomes did not differ between devices. A detailed com- parison of the uni- and multivariable analysis is presented in Table 3. Fig. 3   A Kaplan–Meier curves for the endpoint of all-cause mortal- ity after TTVr. B Kaplan–Meier curves for the endpoint of rehospi- talization for heart failure after TTVr. C Kaplan–Meier Curves for the combined endpoint of all-cause mortality and rehospitalization for heart failure after TTVr ▸ Discussion This study analyzed procedural success, functional capacity, and QoL, as well as mid-term clinical outcomes in patients with symptomatic HF due to TR following TTVr regarding preprocedural RVR. The main findings can be summarized as follows: (i) TTVr is a safe and effective treatment option in patients regardless of RVR; (ii) TTVr resulted in signifi- cant QoL improvement at 1 month; (iii) patients with RVR showed higher 1-year mortality and HF hospitalization; and RVR was independently associated with all-cause mortality and the combined endpoint of mortality or rehospitalization after TTVr. Discussion Clinical Research in Cardiology Table 3   Cox regression analysis for the endpoints of all-cause death, heart failure hospitalization (HFH), and the combination of both endpoints Univariable analysis Multivariable analysis Hazard ratio (95% CI) p-value Hazard ratio (95%CI) p-value Right ventricular remodeling Death 2.6 (1.2–5.6) 0.016 2.3 (1.0–5.0) 0.042 HFH 2.4 (1.1–5.7) 0.038 1.9 (0.8–4.6) 0.136 Combination 2.4 (1.3–4.5) 0.005 2.0 (1.1–3.8) 0.027 Age (years) Death 1.0 (0.9–1.0) 0.108 HFH 1.0 (0.9–1.0) 0.674 Combination 1.0 (0.9–1.0) 0.380 Female sex Death 0.5 (0.3–1.0) 0.052 HFH 0.6 (0.3–1.1) 0.211 Combination 0.7 (0.4–1.1) 0.133 Procedural success Death 0.5 (0.3–0.9) 0.023 0.6 (0.3–1.2) 0.146 HFH 0.5 (0.3–0.9) 0.042 0.6 (0.3–1.3) 0.218 Combination 0.4 (0.2–0.7)  < 0.001 0.5 (0.3–0.9) 0.015 BMI (kg/m2) Death 0.9 (0.9–1.0) 0.294 HFH 1.0 (0.9–1.1) 0.432 Combination 1.0 (0.9–1.1) 0.584 NYHA class III or IV Death 5.0 (0.7–36.9) 0.111 HFH 1.8 (0.6–6.0) 0.326 Combination 2.3 (0.8–6.5) 0.101 eGFR < 60 (ml/min) Death 1.6 (0.6–4.0) 0.354 HFH 1.9 (0.6–6.1) 0.310 Combination 1.3 (0.6–2.7) 0.531 Bilirubine > 1.2 (mg/dl) Death 2.6 (1.3–5.0) 0.005 2.4 (1.2–4.7) 0.010 HFH 1.5 (0.6–3.6) 0.401 Combination 2.0 (1.1–3.5) 0.021 2.3 (1.2–4.2) 0.007 6MWD (m) Death 1.0 (0.9–1.0) 0.138 HFH 1.0 (0.9–1.0) 0.180 Combination 1.0 (0.9–1.0) 0.050 Reduced RV function Death 2.1 (0.9–4.5) 0.068 HFH 2.5 (1.1–5.7) 0.036 1.9 (0.8–4.6) 0.161 Combination 2.2 (1.2–4.0) 0.011 1.7 (0.2–3.2) 0.105 Vena contracta mm Death 1.0 (0.9–1.1) 0.974 HFH 1.0 (0.9–1.1) 0.667 Combination 1.0 (0.9–1.1) 0.830 EF < 50 (%) Death 1.4 (0.7–2.9) 0.334 HFH 3.7 (1.8–7.5)  < 0.001 2.4 (1.1–5.4) 0.034 Combination 2.2 (1.3–3.8) 0.004 1.6 (0.9–3.1) 0.128 Coronary artery disease Death 1.1 (0.6–2.0) 0.849 HFH 2.1 (1.0–4.2) 0.049 1.4 (0.6–3.2) 0.395 Combination 1.5 (0.9–2.6) 0.098 Previous CABG Death 1.7 (0.7–4.0) 0.239 HFH 2.8 (1.1–6.8) 0.025 1.0 (0.3–2.8) 0.980 Combination 2.6 (1.4–5.1) 0.004 1.5 (0.5–4.3) 0.419 Arterial hypertension Death 0.7 (0.4–1.6) 0.441 HFH 2.0 (0.6–6.7) 0.242 Combination 1.2 (0.6–2.3) 0.675 Pulmonary hypertension Death 0.8 (0.4–1.7) 0.609 HFH 2.4 (0.8–6.8) 0.107 Combination 1.6 (0.8–3.0) 0.163 Clinical Research in Cardiology Table 3   (continued) Univariable analysis Multivariable analysis Hazard ratio (95% CI) p-value Hazard ratio (95%CI) p-value Leaflet repair vs. Discussion Procedural success was defined as postprocedural TR grade at discharge was moderate or less (≤ II). Leaflet repair was defined as implantation of a TriClip, MitraClip, or PASCAL. Annuloplasty was defined as implantation of a Cardioband i 6MWD, 6-min walk distance; BMI, body mass index; CABG, coronary artery bypass graft; COPD, chronic obstructive pulmonary disease; EF, ejection fraction; eGFR, estimated glomerular filtration rate; NYHA, New York Heart Association p-values < 0.05 are shown in boldface p-values < 0.05 are shown in boldface Discussion annuloplasty Death 1.4 (0.7–2.7) 0.294 HFH 1.0 (0.5–2.1) 0.896 Combination 1.1 (0.7–1.9) 0.629 Diabetes mellitus Death 1.7 (0.9–3.3) 0.121 HFH 0.9 (0.4–2.1) 0.791 Combination 1.2 (0.7–2.2) 0.476 COPD Death 0.4 (0.7–3.1) 0.380 HFH 2.0 (0.9–4.4) 0.073 Combination 1.5 (0.8–2.9) 0.168 Peripheral artery disease Death 1.3 (0.5–3.6) 0.651 HFH 1.1 (0.3–3.7) 0.863 Combination 1.1 (0.5–2.7) 0.762 Atrial fibrillation Death 0.7 (0.2–2.0) 0.491 HFH 1.0 (0.2–4.0) 0.953 Combination 0.7 (0.3–1.8) 0.485 Prior myocardial infarction Death 0.5 (0.1–3.6) 0.492 HFH 0.8 (0.1–5.6) 0.784 Combination 0.7 (0.2–3.0) 0.666 Prior stroke Death 1.2 (0.5–2.8) 0.699 HFH 1.5 (0.6–3.6) 0.770 Combination 1.1 (0.5–2.2) 0.835 Previous heart surgery Death 1.6 (0.8–3.1) 0.168 HFH 1.4 (0.6–3.1) 0.421 Combination 1.8 (1.0–3.0) 0.044 1.0 (0.4–2.1) 0.923 Pulmonary hypertension was defined as mean pulmonary artery pressure > 20 mmHg. Right ventricular remodeling was defined as RV diam- eter > 35 mm at mid-level. RV dysfunction was defined as FAC < 35% or TAPSE < 17 mm. Procedural success was defined as postprocedural TR grade at discharge was moderate or less (≤ II). Leaflet repair was defined as implantation of a TriClip, MitraClip, or PASCAL. Annuloplasty was defined as implantation of a Cardioband 6MWD, 6-min walk distance; BMI, body mass index; CABG, coronary artery bypass graft; COPD, chronic obstructive pulmonary disease; EF, ejection fraction; eGFR, estimated glomerular filtration rate; NYHA, New York Heart Association p-values < 0.05 are shown in boldface Table 3   (continued) 0.923 Pulmonary hypertension was defined as mean pulmonary artery pressure > 20 mmHg. Right ventricular remodeling was defined as RV diam- eter > 35 mm at mid-level. RV dysfunction was defined as FAC < 35% or TAPSE < 17 mm. Procedural success was defined as postprocedural TR grade at discharge was moderate or less (≤ II). Leaflet repair was defined as implantation of a TriClip, MitraClip, or PASCAL. Annuloplasty was defined as implantation of a Cardioband 6MWD, 6-min walk distance; BMI, body mass index; CABG, coronary artery bypass graft; COPD, chronic obstructive pulmonary disease; EF, ejection fraction; eGFR, estimated glomerular filtration rate; NYHA, New York Heart Association p values<0 05 are shown in boldface Pulmonary hypertension was defined as mean pulmonary artery pressure > 20 mmHg. Right ventricular remodeling was defined as RV diam- eter > 35 mm at mid-level. RV dysfunction was defined as FAC < 35% or TAPSE < 17 mm. Importance of preprocedural RV remodeling in TTVr reported an HR of 6.47 (95% CI, 3.88–10.77) for patients with RVR who underwent isolated TV surgery (vs. HR of 2.6 in our study), which confirmed that RVR is a risk factor for lower survival [13]. In this study, RVR was defined as either RV dilation (basal end-diastolic diameter > 42 mm and/or mid- level diameter > 35 mm) or dysfunction (tricuspid annular plane systolic excursion < 16 mm and/or tissue Doppler S-velocity < 10 cm/s) [13]. Consequently, the comparabil- ity of the results with those of our study may be limited because of differences in the definition of RVR. However, the patient population with RVR defined by our approach (dilation of RV at mid-level with diameter > 35 mm) was included in their definition of RVR. The fact that RVR has a larger impact on outcomes after surgery compared to TTVr has numerous explanations. Because right heart failure is accelerated by myocardial ischemia after cardiopulmonary bypass and suboptimal myocardial protection during sur- gery, the increased strain on the right heart during cardiac surgery compared with TTVr might be an important reason for worse surgical outcomes in patients with preexisting RVR [32]. Another contributing factor of worsening right heart function with cardiac surgery might be the need for excessive blood transfusions and the subsequent increase in right ventricular preload, which further worsens right ventricular function with RVR [32]. Therefore, the reasons mentioned above might be seen as advocacy for the tran- scatheter approach in patients with RVR, even though RVR is still associated with an increase in mortality compared with patients without RVR. with and without preprocedural RVR. Interestingly, the vast majority of unsuccessful device implantations occurred in patients with RVR. The TR degree was significantly reduced after TTVr in patients with and without prior RVR at the 1-month follow- up. The prevalence of procedural success was significantly higher in patients without RVR than in those with RVR (55% vs. 78%, p < 0.001). This is an important finding which could be explained by greater degree of TR and TV deformation including larger valve diameter with larger coaptation gap due to RV dilation and consecutively more challenging anat- omy for catheter-based treatments. In comparison to the considerably less complex mitral valve anatomy, Yoshida et al. Importance of preprocedural RV remodeling in TTVr to the primary pathophysiological processes, we decided to approximate the entity of atrial TR as TR in the absence of RVR, as proposed by Prihadi et al. in 2019 [30]. Conse- quently, RVR was defined as dilation of the RV at the mid- level (> 35 mm diameter, according to current guidelines) [15]. A thorough multiparametric and multimodal investi- gation of the heterogeneous TR population is sophisticated and cumbersome to apply on a daily basis. To this end, the definition was based on only one variable to provide a sim- ple and hands-on parameter for risk assessment of patients undergoing TTVr in everyday clinical practice. The RV basal diameter was not considered a distinct indicator of RVR, as it can be dilated along with the TV annulus in iso- lated RA enlargement [19].i It is of fundamental importance to acknowledge functional TR as a heterogeneous valve disease with distinguishable phenotypes regarding its pathophysiology [10, 11]. The two main phenotypes of functional TR that must be differenti- ated are atrial functional TR, in which the development of regurgitation is primarily due to right atrial dilation, and ventricular functional TR, in which the main mechanism leading to regurgitation is dilation and remodeling of the right ventricle with consequent tethering of valve leaflets [11, 28]. However, in the literature, the definitions of atrial TR vary. Previous definitions of atrial TR mainly based on clinical criteria, whereas there is currently a trend of using echocardiographic parameters to classify TR etiology [1, 11, 29]. For reasons of clinical utilization and with respect As patients with progressive RVR showed significant functional abnormalities, these patients had more frequently Clinical Research in Cardiology reduced FAC (< 35%) (68% vs. 27%, p < 0.001). The role of RVR on outcomes of patients undergoing treatment of TR is largely unknown. TR grade at baseline was significantly higher in patients with RVR than in those without RVR (p = 0.008). Nevertheless, we acknowledge the relation- ship between RVR and severity of TR as part of a continu- ous vicious cycle in which causality cannot be completely clarified, since RVR influences TR severity and vice versa [31]. However, the manifestation of RVR is an important finding that shows an advanced and/or adverse clinical stage in patients with TR. Considering surgery as the only treatment of TR until recently, Calafiore et al. Functional capacity and QoL The present study captured functional capacity and QoL by analyzing NYHA functional class, occurrence, and grade of peripheral edema, and 6 MWD, as well as changes in MLHFQ and SF-36 scores. There was a significant improve- ment in NYHA functional class at the 1-month follow-up compared with baseline, with 66% of patients having NYHA class I or II, which is promising. Smaller series have shown previously that NYHA class can be reduced after TTVr, albeit with off-label use of edge-to-edge device [35]. Here, we did not see a device-related difference in outcomes. Additionally, there was a significant improvement in both surveyed phenotypes, which might indicate a favorable short-term functional outcome of TTVr, regardless of prior RVR or even reversibility of RVR by TTVr. As peripheral edema is a cardinal symptom of TR, we observed changes in the occurrence and grade of edema before and after TTVr. We observed a significant improvement in peripheral edema grade from baseline to 1-month follow-up in both groups, which might imply that even though peripheral edema was still present in some patients, right heart function improved, and volume overload decreased after TTVr in the majority of cases. This is further affirmed by the fact that no up- titration in loop diuretics doses was observed at the 1-month follow-up compared with baseline; therefore, we cannot attribute the edema regression to escalated medical therapy. Nevertheless, peripheral edema was completely absent in 47% and 63% of 1-month follow-up for patients with and without RVR, respectively (p = 0.029), which might indicate Importance of preprocedural RV remodeling in TTVr reported no differences in pro- cedural success and residual regurgitation between patients with atrial functional MR and those with ventricular MR undergoing percutaneous edge-to-edge mitral valve repair [34]. Nevertheless, we acknowledge MR and TR as separate valve diseases that differ in etiology and clinical impact on heart failure progression. Therefore, the results of TMVr regarding ventricular remodeling are not directly transfer- rable to TTVr and are comparable only to a limited extent. Technical and procedural success of TTVr The present study confirmed that TTVr is a safe procedure in a highly symptomatic patient population, which showed a substantial surgical risk profile and was therefore not eli- gible to undergo a surgical approach for TV repair. The rate of technical success we observed was high and compara- ble to that of recent studies conducted in a similar patient population undergoing TTVr [33]. High technical success rates were observed in both surveyed phenotypes of TR, and no significant differences were observed between patients Clinical Research in Cardiology confirm the causality of RVR in the prognosis of patients after TTVr. confirm the causality of RVR in the prognosis of patients after TTVr. that RVR has a negative impact on right heart function after TTVr. Moreover, we assessed functional capacity by com- paring the 6 MWD in all prospectively recruited patients. Overall improvement of 6 MWD was + 39 m (significantly higher compared with baseline, p < 0.001). Small reports on edge-to-edge devices only showed a mean 6 MWD increase of approximately 33 m [36]. However, a clinically relevant increase of at least 50 m was present in only one-third of patients and was not different between groups. Furthermore, the present study demonstrated that TTVr improved QoL by showing significant improvement in MLHFQ and SF-36 scores at the 1-month follow-up, which is comparable to previous studies investigating QoL after TTVr and TMVr, respectively [33, 37]. In addition, we could complement the analysis by showing that there is an improvement in QoL after TTVr regardless of RVR presence. Supplementary Information  The online version contains supplemen- tary material available at https://​doi.​org/​10.​1007/​s00392-​024-​02428-z. Supplementary Information  The online version contains supplemen- tary material available at https://​doi.​org/​10.​1007/​s00392-​024-​02428-z. Acknowledgements  This project has been supported by Koeln For- tune Program/Faculty of Medicine, University of Cologne. The graphic abstract has been partly generated using Servier Medical Art, pro- vided by Servier, licensed under a Creative Commons Attribution 3.0 unported license. Considering the role of maladaptive RVR in unfavorable clinical outcomes, concerns have been raised in TMVr [40]. Similar results were observed in patients who underwent TV surgery, as mentioned earlier [13]. Additionally, we observed that patients with RVR showed higher prevalence of the combined endpoint of one-year mortality and HF hos- pitalization. Most importantly, RVR was independently asso- ciated with all-cause mortality and the combined endpoint of mortality or rehospitalization after TTVr. Consequently, even though our data suggest an improvement in functional capacity and quality of life regardless of TR etiology already after 1 month, mid-term clinical outcomes and survival in particular seem to be negatively affected by the presence of preprocedural RVR. This hypothesis-generating finding should be further analyzed in larger controlled studies to Author contribution  The authors take responsibility for all aspects of the reliability and freedom from bias of the data presented and their discussed interpretation. All authors have read and approved the manuscript. Funding  Open Access funding enabled and organized by Projekt DEAL. Limitations Several limitations should be acknowledged. First, this is an observational single-center study, and our results are hypothesis-generating. Second, imaging quality was not high enough in all patients for 3D image acquisition; thus, more elaborate echo measures on RV anatomy and function are lacking. Third, there was no adjudication committee for the reported clinical outcomes. Fourth, differentiation of TR phenotype did not lie in the scope of this manuscript as our goal was to provide a hands-on parameter for risk assessment in everyday clinical practice. Nevertheless, fur- ther research in this direction should be promoted to fully elucidate the complex pathophysiology and associated risk in patients with TR. Finally, we analyzed not only one but several devices which have been implanted in consecutive patients. However, outcomes between devices were not different, and additionally reporting real-world outcomes should be considered as a strength of this study. In summary, our results support the conclusion that TTVr improves functional capacity and QoL in patients with heart failure. Moreover, it seems to do so without regard to the presence of RVR, even though there might be indications of at least some impact of RVR on right heart function after TTVr. Conclusion Despite the refinement of functional capacity and QoL, the association with mid-term survival is still largely unknown in the context of TTVr. The only randomized trial so far in this field showed no survival benefit after edge-to-edge TR repair [10]. Compared with this study, our overall observed rate of all-cause mortality at the 12-month follow-up was higher (19%), which can probably relate to a sicker popula- tion and higher prevalence of severe comorbidities (more patients with diabetes, kidney disease, chronic obstructive lung disease, and NYHA class III/IV for instance). On the contrary, other early experience observational studies had similar one-year outcomes after TTVr and TMVr [38, 39]. Considering the role of maladaptive RVR in unfavorable clinical outcomes, concerns have been raised in TMVr [40]. Similar results were observed in patients who underwent TV surgery, as mentioned earlier [13]. Additionally, we observed that patients with RVR showed higher prevalence of the combined endpoint of one-year mortality and HF hos- pitalization. Most importantly, RVR was independently asso- ciated with all-cause mortality and the combined endpoint of mortality or rehospitalization after TTVr. Consequently, even though our data suggest an improvement in functional capacity and quality of life regardless of TR etiology already after 1 month, mid-term clinical outcomes and survival in particular seem to be negatively affected by the presence of preprocedural RVR. This hypothesis-generating finding should be further analyzed in larger controlled studies to Despite the refinement of functional capacity and QoL, the association with mid-term survival is still largely unknown in the context of TTVr. The only randomized trial so far in this field showed no survival benefit after edge-to-edge TR repair [10]. Compared with this study, our overall observed rate of all-cause mortality at the 12-month follow-up was higher (19%), which can probably relate to a sicker popula- tion and higher prevalence of severe comorbidities (more patients with diabetes, kidney disease, chronic obstructive lung disease, and NYHA class III/IV for instance). On the contrary, other early experience observational studies had similar one-year outcomes after TTVr and TMVr [38, 39]. TTVr resulted in significant QoL improvement already after 1 month, irrespective of RVR. Patients with RVR showed higher 1-year mortality and HF hospitalization rates; RVR was independently associated with all-cause mortality and the combined endpoint of mortality or rehospitalization after TTVr. References g g 20. Du H, Wonggom P, Tongpeth J et al (2017) Six-minute walk test for assessing physical functional capacity in chronic heart failure. Curr Heart Fail Rep 14:158–166 1. Fender EA, Zack CJ, Nishimura RA (2018) Isolated tricuspid regurgitation: outcomes and therapeutic interventions. Heart 104:798–806 21. Navas JP, Martinez-Maldonado M (1993) Pathophysiology of edema in congestive heart failure. Heart Dis Stroke 2:325–329 2. 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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/. 16. Lancellotti P, Tribouilloy C, Hagendorff A et al (2013) Recommenda- tions for the echocardiographic assessment of native valvular regurgi- tation: an executive summary from the European Association of Car- diovascular Imaging. Eur Heart J Cardiovasc Imaging 14:611–644 17. Hahn RT, Zamorano JL (2017) The need for a new tricuspid regurgitation grading scheme. Eur Heart J Cardiovasc Imaging 18:1342–1343i 18 Humbert M, Kovacs G, Hoeper MM, ESC/ERS Scientific Docu- ment Group et al (2022) ESC/ERS guidelines for the diagnosis and treatment of pulmonary hypertension. Eur Heart J 43:3618–3731 19. 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Hypocholesterolemic Effect of Mature Leaf Extract of Sugarcane, Saccharum officinarum (Linnaeus, 1753), in Induced Rats
Asean Journal Of Science And Engineering
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Journal homepage: http://ejournal.upi.edu/index.php/AJSE/ Journal homepage: http://ejournal.upi.edu/index.php/AJSE/ Hypocholesterolemic Effect of Mature Leaf Extract of Sugarcane, Saccharum Officinarum (Linnaeus, 1753), in Induced Rats Raamah Rosales1* 1College of Arts and Sciences, Cebu Technological University-Main Campus, Cebu City 600, the Philippines Correspondence: E-mail: raamah.rosales@ctu.edu.ph ASEAN Journal of Science and Engineering 1(3) (2021)199-206 ASEAN Journal of Science and Engineering 1(3) (2021)199-206 © 2021 Universitas Pendidikan Indonesia Rosales., Hypocholesterolemic Effect of Mature Leaf Extract of … | 200 Rosales., Hypocholesterolemic Effect of Mature Leaf Extract of … | 200 Rosales., Hypocholesterolemic Effect of Mature Leaf Extract of … | 200 A R T I C L E I N F O ____________________ Keyword: Hypocholesterolemic, Mature, Rats. Article History: Submitted/Received 09 Jun 2021 First revised 07 Jul 2021 Accepted 09 Sep 2021 First available online 11 Sep 2021 Publication date 01 Des 2021 ____________________ Keyword: Hypocholesterolemic, Mature, Rats. Article History: Submitted/Received 09 Jun 2021 First revised 07 Jul 2021 Accepted 09 Sep 2021 First available online 11 Sep 2021 Publication date 01 Des 2021 Article History: Submitted/Received 09 Jun 2021 First revised 07 Jul 2021 Accepted 09 Sep 2021 First available online 11 Sep 2021 Publication date 01 Des 2021 Hypercholesterolemia is a common health risk factor for various cardiovascular diseases. The use of the medicinal plant as an alternative treatment is gaining much attention due to the reported adverse effects of statins on liver and muscle toxicity. This study aimed to determine the hypocholesterolemic properties of the mature leaf of the sugarcane plant using hypercholesterolemia-induced rats. Thirty-two Sprague Dawley rats were divided into 4 groups and fed with a normal diet and a 4% high cholesterol diet. The dose conversion was based on the human dose to surface area ratio of 0.025 at 1mg/20g rat body weight. The treatment with mature leaf extract of S. officinarum significantly lowered the cholesterol levels. The hypocholesterolemic activity and percent reduction of the plant extract were comparable to the effects of a synthetic drug, Fluvastatin, in lowering the cholesterol levels. This study presents the potential of mature leaf extract of S. officinarum as an alternative therapeutic strategy for hypercholesterolemia. Keyword: Hypocholesterolemic, Mature, Rats. Keyword: Hypocholesterolemic, Mature, Rats. © 2021 Universitas Pendidikan Indonesia 200 1. INTRODUCTION Hypercholesterolemia is a health risk condition that plays a role in developing cardiovascular diseases, including atherosclerosis, high blood pressure, or acute myocardial infarction. Hypercholesterolemia is a lipoprotein metabolic syndrome characterized by elevated serum concentration of low-density lipoprotein (LDL) and blood cholesterol (Yokozawa et al., 2006). Various hypocholesterolemic drugs are known to lower cholesterol levels but may have long-term adverse effects such as muscle and liver damage, renal failure, and myopathy. The usage of herbal medicine may provide a safer strategy in treating human diseases. Plant medicinal potential largely depends on biological compounds derived from secondary metabolites (Masa et al., 2016). These metabolites vary quantitatively among species, between populations of the same species, and between the developmental stages of plant organs such as leaf (Achakzai et al., 2009; Azam et al., 2013; El Sawi et al., 2013; Del Valle et al., 2015). Masa et al., (2016) quantified flavonoids and diterpenes in leaves of Cistus ladanifer L. at different ages. The result showed varying phenolic contents in young, mature leaves and near senescent ones. Watanabe et al., (2016) explained that leafage, either young or mature, affects the distribution of minerals in a different leaf of vegetables cultivated in an open field. Sugarcane is an economically important agricultural crop in the Philippines. Annual sugar production produces a massive volume of sugarcane leaves that are not fully utilized for other significant purposes. There are no reports in any published literature in a scientific journal on the hypocholesterolemic effects specific to the mature leaf of the sugarcane plant to date. To address this gap, this study aimed at investigating the hypocholesterolemic effect of the mature leaf of sugarcane plant in induced rats. 2.1 Plant Material Mature leaf samples were collected from sugarcane plantations in Northern Cebu Island, Philippines (11.1491° N, 123.9861° E) between March to June 2018 and authenticated by the Department of Biology, University of San Carlos, Cebu Philippines. Mature sugarcane leaf is fully expanded and highly exposed to sunlight (Gianotto et al., 2011) (see Figure 1). Samples collected were designated as leaf +2, +3, and +4 following the Kujiper (1915) numbering system for the sugarcane plant (Gianotto et al., 2011; Zhao et al., 2012). DOI: http://dx.doi.org/10. 17509/xxxx.xxxx p- ISSN 2776-6098 e- ISSN 2776-2538 201 | ASEAN Journal of Science and Engineering, Volume 1 Issue 3, December 2021 Hal 199-206 201 | ASEAN Journal of Science and Engineering, Volume 1 Issue 3, December 2021 Hal 199-206 Figure 1. Kujiper's leaf numbering system on sugarcane plant (1915). J f g g, 3, 99 Figure 1. Kujiper's leaf numbering system on sugarcane plant (1915). 2.4 Preparation of Positive and Negative Control Forty (40) mg of Fluvastatin sodium tablet was used as the positive control. The tablet was powdered using a mortar and dissolved with a sufficient amount of distilled water to make a 50 mL solution. Distilled water was used for the negative control and administered with the same volume as the positive control. 2.2 Preparation and Extraction of Plant Samples A total of 10 kg air-dried and finely chopped leaves macerated with 95 % ethanol were placed in glass containers. The containers were thoroughly shaken and allowed to stand at room temperature with occasional manual agitation using a stirring rod. The mixture was allowed to stand for 72 hr to ensure complete extraction, then strained and the marc discarded. The menstruum was filtered using sterile Whatman no. I filtered paper and concentrated using a rotary evaporator (Heidolph Hei-VAP Platinum 2 Rotary Evaporator, Germany) at 60-65ºC temperature range. The filtrate was placed in a water bath at 75-80ºC temperature range to evaporate to dryness and obtain solvent-free crude extract—a total of 430 g with a percent yield of 4.2 % extracted after the entire concentration process. 2.3 Preparation of Test Solution Five (5) g of sugarcane leaf extract was dissolved in enough water to make a 10 mL solution. The concentration of this solution reached 50%. 2.5 Ethical Review The methods were submitted for ethical review and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of San Carlos-Talamban Campus (USC-TC), Cebu, Philippines, with the approved protocol number: 2018-029-024. 2.8 Induction of Hypercholesterolemia The rats were fed 4% of cholesterol powder technical grade once per day for seven consecutive days. 2.6 Preparation of Experimental Animal Before experiments, male Sprague Dawley rats weighing between 180-200 g of ten (10) days old were obtained from the USC-TC Animal Facility. The rats were acclimated for seven DOI: http://dx.doi.org/10.17509/xxxx.vxix p- ISSN NomorISSN e- ISSN NomorISSN DOI: http://dx.doi.org/10.17509/xxxx.vxix p- ISSN NomorISSN e- ISSN NomorISSN DOI: http://dx.doi.org/10.17509/xxxx.vxix p- ISSN NomorISSN e- ISSN NomorISSN Rosales., Hypocholesterolemic Effect of Mature Leaf Extract of … | 202 Rosales., Hypocholesterolemic Effect of Mature Leaf Extract of … | 202 (7) days in an air-conditioned room with an average temperature of 25∘C and 55% humidity. The rats were provided free access to food and water on a 12 h light-dark cycle. (7) days in an air-conditioned room with an average temperature of 25∘C and 55% humidity. The rats were provided free access to food and water on a 12 h light-dark cycle. Block randomization allocated the test animals through an odd/even scheme. All thirty- two healthy laboratory rats numbered randomly in odd/even. Each of the test groups has an equal number of test animals. The first eight (8) even-numbered rats were assigned to the positive control group, while the first eight (8) odd-numbered rats were assigned to the test group for the crude extract treatment. The same procedure applies to the negative and untreated groups (Conforti et al., 2007). During the experiments, the rats were in separate cages with eight members big enough for the rats to move around while maintaining the standard conditions to acclimatize them (Kanda et al., 2015). The rats fed with the standard animal pellet diet with 225 grams pellets (3.0 grams pellets per rat) and distilled water was given once a day for one week during the entire course of the experiment (Kanda et al., 2015). The test animals acclimatize in the laboratory for seven (7) days. The test animals again acclimatize for 10 min before the beginning of testing. The first measurement (T0) was taken once the acclimation period concluded. 2.7 Determination of Baseline Cholesterol Level The rats fasted for twelve hours from 8:00 pm – 8:00 am. The body weights of each rat were determined and recorded using a digital weighing scale. A Beaker of predetermined weight was used to contain the rat to facilitate weighing. Topical lidocaine cream was applied prior to tail clipping to prevent pain. After obtaining the weights of each rat, the tail was swabbed with 95% alcohol and then cut at the tip; a drop of blood was directly placed in the test strips of the cholesterol tester (EasyLife GCU Plus with CE 0197, model: ET-3431, Taiwan) to obtain the baseline output of cholesterol. 2.9 Determination of Blood Cholesterol Levels After seven consecutive days of feeding the rats with 4% of cholesterol powder and distilled water three times a day, the cholesterol level of rats was taken as the hypercholesterolemic value. DOI: http://dx.doi.org/10. 17509/xxxx.xxxx p- ISSN 2776-6098 e- ISSN 2776-2538 203 | ASEAN Journal of Science and Engineering, Volume 1 Issue 3, December 2021 Hal 199-206 03 | ASEAN Journal of Science and Engineering, Volume 1 Issue 3, December 2021 Hal 199-206 203 | ASEAN Journal of Science and Engineering, Volume 1 Issue 3, December 2021 Hal 199-206 2.11 Statistical Treatment Values for activity and reduction were presented as mean ± S.D. Tukey HSD tested the statistical difference between the treatment and the controls to elucidate the variation between test groups. 2.10 Administration of Test Solution and Controls The thirty-two rats were divided into four (4) groups: positive control, test group for crude extract, negative control, and untreated. Test animals for positive control were given 0.13mL/20g Fluvastatin Sodium. Test animals for negative control were given distilled water. Test animals for test groups using the crude extract were given 0.8ml/20g rats of Test Solutions. The untreated group was not administered with test drugs for the sole reason of determining the highest possible cholesterol level. The test solutions and the controls were administered orally to rats three times a day at 8:00 am, 12:00 noon, and 6:00 pm for three consecutive days. Cholesterol levels were recorded twelve hours after the third treatment each day. Oral administration was carried out using oral gavage. Dose conversion was based on the human dose to surface area ratio of rats of 0.025 at 1mg/20g body weight. DOI: http://dx.doi.org/10. 17509/xxxx.xxxx p- ISSN 2776-6098 e- ISSN 2776-2538 3.1 Hypocholesterolemic Mean Activity and Reduction The mean percent hypocholesterolemic activity varies among the test groups (Figure 2). Positive control has the highest mean value of 100%, while the untreated group and negative control have no hypocholesterolemic activity. The test group for crude extract indicated 80.76 % hypocholesterolemic, relatively close to the mean value of the positive control. Figure 2. Mean percent hypocholesterolemic activity 0% 20% 40% 60% 80% 100% 120% Postive Control Test Group Negative Control Untreated % Hypocholeseric Activity Test Groups Figure 2. Mean percent hypocholesterolemic activity Table 1 shows the Tukey HSD test results. The comparison of all groups was significant except between positive control vs. test group for crude extract and negative control vs. untreated group. These groups may have indicated the same % activity and reduction. Table 1. Post-hoc analysis between each test group for hypocholesterolemic activity. Tukey HSD Description Mean Difference Std. Error Sig. 99% Confidence Interval Lower Bound Upper Bound (+) Control Test Group 19.24 4.33 0.020 -38.5378 0.0538 (-) Control 110.63 14.34 0.000 -129.9307 -91.3391 Untreated 116.94 15.17 0.000 -136.2347 -97.6431 Test Group (+) Control 91.39 12.39 0.000 -110.6887 -72.0971 Untreated 97.69 13.19 0.000 -116.9926 -78.4010 (-) Control Untreated 6.30 2.13 0.145 -25.5998 12.9910 Table 1. Post-hoc analysis between each test group for hypocholesterolemic activity. This study presents the hypocholesterolemic effect of the mature leaf extract of S. officinarum in hypercholesterolemia-induced Sprague Dawley rats. Fluvastatin was used as a positive control because it is widely used as a synthetic drug in lowering blood cholesterol DOI: http://dx.doi.org/10.17509/xxxx.vxix p- ISSN NomorISSN e- ISSN NomorISSN p g p- ISSN NomorISSN e- ISSN NomorISSN 204 Rosales., Hypocholesterolemic Effect of Mature Leaf Extract of … | 2 levels. The result showed that positive control posted the highest mean % hypocholesterolemic activity value of 100 %, while the test group for crude extract indicated 80.76 % hypocholesterolemic activity. Interestingly, the % hypocholesterolemic activity of the crude extract was relatively close to the synthetic drug. The crude extract has a mean percent reduction of 95.26%, comparable to the 103.94% of the positive control. At a given dose, the result of the crude extract's mean percent reduction and activity showed its potential in lowering blood cholesterol levels. The hypocholesterolemic properties of S. officinarum may be due to either the individual or the synergistic action of the phenolic components of the mature plant leaf (Chuerfa & Allen, 2015). 4. CONCLUSION In conclusion, this study demonstrates the hypocholesterolemic potential of the mature leaf of S. officinarum. It showed hypocholesterolemic activity and reduction comparable to the synthetic drug used in this study. The result explicitly represents the hypocholesterolemic effect of the mature leaf of the sugarcane plant. Future studies may be conducted to confirm these results further. The author declares no conflict of interest and the article paper is free of plagiarism. The author declares no conflict of interest and the article paper is free of plagiarism. 3.1 Hypocholesterolemic Mean Activity and Reduction Physiological mechanisms of different tissues, such as hydrolytic control of specific lipoproteins and their selective uptake and metabolism, can influence lipidemic profile (Rouhi-Boroujeni et al., 2015). Sugarcane wax contains phenolic components, including flavonoids, known to possess hypocholesterolemic properties (Singh et al., 2015). Flavonoids can regulate blood lipids by increasing the activity of lecithin acyltransferase (LCAT) (You et al., 2008). LCAT is responsible for the inclusion of free cholesterol in the high-density lipoprotein and its transfer back to very-low-density lipoprotein, which is returned later in liver cells (Dobiásová & Frohlich, 1999). 5. ACKNOWLEDGMENT The author would like to thank the Department of Science and Technology – Scholarships Education Institute (DOST-SEI) for providing funds for this study. The author would also like to thank Glen Mark Piañar for his assistance in the statistical analysis of the data. 7. REFERENCES Achakzai, A.K.K., Achakzai, P., Masood, A., Alikayani, S., and Tareen, R.B. (2009). Response of plants and age on the distribution of secondary metabolites on plants found in Quetta. Pakistan Journal of Botany. 41, 2129-2135. Azam, M., Zhang, B., Jiang, Q., and Chen, K. (2013). Citrus leaf volatiles as affected by developmental stage and genetic type. International Journal of Molecular Sciences, 14, 17744-17766. Kanda, I. (2015). Exotic Animal Formulary. The Canadian Veterinary Journal, 56(7), 736. Chuerfa, M., and Allem, R. (2015). Study of hypocholesterolemic activity of Algerian Pistacia lentiscus leaves extracts in vivo. Revista Brasileira Farmacognosia, 25, 142-144. DOI: http://dx.doi.org/10. 17509/xxxx.xxxx p- ISSN 2776-6098 e- ISSN 2776-2538 205 | ASEAN Journal of Science and Engineering, Volume 1 Issue 3, December 2021 Hal 199-206 Conforti, A., Bellavite, P., Bertani, S., Chiarotti, F., Menniti-Ippolito, F., and Raschetti, R. (2007). Rat models of acute inflammation: a randomized controlled study on the effects of homeopathic remedies. Business Model Canvas Complementary and Alternative Medicine, 7(1), 1-10. Del Valle, J. C., Buide, M. L., Casimiro-Soriguer, I., Whittall, J. B., and Narbona, E. (2015). On flavonoid accumulation in different plant parts: Variation patterns among individuals and populations in the shore campion (Silene littorea). Frontiers in Plant Science, 6, 939. Dobiásová, M., and Frohlich, J. (1999). Advances in the understanding of the role of lecithin cholesterol acyltransferase (LCAT) in cholesterol transport. Clinical Chimica Acta, 286, 257- 271. El Sawi, S.A.M., Mahmoud, D.M.M., and El Alfy, T.S.M.A. (2013). Phytochemical screening and qualitative analysis of the polysaccharide contents of Chorisia insignis. Journal of Herbs, Spices, and Medicinal Plants, 19, 313-320. Gerhardt, A.L., and Gallo, N.N. (1998). Full-fat rice and oat bran similarly reduce hypercholesterolemia in humans. The Journal of Nutrition, 128, 865-869. Gianotto, A.C., Bespalhok, J., Arruda, P., and Burnquist, W. (2011). Sugarcane (Saccharum X officinarum): A reference study for the regulation of genetically modified cultivars in Brazilian Tropical Plant Biology. Tropical Plant Biology, 4, 62-89. Kujiper, J. (1915). Degroei van bladschijf, bladscheede em stengel van het suikerriet. Arch Suikerind Ned Indi, 23, 528–556. Masa, C. V., Sosa Díaz, T., Alías Gallego, J. C., and Chaves Lobón, N. (2016). Quantitative variation of flavonoids and diterpenes in leaves and stems of Cistus ladanifer L. at different ages. Molecules, 21(3), 275. Nair, A.B., and Jacob, S. (2016). A simple guide for dose conversion between animals and humans. Journal of Basic Clinical Pharmacy, 7, 27-31. Rouhi-Boroujeni, H., Heidarian, E., Mohammadizzadeh, E., and Rafieian-Kopaei, M. DOI: http://dx.doi.org/10. 17509/xxxx.xxxx p- ISSN 2776-6098 e- ISSN 2776-2538 Zhao, D., Glynn, N.C., Glaz, B., Comstock, J.C., and Johnson, R.M. (2012). Development of leaf spectral models for evaluating large numbers of sugarcane genotypes. Crop Science, 52, 1837-1847. yp Rosales., Hypocholesterolemic Effect of Mature Leaf Extract of … | 206 7. REFERENCES (2015). Herbs with anti-lipid effects and their interactions with a statin as a chemical anti- hyperlipidemia group drugs: A systematic review. ARYA Atheroscler, 11, 244-251. Singh, A., Lal, U.R., Mukhtar, M., Mukhtar, H.M., Singh, P.S., and Shah, R.K. (2015). Phytochemical profile of sugarcane and its potential aspects. Pharmacognosy Reviews, 9, 45-54. Watanabe, T., Maejima, E., Urayama, M., Owado, M., and Yamauchi, A. (2016). Distribution of minerals in young and mature leaves of different leaf vegetable crops cultivated in a field. Open Journal of Plant Science, 1, 005-009. Yokozawa, T., Cho, E.J., Satoh, A., Okamoto, T., and Sei, Y. (2006). The protective role of Chinese prescription Kangen-karyu extracts on diet-induced hypercholesterolemia in rats. Biological and Pharmaceutical Bulletin, 29, 760-765. You, C.L., Su, P.Q., and Zhou, X.X. (2008). Study on effect and mechanism of Scutellaria baicalensis stem-leaf total flavonoids in regulating lipid metabolism. Zhongguo Zhong Yao ZA Zhi, 33, 1064-1066. DOI: http://dx.doi.org/10.17509/xxxx.xxxx p- ISSN 2775-6793 e- ISSN 2775-6815
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https://figshare.com/articles/journal_contribution/Supplementary_Figure_Legends_from_YC-1_Induces_S_Cell_Cycle_Arrest_and_Apoptosis_by_Activating_Checkpoint_Kinases/22364549/1/files/39808817.pdf
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Supplementary Figure Legends from YC-1 Induces S Cell Cycle Arrest and Apoptosis by Activating Checkpoint Kinases
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Supplementary Figure Legends Fig. 1. YC-1-induced S-phase arrest in synchronized cells. To synchronize the cell cycle, HEK293 cells were treated with 1mM hydroxyurea for 24 hours, and re-entry into the cell cycle was initiated by washing-out the hydroxyurea. Cells were treated with DMSO (- YC-1) or 1 µM YC-1 (+ YC-1) immediately after the washing-out procedure. Cell cycles were analyzed at the indicated times after washing-out. Fig. 2. Effects of YC-1 on DNA breakage, topoisomerase I and topoisomerase II. (A) YC-1 does not induce DNA breakage. DNA breakage was analyzed by using Comet assays. Hep3B cells were treated with YC-1 (1 or 2 µM) or 100 µM H2O2 for (a positive control) for 12 hours. Cells embedded in agarose gels, lysed and electrophoresed. DNAs were visualized by staining with SYBR green dye. (B) YC-1 does not inhibit topoisomerase I activity. Topoisomerase I activity was measured by using DNA relaxation assays. Hep3B cells were treated with DMSO (Con) or various concentrations of YC-1 for 12 hours. Nuclear proteins were extracted as described in Methods and used for assays. The enzyme reaction was initiated with pcDNA (substrate), and products were electrophoresed on 1% agarose gels, and stained with ethidium bromide. Wound pcDNA was converted to its unwound form by topoisomerase I. (C) YC-1 does not inhibit topoisomerase II activity. Topoisomerase II activity was analyzed by examining the ATP-dependent decatenation of Kinetoplast DNA (kDNA). Nuclear proteins were extracted from Hep3B cells incubated with DMSO (Con) or YC-1 (1 or 2 µM) for 12 hours, and the enzymatic reaction was initiated with nuclear extract and kDNA. DNA samples were electrophoresed in 1% agarose gels, and stained with ethidium bromide. Decatenated and catenated kDNAs (provided by TopoGEN Inc.) were loaded as standards.
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https://europepmc.org/articles/pmc7082841?pdf=render
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The Cytoprotective Enzyme Heme Oxygenase-1 Suppresses Pseudorabies Virus Replication in vitro
Frontiers in microbiology
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Edited by: Chunfu Zheng, Fujian Medical University, China Reviewed by: Yi-Quan Wu, National Cancer Institute (NCI), United States Qi Wang, Harbin Veterinary Research Institute (CAAS), China *Correspondence: Yongkun Du mututushen@163.com Gaiping Zhang zhanggaiping2003@163.com Specialty section: This article was submitted to Virology, a section of the journal Frontiers in Microbiology Specialty section: This article was submitted to Virology, a section of the journal Frontiers in Microbiology Received: 17 December 2019 Accepted: 27 February 2020 Published: 13 March 2020 Citation: Zhang A, Wan B, Jiang D, Wu Y, Ji P, Du Y and Zhang G (2020) The Cytoprotective Enzyme Heme Oxygenase-1 Suppresses Pseudorabies Virus Replication in vitro. Front. Microbiol. 11:412. doi: 10.3389/fmicb.2020.00412 Keywords: heme oxygenase-1, carbon monoxide, biliverdin, antiviral effect, pseudorabies virus The Cytoprotective Enzyme Heme Oxygenase-1 Suppresses Pseudorabies Virus Replication in vitro Angke Zhang, Bo Wan, Dawei Jiang, Yanan Wu, Pengchao Ji, Yongkun Du* and Gaiping Zhang* Angke Zhang, Bo Wan, Dawei Jiang, Yanan Wu, Pengchao Ji, Yongkun Du* and Gaiping Zhang* College of Animal Sciences and Veterinary Medicine, Henan Agricultural University, Zhengzhou, China Pseudorabies virus (PRV) infection brings about great economic losses to the swine industry worldwide, as there are currently no effective therapeutic agents or vaccines against this disease, and mutations in endemic wild virulent PRV strains result in immune failure of traditional vaccines. Heme oxygenase-1 (HO-1) catalyzes the conversion of heme into biliverdin (BV), iron and carbon monoxide (CO), all of which have been demonstrated to protect cells from various stressors. However, the role of HO-1 in PRV replication remains unknown. Thus, the present study aimed to investigate the effect of HO-1 on PRV replication and determine its underlying molecular mechanisms. The results demonstrated that induction of HO-1 via cobalt-protoporphyrin (CoPP) markedly suppressed PRV replication, while HO-1 specific small interfering RNA or inhibitor zinc-protoporphyrin partially reversed the inhibitory effect of CoPP on PRV replication. Furthermore, overexpression of HO-1 notably inhibited PRV replication, while knockdown of endogenous HO-1 expression promoted PRV replication. Mechanism analyses indicated that the HO-1 downstream metabolites, CO and BV/BR partially mediated the virus suppressive effect of HO-1. Taken together, the results of the present study suggest that HO-1 may be developed as a novel endogenous antiviral factor against PRV, and the HO-1/BV/CO system may constitute a unique antiviral protection network during PRV infection and interaction with host cells. Keywords: heme oxygenase-1, carbon monoxide, biliverdin, antiviral effect, pseudorabies virus ORIGINAL RESEARCH published: 13 March 2020 doi: 10.3389/fmicb.2020.00412 INTRODUCTION Received: 17 December 2019 Accepted: 27 February 2020 Published: 13 March 2020 Pseudorabies (PR), caused by the pseudorabies virus (PRV) and characterized by severe reproductive, respiratory and neurological disorders, is one of the most devastating infectious diseases in pigs that brings about great economic losses to the swine industry worldwide (Nauwynck et al., 2007; Freuling et al., 2017). PRV belongs to the Herpesviridae family, α-herpesvirinae subfamily and varicellovirus genus (Mettenleiter, 2000). Although the virus has been reported to infect a variety of mammals, including ruminants, carnivores and rodents and cause ∼100% mortality, pigs are the only natural hosts for PRV in which latency is established (Mettenleiter, 1991). Since the 2011 outbreak of PRV in some farms of China, in which pigs were vaccinated with Bartha-K61, subsequent mutations in PRV strains have introduced great challenges in the Citation: The present study aimed to investigate the role of HO-1 activity on the infection and replication of PRV in porcine kidney (PK)- 15 cells and swine testis (ST) cells, and determine the potential molecular mechanisms involved in this process. The results demonstrated for the first time that induction or overexpression of HO-1 markedly inhibits PRV replication, while knockdown of endogenous HO-1 expression facilitates PRV replication, indicating that HO-1 may act as an effective endogenous antiviral factor. Further mechanistic studies revealed that the downstream metabolites of HO-1 (BV/BR and CO) partially mediate the antiviral activity of this enzyme. Taken together, the results of the present study suggest that HO-1 and its products (BV and CO) may function in suppressing PRV replication, thus act as novel therapeutic targets against PRV infection. gg g y CO is another metabolite of HO-1, which has been demonstrated to exert anti-inflammatory, antiapoptotic and cytoprotective effects in several types of diseases (Otterbein, 2002; Chung et al., 2008). CO has been reported to inhibit the expression of proinflammatory molecules on the cell surface (Riquelme et al., 2015a), regulation of mitochondrial function (Riquelme et al., 2015b) and inhibit T cell activation (Mackern- Oberti et al., 2015). With relevance to the present study, previous findings have reported an association between CO and viral replication in host cells. For example, CO has been demonstrated to suppress ROS generation in EV71- infected cells, thus restraining viral replication in host cells (Tung et al., 2011). Another study reported that HO-1-derived CO suppresses the NF-κB signaling pathway, while activating the cGMP/PKG cascade to impede porcine reproductive and respiratory syndrome virus (PRRSV) replication in its permissive cells (Zhang et al., 2017). The discovery of CO-releasing molecules (CORMs) provides pharmacological tool to further determine the bioactive properties of CO (Motterlini et al., 2002). p y Heme oxygenase-1 (HO-1) is ubiquitously expressed in mammalian cells and catalyzes the conversion of heme into carbon monoxide (CO), iron and biliverdin (BV), which is subsequently converted into bilirubin (BR) via NADPH- dependent biliverdin reductase (BVR) (Tenhunen et al., 1969). HO-1 is considered to exert its cytoprotective effect via the antioxidant, antiapoptotic and anti-inflammatory activities of its downstream metabolites (Ryter and Choi, 2009, 2016). Previous studies have reported that HO-1 exerts a protective role against oxidative stress (Lee J.C. et al., 2014). Citation: Virus infection of host cells is often accompanied by oxidative stress stimulation, which means the potential role of HO-1 during virus infection has recently been gaining recognition. Thus far, a number of studies have demonstrated the antiviral activity of HO-1. For example, induction or overexpression of HO-1 has been reported to inhibit infection of influenza (Hashiba et al., 2001), human immunodeficiency virus (HIV) (Devadas and Dhawan, 2006), hepatitis B virus (HBV) (Protzer et al., 2007), dengue virus (DENV) (Tseng et al., 2016), and human respiratory syncytial virus (RSV) (Espinoza et al., 2017). HO-1 has also exhibited antiviral properties within viruses effecting domestic animals. For instance, HO-1 can suppress the replication of bovine viral diarrhea virus (BVDV) in vitro (Xiao et al., 2014). These previous studies emphasize the potential use of this cytoprotective enzyme as a virucidal agent. However, the molecular mechanism underlying the antiviral effect of HO-1 still remains largely unknown. determine the bioactive properties of CO (Motterlini et al., 2002). As mentioned above, the role of HO-1 and its underlying molecular mechanisms during PRV infection remain unclear. Given that the HO-1 products, BV and CO, play key roles in mediating the cytoprotective function of this molecule that exerts antiviral effects, studying the role of this enzyme in relation to PRV replication may help identify novel therapeutic approaches to overcome viral infection. The present study aimed to investigate the role of HO-1 activity on the infection and replication of PRV in porcine kidney (PK)- 15 cells and swine testis (ST) cells, and determine the potential molecular mechanisms involved in this process. The results demonstrated for the first time that induction or overexpression of HO-1 markedly inhibits PRV replication, while knockdown of endogenous HO-1 expression facilitates PRV replication, indicating that HO-1 may act as an effective endogenous antiviral factor. Further mechanistic studies revealed that the downstream metabolites of HO-1 (BV/BR and CO) partially mediate the antiviral activity of this enzyme. Taken together, the results of the present study suggest that HO-1 and its products (BV and CO) may function in suppressing PRV replication, thus act as novel therapeutic targets against PRV infection. BV and BVR are well characterized signaling cascades and the only metabolic pathway producing BR (Maines, 2005; Kapitulnik and Maines, 2009). The conversion of BV to BR via BVR is a physiological process, synchronized with the metabolism of heme (Greenberg, 2002; Bach, 2006). Citation: Previous studies have demonstrated that the BV/BVR system displays effective anti-inflammatory and antiviral activities (Sass et al., 2004; Wegiel et al., 2011). For example, as a downstream metabolite of HO-1, BV has been demonstrated to act as a key effector against HCV replication by activating the antiviral IFN response and inhibiting the NS3/4A protease activity of HCV (Lehmann et al., 2010; Zhu et al., 2010). HO-1 derived from BR has been reported to suppress human herpes simplex type 1 virus (HSV-1) and EV71 infection, as well as protease activity of DENV and HIV (Santangelo et al., 2012; Olagnier et al., 2014; Liu et al., 2016). Iron ions derived from HO-1 have been demonstrated to participate in key cellular processes that are dependent on this metal, either promoting or suppressing the translation of particular mRNAs, depending on its concentrations (Eisenstein et al., 1991). Elevated intracellular iron concentrations have been implicated in the activation of the cytoprotective NF-κB signaling pathway, which can effectively reverse Fas-mediated cell apoptosis (Choi et al., 2004). Notably, HO-1-derived iron was reported to suppress Citation: Zhang A, Wan B, Jiang D, Wu Y, Ji P, Du Y and Zhang G (2020) The Cytoprotective Enzyme Heme Oxygenase-1 Suppresses Pseudorabies Virus Replication in vitro. Front. Microbiol. 11:412. doi: 10.3389/fmicb.2020.00412 March 2020 | Volume 11 | Article 412 1 Frontiers in Microbiology | www.frontiersin.org HO-1 Suppresses PRV Replication in vitro Zhang et al. subgenomic replication of HCV by inactivating its NS5B RNA- dependent RNA polymerase activity (Fillebeen et al., 2005), suggesting its potential antiviral activity. subgenomic replication of HCV by inactivating its NS5B RNA- dependent RNA polymerase activity (Fillebeen et al., 2005), suggesting its potential antiviral activity. prevention and control of PR (An et al., 2013; Yu et al., 2014). To date, several precautionary measures have been taken to prevent and control PR in China; however, due to the sophisticated factors associated with the virus biology, transmission and infection of PRV remains difficult to completely control. gg g p y CO is another metabolite of HO-1, which has been demonstrated to exert anti-inflammatory, antiapoptotic and cytoprotective effects in several types of diseases (Otterbein, 2002; Chung et al., 2008). CO has been reported to inhibit the expression of proinflammatory molecules on the cell surface (Riquelme et al., 2015a), regulation of mitochondrial function (Riquelme et al., 2015b) and inhibit T cell activation (Mackern- Oberti et al., 2015). With relevance to the present study, previous findings have reported an association between CO and viral replication in host cells. For example, CO has been demonstrated to suppress ROS generation in EV71- infected cells, thus restraining viral replication in host cells (Tung et al., 2011). Another study reported that HO-1-derived CO suppresses the NF-κB signaling pathway, while activating the cGMP/PKG cascade to impede porcine reproductive and respiratory syndrome virus (PRRSV) replication in its permissive cells (Zhang et al., 2017). The discovery of CO-releasing molecules (CORMs) provides pharmacological tool to further determine the bioactive properties of CO (Motterlini et al., 2002). As mentioned above, the role of HO-1 and its underlying molecular mechanisms during PRV infection remain unclear. Given that the HO-1 products, BV and CO, play key roles in mediating the cytoprotective function of this molecule that exerts antiviral effects, studying the role of this enzyme in relation to PRV replication may help identify novel therapeutic approaches to overcome viral infection. Cell Viability and Cytotoxicity Assay Cell Viability and Cytotoxicity Assay Cell viability and cytotoxicity were assessed via Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology), according to the manufacturer’s protocol. Briefly, PK-15 or ST cells were seeded into 96-well plates at a density of 1 × 104 cells/well. Cells were cultured with 10% FBS + DMEM for 24 h at 37◦C in 5% CO2, prior to replacing the culture medium with 3% FBS + DMEM supplemented with varying concentrations of CoPP, BV or CORM-2 and incubated for 36 h at 37◦C. CCK- 8 reagent (10 µl) was added to each well containing 100 µl of DMEM and incubated for 2 h at 37◦C, and cell viability was subsequently analyzed at a wavelength of 450 nm, using an epoch microplate spectrophotometer (BioTek Instruments, Inc.). The 50% cytotoxic concentration (CC50), defined as the concentration that causes visible changes in 50% of intact cells, was determined by comparing the CoPP-treated group with the untreated group, using GraphPad Prism software (version 5.0; GraphPad Software, Inc.). The maximal non-cytotoxic concentration (MNCC) was defined as the maximal concentration of CoPP that did not exhibit toxic effects via CCK-8 analysis. Cells and Virus Strain Vero cells derived from African green monkey kidney cells and PK-15 cells were both obtained from China Center for Type Culture Collection (CCTCC) and were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 0.1 mg/ml streptomycin (Thermo Fisher Scientific, Inc.) at 37◦C in 5% CO2. ST cells (obtained from CCTCC) were maintained in 10% FBS + MEM (Gibco; Thermo Fisher Scientific, Inc.), March 2020 | Volume 11 | Article 412 Frontiers in Microbiology | www.frontiersin.org 2 HO-1 Suppresses PRV Replication in vitro Zhang et al. followed by the aforementioned culture conditions. The Bartha- K61 PRV strain (GenBank ID: JF797217.1) was kindly provided by Professor Jiang Wang of Henan Agricultural University. denaturation at 95◦C for 10 min; 40 cycles at 95◦C for 20 s, 55◦C for 30 s and 72◦C for 20 s. Relative mRNA levels were quantified using the 2−11Ct method and normalized to the internal reference gene β-actin. siRNA Treatment siRNAs targeting the swine HO-1 coding sequences (GenBank ID: NM_001004027.1) were synthesized by Shanghai GenePharma Co., Ltd., and scrambled sequences were used as the non-targeted siRNA control. The HO-1 siRNA sequences were as follows: Sense, 5′-CGUCCUUGUACCACAUCUAdTdT-3′ and anti-sense, 5′-UAGAUGUGGUACAAGGACGdTdT-3′; and BVR sense, 5′-UCCUC AGCGUUCCUGAACCUGTT-3′ and BVR anti-sense, 5′-CAGGUUCAGGAACGC UGAGGATT-3′. siRNAs were transfected into PK-15 cells at a final concentration of 100 nM, using the X-tremeGENE HP siRNA transfection reagent (Roche Applied Science) and incubated for 12 h at 37◦C. Subsequently, cells were infected with 0.01 MOI of PRV prior to treatment with 50 µM of CoPP or BV, from 1 hpi onwards. HO-1 or BVR knockdown efficiency was assessed via western blotting, and the effect of HO-1 or BVR knockdown on PRV infection was determined as described for the supernatants virus copies and PRV gB expression detection. Antibodies and Other Reagents Regarding detection of supernatant virus copies, the PRV gB segment was cloned into the pEASY-T1 vector (TransGen Biotechnology Co., Ltd.), in order to construct the pEASY-T1- gB recombinant plasmid and generate a standard curve. The standard curve was plotted from the results of parallel PCRs performed on serial dilutions of standard DNA, while absolute quantities of supernatant RNA were calculated by normalization to the standard curve. Rabbit polyclonal antibody directly against PRV glycoprotein B (gB) and mouse anti-HO-1 monoclonal antibody were both purchased from Abcam. Mouse anti-α-tubulin monoclonal antibody was obtained from Sigma-Aldrich; while rabbit anti- BVR monoclonal antibody was purchased from Cell Signaling Technology, Inc. Cobalt-protoporphyrin (CoPP), a long-established HO-1 inducer; zinc-protoporphyrin (ZnPP), a HO-1 inhibitor; CO- releasing molecule-2 (CORM-2), a transition metal carbonyl that can liberate CO to elicit direct biological activities and BV were all purchased from Sigma-Aldrich; while CO scavenger hemoglobin (Hb) was purchased from Beyotime Institute of Biotechnology. Inactive (i)CORM-2 was prepared by incubating CORM-2 dissolved in DMSO for 24 h at 37◦C in 5% CO2 to release CO. Small interfering (si)BVR and siHO-1, and their corresponding negative controls (NCs) were synthesized by Shanghai GenePharma Co., Ltd. Modulation of HO-1 Activity and the Contents of CO and BV in Cells PK-15 or ST cells were inoculated with PRV at a multiplicity of infection (MOI) of 0.01, in DMEM at 37◦C for 1 h. Cells were subsequently washed three times with PBS and cultured in 3% FBS + DMEM supplemented with 0, 10, 25, and 50 µM of CoPP (dissolved in 0.1 M of NaOH) for 36 h at 37◦C. CORM- 2 (dissolved in DMSO) was used as a CO donor, and the final concentrations were identical to that of CoPP, which was added 1 h post-infection (hpi), while 50 µM of iCORM-2 was used as a control. Exogenous BV (dissolved in 0.1 of M NaOH) was used to assess the effect of BV on PRV infection, and the concentrations were identical to that of CORM-2. Reverse Transcription-Quantitative PCR (RT-qPCR) Following treatment, cells were harvested and total RNA were extracted using RNAiso plus reagent (Takara Biotechnology Co., Ltd.), according to the manufacturer’s protocol. Total RNA (500 ng) was reverse transcribed into cDNA using the PrimeScript R⃝RT reagent kit (Takara Biotechnology Co., Ltd.). qPCR was subsequently performed on a Light Cycler 480 SYBR Green I Master fluorescence quantitative PCR instrument, using FastStart Universal SYBR green master (Roche Diagnostics GmbH). The following primer sequences were used for qPCR: pHO-1 forward, 5′-GGCTGAGAATGCCGAGTT-3′ and reverse, 5′-ATGTAGCGGGTGTA GGCGTGGG-3′; gPRV forward, 5′-GGCGTACTGGCGCACTCTG-3′ and reverse, 5′-ATGTC CCCGACGATGAAGC-3′; gB forward, 5′-CCTCGTCCACG TCGTC CTC-3′ and reverse, 5′-GGCATCGCCAACTTCTTCC- 3′; and β-actin forward, 5′-T CCCTGGAGAAGAGCTACGA-3′ and reverse, 5′-AGCACTGTGTTGGCGTA CAG-3′. The following thermocycling conditions were used for qPCR: Initial Cytopathic Effect (CPE) Inhibition Assay The antiviral activity of CoPP against PRV was determined via the CPE inhibition assay. Briefly, PK-15 or ST cells were seeded into 96-well plates at a density of 1 × 104 cells/well about Indirect Immunofluorescence Assay (IFA) Indirect Immunofluorescence Assay (IFA) PK-15 or ST cells were seeded into 24-well plates at a density of 5x104 cells/well, and then infected with 0.01 MOI of PRV for 24 h at 37◦C, in the presence or absence of 10, 25, and 50 µM of CoPP. The infected cells were fixed using 4% paraformaldehyde for 30 min at room temperature, washed three times in PBS and permeabilized with 0.5% Triton X- 100 for 30 min at room temperature. The fixed cells were subsequently blocked with 1% bovine serum albumin (BSA; Beyotime Institute of Biotechnology) for 1 h at room temperature and incubated with rabbit anti-PRV gB or mouse anti-HO-1 antibodies (1:300; Abcam), diluted in 1% BSA supplemented with PBS, for 1 h at room temperature. Cells were washed three times with PBS, prior to incubation with goat anti-mouse IgG H&L (Alexa Fluor 594) or goat anti-rabbit IgG H&L (Alexa Fluor 488) (both 1:300; Abcam), diluted in 1% BSA supplemented with PBS, for 1 h at room temperature. Cell nuclei were counterstained with DAPI (Beyotime Institute of Biotechnology) for 5 min at room temperature. Samples were washed three times with PBS and the positively stained cells were observed under a confocal microscope with a magnification of 100 x (Leica Microsystems GmbH). ZnPP Treatment of Target Cells g In order to determine whether HO-1 enzyme activity was essential for its antiviral function, PK-15 or ST cells with 80% confluence were treated with 0, 20, 40, and 60 µM of ZnPP for 12 h at 37◦C. Cells were washed three times with PBS and subsequently infected with 0.01 MOI of PRV for 1 h at 37◦C. The viral suspension was replaced with 3% FBS + DMEM containing 50 µM of CoPP. Cells were harvested to determine gB expression via western blotting, while supernatants virus copies were detected via RT-qPCR analysis, at 36 hpi. PK-15 or ST cells with 80% confluence were treated with 0, 20, 40, and 60 µM of ZnPP for 24 h at 37◦C, in order to investigate the effect of ZnPP on HO-1 protein expression. HO-1 knockdown in PK-15 and ST cell lines was constructed by cloning the short hairpin (sh)RNA sequence of HO-1 into pLKO.1-EGFP-puro expression plasmid. The shRNA sequences targeting HO-1 were as follows: Sense, 5′-CCGGC GTCCTTGTACCACATCTACTCGAGTAGATGTGGTACAAG GACGTTTTTG-3′; and shRNA-control, 5′-GCACTACCA GAGCTAACTCAGATAGTACT-3′. Stable cell lines were established via the aforementioned process and those with HO-1 knockdown were detected via RT-qPCR and western blotting analyses. HO-1 knockdown in PK-15 and ST cell lines was constructed by cloning the short hairpin (sh)RNA sequence of HO-1 into pLKO.1-EGFP-puro expression plasmid. The shRNA sequences targeting HO-1 were as follows: Sense, 5′-CCGGC GTCCTTGTACCACATCTACTCGAGTAGATGTGGTACAAG GACGTTTTTG-3′; and shRNA-control, 5′-GCACTACCA GAGCTAACTCAGATAGTACT-3′. Stable cell lines were established via the aforementioned process and those with HO-1 knockdown were detected via RT-qPCR and western blotting analyses. Cytopathic Effect (CPE) Inhibition Assay March 2020 | Volume 11 | Article 412 Frontiers in Microbiology | www.frontiersin.org 3 HO-1 Suppresses PRV Replication in vitro Zhang et al. 24 h before infecting with equal volumes of PRV (0.01 MOI), following by the addition of twofold serial dilutions of CoPP to each well. The MNCC was set as the highest concentration for this experiment. Following cell culture for 72 h at 37◦C, the CPE was observed in each well. PRV-infected PK-15 or ST cells without CoPP treatment were used as the control. The CoPP concentration decreasing CPE by 50%, compared with the control group, was estimated using GraphPad Prism software and defined as the half maximal inhibitory concentration (IC50), expressed in µmol/l. The selectivity index (SI) was calculated from the ratio of CC50 to IC50. into 6-well plates at a density of 2 × 105 cells/well 24 h prior to exposure with PRV (MOI of 0.01). At 24 hpi, the virus supernatant was discarded, cells were washed three times with PBS and incubated with DMEM supplemented with 10 µM/l of DCFH-DA probe at 37◦C for 20 min. Subsequently, cells were re-washed three times with PBS. Cells were observed under a confocal fluorescence microscope (100 x; Leica Microsystems GmbH) and assessed via flow cytometry analysis. For analysis of intracellular RNS level, PK-15 cells were plated in 6-well plates at a density of 2 × 105 cells/well 1 day before and followed by infecting with 0.01 MOI of PRV for 24 h. Then the old medium was replaced with fresh serum free DMEM containing 5 µM/l DAF-FM DA probe and inubated for 20 min at 37◦C. After washing using PBS for 3 times, cells were subjected to flow cytometry analysis. PK-15 cells not infected with PRV were used as the control in both experiments. Generation of Stable Cell Lines In order to generate cell line stably expressing porcine HO-1, the HO-1 gene was cloned into the lentiviral expression vector, pTRIP-puro to construct pTRIP-pHO-1-puro. Subsequently, the recombinant plasmids (0.9 µg) were co-transfected into 293T cells, with pMDG.2 (1.0 µg) and psPAX2 (1.8 µg) plasmids, using X-tremeGENE HP DNA transfection reagent and Opti- MEM to construct pseudotyped lentiviral vectors. Supernatants containing the pseudotyped lentiviruses were collected at 48 h post-transfection and centrifuged at 12,000 × g for 10 min at 4◦C to remove cell debris. Subsequently, the supernatants were filtered with 0.22 µM filter and stored at −80◦C for subsequent experimentation. PK-15 or ST cells that reached 80% confluence were transduced with the pseudotyped lentiviruses expressing HO-1 and supplemented with 1 µg/ml of polybrene (Sigma-Aldrich; Merck KGaA). After 24 h, the culture medium was replaced with 10% FBS + DMEM/MEM supplemented with 8 µg/ml of puromycin (Sigma-Aldrich; Merck KGaA), which was changed every 48 h. mRNA and protein expression levels of PK-15 or ST cell with upregulated HO-1 were determined via RT-qPCR and western blotting, respectively. PK-15 or ST cells that reached 80% confluence were transduced with the pseudotyped lentiviruses expressing HO-1 and supplemented with 1 µg/ml of polybrene (Sigma-Aldrich; Merck KGaA). After 24 h, the culture medium was replaced with 10% FBS + DMEM/MEM supplemented with 8 µg/ml of puromycin (Sigma-Aldrich; Merck KGaA), which was changed every 48 h. mRNA and protein expression levels of PK-15 or ST cell with upregulated HO-1 were determined via RT-qPCR and western blotting, respectively. RESULTS PK-15 or ST cells were seeded into 6-well plates at a density of 2 × 105 cells/well 24 h prior to treatment with 0, 10, 25, 50 µM of CoPP for 12 h at 37◦C, and followed by washing three times with PBS to remove residual CoPP. Cells were subsequently pre-chilled on ice for 30 min, followed by inoculation with ice- cold PRV (1.0 MOI) for 1 h at 4◦C to allow abundant virus adsorption without internalization. Cells were washed extensively with ice-cold PBS to remove unabsorbed viruses and analyzed via RT-qPCR or western blotting. Enzyme-Linked Immunosorbent Assay (ELISA) Intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) production in PK-15 cells before and after PRV infection was assessed using DCFH-DA and DAF-FM DA fluorescence probe (Beyotime Institute of Biotechnology), according to the manufacturer’s protocol. PK-15 cells were seeded In order to determine whether induction of HO-1 or PRV infection leads to abnormal changes of intracellular BV, the amount of BR was initially assessed as a reflection of intracellular BV levels after treatment with CoPP or PRV infection of host March 2020 | Volume 11 | Article 412 Frontiers in Microbiology | www.frontiersin.org 4 HO-1 Suppresses PRV Replication in vitro Zhang et al. and quantified using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). A total of 50 µg protein/lane was separated via SDS-PAGE on a 12% gel. The separated proteins were subsequently transferred onto a polyvinylidene difluoride membrane (EMD Millipore) and blocked with 2.5% skim milk (m/v) supplemented with TBST for 1 h at room temperature. The membranes were incubated with primary antibodies against: PRV gB protein (1:1,000), pHO- 1 (1:1,000), BVR (1:1,000) and α-tubulin (1:5,000) overnight at 4◦C. Membranes were washed three times with TBST. Following the primary incubation, membranes were incubated with HRP-conjugated goat anti-mouse or -rabbit IgG secondary antibodies for 1 h at room temperature. Protein bands were visualized using an ECL chemiluminescent detection system (Pierce; Thermo Fisher Scientific, Inc.), according to the manufacturer’s protocol. cells. PK-15 or ST cells were seeded into 6-well plates at a density of 2 × 105 cells/well. Following cell culture for 24 h at 37 ◦C, the medium was changed to 3% FBS + DMEM, with or without 50 µM of CoPP, or cells were infected with 0.01 MOI of PRV. Cells and supernatants were harvested at 36 h following treatment with CoPP, or at 12, 24, 36, 48 h post- PRV infection, and subsequently frozen and thawed three times. Cell lysates were centrifuged at 12,000 × g for 10 min at 4◦C and supernatants were collected for BR detection using a BR ELISA kit (Elabscience Biotechnology Co., Ltd.), according to the manufacturer’s protocol. Samples were subsequently analyzed at a wavelength of 540 nm, using an epoch microplate spectrophotometer. Statistical Analysis Statistical analysis was performed using GraphPad Prism software. Data are presented as the mean values ± standard deviation. Differences between two groups was assessed using unpaired Student’s t-test, while one-way analysis of variance (ANOVA) was used to compare differences in three or more groups. P < 0.05 was considered to indicate a statistical difference. All experiments were performed in triplicate. PRV Infection Downregulates HO-1 Expression While Stimulates Oxidative Stress Response in Both PK-15 and ST Cells In order to understand the role of the stress-induced protective enzyme against PRV infection, HO-1 expression was assessed in PK-15 and ST cells during PRV infection. PRV (0.01 MOI) was used to infect PK-15 and ST cells, and mRNA and protein expression levels of HO-1 or gB were analyzed via RT-qPCR and western blotting, respectively. Infection with PRV markedly downregulated HO-1 mRNA expression in both PK-15 and ST cells, as the infection process progressed from 12, 24, 36, and 48 hpi compared with the 0 hpi (Figures 1A,B). In accordance with RT-qPCR analysis, western blotting demonstrated decreased HO-1 protein expression in both PK-15 and ST cells at 12, 24, 36, and 48 hpi compared with the 0 hpi (Figures 1C,D). Furthermore, downregulated HO-1 expression was indicated to be dependent of PRV replication, as UV-inactivated PRV failed to affect HO-1 expression compared with the 0 hpi group (Figures 1A–D). Virus infection of host cells was often associated with oxidative stress stimulation. Thus we determined whether PRV infection affected intracellular oxidative stress using DCFH-DA probe for ROS detection and DAF-FM DA probe for RNS detection. Flow cytometry results revealed that PRV infection markedly promoted intracellular ROS (Figures 1E,F) and RNS (Figures 1G,H) levels compared with For the entry assay, PK-15 cells were treated with 0, 10, 25, 50 µM of CoPP for 12 h at 37◦C. After washing with PBS for three times, pre-cold cells were inoculated with PRV (1.0 MOI) for 1 h at 4◦C to allow viruses to bind without internalizing into cells. Viruses were subsequently discarded and washed three times with ice-cold PBS. Cells were transferred to 37◦C for 1 h to promote virus entry into cells. Following extensive washing with PBS, cells were treated with trypsin for 30 s at 37◦C and washed three times with PBS to remove the non-internalized virions on the cell surface. Subsequently, cells were harvested for viral genome abundance and gB protein detection via RT-qPCR and western blotting, respectively. Enzyme-Linked Immunosorbent Assay (ELISA) As Hb has a high affinity for CO (forming HbCO) (Tung et al., 2011), the intracellular CO levels were determined following HO-1 induction or PRV infection using a commercially available HbCO ELISA kit (Elabscience Biotechnology Co., Ltd.), according to the manufacturer’s protocol. Briefly, PK-15 or ST cells were seeded into 6-well plates at a density of 2 × 105 cells/well. After 24 h, cells were treated with 3% FBS + DMEM supplemented with 50 µM of CoPP, with or without 50 µg/ml of Hb, or inoculated with PRV as aforementioned. Cells and supernatants were harvested at 36 h post-treatment or at 12, 24, 36, 48 h post-PRV infection to detect HbCO via ELISA. Samples were subsequently analyzed at a wavelength of 450 nm, using an epoch microplate spectrophotometer. Western Blotting Western blotting was performed to determine HO-1 expression and viral proteins under different processing conditions. Treated cells were harvested and lysed on ice for 30 min using NP40 lysis buffer supplemented with protease inhibitor (Beyotime Institute of Biotechnology). Total protein was extracted via centrifugation at 12,000 × g for 10 min at 4◦C March 2020 | Volume 11 | Article 412 Frontiers in Microbiology | www.frontiersin.org 5 Zhang et al. HO-1 Suppresses PRV Replication in vitro FIGURE 1 | PRV infection suppresses HO-1 expression while stimulates oxidative stress response in both PK-15 and ST cells. (A,C) PK-15 or (B,D) ST cells were infected with PRV at a MOI of 0.01 for 1 h at 37◦C. Cells samples were collected at 0, 12, 24, 36, and 48 hpi to detect HO-1 and PRV gB mRNA and protein levels via RT-qPCR and western blotting, respectively. (E,F) PK-15 cells were infected with 0.01 MOI of PRV for 24 h. Then cells were incubated with DMEM supplemented with 10 µM/l of DCFH-DA at 37◦C for 20 min. Cells were observed under a confocal fluorescence microscope (100x) and assessed via flow cytometry analysis. (G,H) PK-15 cells infected with 0.01 MOI of PRV for 24 h were incubated with DMEM containing 5 µM/l DAF-FM DA and incubated for 20 min at 37◦C. After washing using PBS for three times, cells were subjected to flow cytometry analysis. PK-15 cells mock infected with PRV were included as a control in both experiments. Data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. PRV, pseudorabies virus; HO-1, heme oxygenase-1; RT-qPCR, reverse transcription-quantitative PCR. FIGURE 1 | PRV infection suppresses HO-1 expression while stimulates oxidative stress response in both PK-15 and ST cells. (A,C) PK-15 or (B,D) ST cells were infected with PRV at a MOI of 0.01 for 1 h at 37◦C. Cells samples were collected at 0, 12, 24, 36, and 48 hpi to detect HO-1 and PRV gB mRNA and protein levels via RT-qPCR and western blotting, respectively. (E,F) PK-15 cells were infected with 0.01 MOI of PRV for 24 h. Then cells were incubated with DMEM supplemented with 10 µM/l of DCFH-DA at 37◦C for 20 min. Cells were observed under a confocal fluorescence microscope (100x) and assessed via flow cytometry analysis. Western Blotting (G,H) PK-15 cells infected with 0.01 MOI of PRV for 24 h were incubated with DMEM containing 5 µM/l DAF-FM DA and incubated for 20 min at 37◦C. After washing using PBS for three times, cells were subjected to flow cytometry analysis. PK-15 cells mock infected with PRV were included as a control in both experiments. Data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. PRV, pseudorabies virus; HO-1, heme oxygenase-1; RT-qPCR, reverse transcription-quantitative PCR. replication were subsequently analyzed via western blotting and by assessing the median tissue culture infectious dose (TCID50). CoPP increased HO-1 protein expression, while decreasing PRV gB protein expression in both PK-15 and ST cells, in a dose-dependent manner (Figures 2A,C). Furthermore, CoPP decreased supernatants progeny virus titers in a dose-dependent manner (Figures 2B,D). In order to confirm the antiviral activity of HO-1 on PRV infection, IFA was performed to assess PRV gB protein expression, in the presence or absence of 10, 25, and 50 µM of CoPP. IFA analysis demonstrated that induction of HO-1 via CoPP markedly inhibited PRV gB expression (Figure 2E). Taken together, these results suggest that HO-1 may play a key role in suppressing PRV replication in vitro. mock infected cells, indicating that PRV infection can stimulate cell oxidative stress. Induction of HO-1 Inhibits PRV Replication in vitro (F,G) PK-15 cells were transfected with 100 n HO-1 or siNC for 12 h, then cells were infected with PRV at a MOI of 0.01, in the presence or absence of 50 µM of CoPP. Cells were harvested to detect gB O-1 mRNA and protein expression levels via RT-qPCR and western blotting at 36 hpi, respectively. Data are presented as the mean ± standard deviation of t dependent experiments. **P < 0.01, ***P < 0.001. HO-1, heme oxygenase-1; PRV, pseudorabies virus; MOI, multiplicity of infection; CoPP, cobalt-protoporp C, negative control; RT-qPCR, reverse transcription-quantitative PCR. FIGURE 2 | Induction of HO-1 inhibits PRV replication. (A,B) PK-15 or (C,D) ST cells were inoculated with PRV at a MOI of 0.01 for 1 h at 37◦C, followed by treatment with 0, 10, 25, 50 µM of CoPP. Cells and supernatants were harvested to detect HO-1 and gB protein expression via western blotting, and measure supernatant progeny virus titers using TCID50, at 36 hpi. (E) Indirect immunofluorescence assay was performed. (F,G) PK-15 cells were transfected with 100 nM of siHO-1 or siNC for 12 h, then cells were infected with PRV at a MOI of 0.01, in the presence or absence of 50 µM of CoPP. Cells were harvested to detect gB and HO-1 mRNA and protein expression levels via RT-qPCR and western blotting at 36 hpi, respectively. Data are presented as the mean ± standard deviation of three independent experiments. **P < 0.01, ***P < 0.001. HO-1, heme oxygenase-1; PRV, pseudorabies virus; MOI, multiplicity of infection; CoPP, cobalt-protoporphyrin; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR. FIGURE 2 | Induction of HO-1 inhibits PRV replication. (A,B) PK-15 or (C,D) ST cells were inoculated with PRV at a MOI of 0.01 for 1 h at 37◦C, followed by treatment with 0, 10, 25, 50 µM of CoPP. Cells and supernatants were harvested to detect HO-1 and gB protein expression via western blotting, and measure supernatant progeny virus titers using TCID50, at 36 hpi. (E) Indirect immunofluorescence assay was performed. (F,G) PK-15 cells were transfected with 100 nM of siHO-1 or siNC for 12 h, then cells were infected with PRV at a MOI of 0.01, in the presence or absence of 50 µM of CoPP. Cells were harvested to detect gB and HO-1 mRNA and protein expression levels via RT-qPCR and western blotting at 36 hpi, respectively. Induction of HO-1 Inhibits PRV Replication in vitro p Since PRV infection notably aberrated HO-1 expression, the effect of HO-1 on PRV infection and replication was further investigated. First, PK-15 and ST cells were treated with 0, 10, 25, 50 µM of CoPP to evaluate its effect on HO-1 expression. As expected, treatment with CoPP significantly induced HO- 1 mRNA and protein expression levels in a dose-dependent manner, in both PK-15 and ST cells (Supplementary Figures 1A– D). In order to investigate the effect of HO-1 on PRV infection and replication, PRV-infected PK-15 and ST cells were treated with 0, 10, 25, 50 µM of CoPP, and PRV infection and Subsequently, PK-15 cells were transfected with siHO-1 or siNC, followed by infection with PRV and treatment with 50 µM Frontiers in Microbiology | www.frontiersin.org March 2020 | Volume 11 | Article 412 6 g et al. HO-1 Suppresses PRV Replication in v GURE 2 | Induction of HO-1 inhibits PRV replication. (A,B) PK-15 or (C,D) ST cells were inoculated with PRV at a MOI of 0.01 for 1 h at 37◦C, followed by atment with 0, 10, 25, 50 µM of CoPP. Cells and supernatants were harvested to detect HO-1 and gB protein expression via western blotting, and measure pernatant progeny virus titers using TCID50, at 36 hpi. (E) Indirect immunofluorescence assay was performed. (F,G) PK-15 cells were transfected with 100 nM of O-1 or siNC for 12 h, then cells were infected with PRV at a MOI of 0.01, in the presence or absence of 50 µM of CoPP. Cells were harvested to detect gB and -1 mRNA and protein expression levels via RT-qPCR and western blotting at 36 hpi, respectively. Data are presented as the mean ± standard deviation of three d t i t **P 0 01 ***P 0 001 HO 1 h 1 PRV d bi i MOI lti li it f i f ti C PP b lt t h i HO-1 Suppresses PRV Replication in vitro Zhang et al. GURE 2 | Induction of HO-1 inhibits PRV replication. (A,B) PK-15 or (C,D) ST cells were inoculated with PRV at a MOI of 0.01 for 1 h at 37◦C, followed by atment with 0, 10, 25, 50 µM of CoPP. Cells and supernatants were harvested to detect HO-1 and gB protein expression via western blotting, and measure pernatant progeny virus titers using TCID50, at 36 hpi. (E) Indirect immunofluorescence assay was performed. Knockdown of Endogenous HO-1 Expression Promotes PRV Replication of CoPP, in order to confirm whether the antiviral activity of CoPP was specifically mediated by HO-1. Samples were harvested and the expression levels of PRV gB and HO-1 were determined. Knockdown of HO-1 partially reversed decreased PRV gB mRNA expression (Figure 2F), as well as the protein (Figure 2G) caused by CoPP treatment compared with the siNC transfection group. These results indicate that HO-1 specifically mediates the antiviral effect of CoPP. Expression Promotes PRV Replication Since upregulation of HO-1 suppressed PRV infection and replication, the association between endogenous basal HO-1 and PRV replication was subsequently investigated. First, PK- 15 and ST recombinant cell lines with downregulated HO- 1 expression were identified. RT-qPCR analysis demonstrated that HO-1 mRNA expression was markedly downregulated in both PK-15shHO−1 (decreased by 62.8%) and STshHO−1 (decreased by 65.3%) cells compared with the control cells (Figure 5A), while western blot analysis indicated downregulated HO-1 protein expression (Figure 5B). PK-15shNC, PK-15shHO−1, STshNC, or STshHO−1 cells were incubated with PRV (0.01 MOI) and the viral replication kinetics was analyzed. The results demonstrated that shHO-1 markedly aberrated basal level HO-1 protein expression, while simultaneously enhancing PRV gB expression in both PK-15 (Figure 5C) and ST cells (Figure 5D), at 12, 24, 36, and 48 hpi. Furthermore, knockdown of basal level HO-1 expression increased the supernatant progeny virus copies compared with PK-15shNC and STshNC cells (Figures 5E,F), suggesting that endogenous HO-1 plays an antiviral role during the infection and replication of PRV in vitro. Enzyme Activity of HO-1 Is Necessary for Its Antiviral Function ZnPP, a phyrin that upregulates HO-1 protein expression, while inhibiting HO-1 enzymatic activity was used to assess whether it can attenuate PRV inhibition caused by CoPP, in order to determine whether HO-1 enzymatic activity is necessary for CoPP-mediated attenuation of PRV replication. PK-15 or ST cells were treated with 0, 20, 40, 60 µM of ZnPP for 12 h, subsequently cells were harvested and HO- 1 expression was determined. The results demonstrated that treatment with ZnPP induced HO-1 protein expression in both cell lines, in a dose-dependent manner (Figures 3A,B). Subsequently, PK-15 or ST cells were treated with varying concentrations of ZnPP for 12 h, followed by infection with PRV and treatment with 50 µM of CoPP for 24 h. As expected, pretreatment with ZnPP partially reversed the inhibitory effect of CoPP on PRV gB expression (Figures 3C,D) and increased supernatants virus copies compared with the CoPP treatment group (Figures 3E,F). Treatment with ZnPP alone slightly induced PRV gB expression, as well as supernatant virus copies (Figures 3C–F). Taken together, these results suggest that HO-1 enzymatic activity is essential for this antiviral effect, and endogenous HO-1 plays a role in restricting virus replication as well. CoPP Treatment Decreases Viral Infectivity, Without Inducing Cytotoxicity or Interfering With Virus Binding and Entry Into Cells y To understand the molecular mechanism underlying HO-1 activity of viral protein expression and PRV replication in CoPP-treated cells, a virus-binding assay was performed as previously described by Cheshenko et al. (2013), in order to determine whether equal amounts of virus bound to the surface of CoPP-treated and -untreated PK-15 and ST cells. RT-qPCR analysis indicated that treatment with CoPP did not affect the virus genome content compared with the untreated group, in both PK-15 and ST cells (Figures 6A,B). Western blotting analysis of gB expression demonstrated that equal amounts of virus bound to the surface of untreated and CoPP-treated PK-15 and ST cells, respectively (Figures 6C,D), suggesting that treatment with CoPP does not interfere with virus binding to these cells. Induction of HO-1 Inhibits PRV Replication in vitro Data are presented as the mean ± standard deviation of three independent experiments. **P < 0.01, ***P < 0.001. HO-1, heme oxygenase-1; PRV, pseudorabies virus; MOI, multiplicity of infection; CoPP, cobalt-protoporphyrin; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR. 7 March 2020 | Volume 11 | Article 412 Frontiers in Microbiology | www.frontiersin.org Zhang et al. HO-1 Suppresses PRV Replication in vitro Overexpression of HO-1 Suppresses PRV Replication (C,D) Cells were collected to detect PRV gB protein expression via western blotting, while (E,F) supernatant progeny copies were assessed via RT-qPCR, at 36 hpi. Data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. HO-1, heme oxygenase-1; ZnPP, zinc-protoporphyrin; CoPP, cobalt-protoporphyrin; PRV, pseudorabies virus; RT-qPCR, reverse transcription-quantitative PCR. of CoPP on PRV. (A) PK-15 or (B) ST cells were treated with 0, 20, 40, 60 µM of tern blotting PK 15 or ST cells were treated with 0 20 40 and 60 µM of ZnPP for FIGURE 3 | HO-1 specific inhibitor ZnPP partially reverses the inhibitory effect of CoPP on PRV. (A) PK-15 or (B) ST cells were treated with 0, 20, 40, 60 µM of ZnPP for 24 h. Cells were harvested to detect HO-1 protein expression via western blotting. PK-15 or ST cells were treated with 0, 20, 40, and 60 µM of ZnPP for 12 h at 37◦C. Then cells were infected with 0.01 MOI of PRV and followed by incubating with 50 µM of CoPP from 1 hpi onwards. (C,D) Cells were collected to detect PRV gB protein expression via western blotting, while (E,F) supernatant progeny copies were assessed via RT-qPCR, at 36 hpi. Data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. HO-1, heme oxygenase-1; ZnPP, zinc-protoporphyrin; CoPP, cobalt-protoporphyrin; PRV, pseudorabies virus; RT-qPCR, reverse transcription-quantitative PCR. In order to determine whether CoPP affected PRV virulence via its virucidal effect, virus inocula were incubated with varying concentrations of CoPP for 1 h at 37◦C, subsequently added to Vero cells and washed to remove CoPP at 1 hpi. Western blot analysis demonstrated that PRV gB expression gradually decreased in CoPP-treated PRV cells compared with the untreated virus control (Figure 6G). Furthermore, supernatants virus titers indicated a downward trend between virus treated with vehicle and virus treated with CoPP (Figure 6H). To eliminate the possibility that the antiviral activity of CoPP was due to its cytotoxicity effect, the CC50 and IC50 values of CoPP were determined. The IC50 and CC50 values of CoPP in PK-15 cells were 31.6 and 99.2 µM, respectively, while the SI (CC50/IC50) value of CoPP was 3.14. Overexpression of HO-1 Suppresses PRV Replication p A recombinant lentivirus overexpressing HO-1 was used to establish cell lines in both PK-15 and ST cells to further verify the effect of HO-1 on PRV replication. First, stable cell lines overexpressing HO-1 were validated, whereby both RT- qPCR and western blotting analyses demonstrated that HO-1 mRNA and protein expression levels were notably upregulated in recombinant cell lines (PK-15vector, PK-15HO−1, STvector and STHO−1) compared with the empty vector control group, in both cell types (Figures 4A,B). Subsequently, viral infection characteristics of all cell lines were evaluated at different time points via western blotting and RT-qPCR. Overexpression of HO-1 markedly aberrated gB protein expression and decreased supernatant virus copies compared with the control group at each assessed time point (Figures 4C–F). Furthermore, IFA analysis indicated that overexpression of HO-1 notably abrogated PRV gB expression, while the control groups exhibited apparent more viral infection in both PK-15 and ST cells (Supplementary Figure 2). Taken together, these results suggest that overexpression of HO-1 suppresses PRV replication in vitro. In order to determine how upregulated HO-1, with CoPP prohibited PRV replication, PK-15 cells were inoculated with PRV (0.01 MOI) for 1 h at 4◦C to allow viruses to bind without internalizing into cells. Viruses were subsequently discarded and washed three times with PBS. Cells were treated with DMEM containing 0, 10, 25, 50 µM of CoPP for 1 h at 37◦C. Subsequently, cells were washed and treated with trypsin prior to harvesting for RT-qPCR and western blotting analyses. No significant differences were observed in PRV gB expression and virus genome content between CoPP-treated and -untreated cells (Figures 6E,F), indicating that treatment with CoPP does not affect virus entry into host cells. March 2020 | Volume 11 | Article 412 Frontiers in Microbiology | www.frontiersin.org 8 Zhang et al. HO-1 Suppresses PRV Replication in vitro FIGURE 3 | HO-1 specific inhibitor ZnPP partially reverses the inhibitory effect of CoPP on PRV. (A) PK-15 or (B) ST cells were treated with 0, 20, 40, 60 µM of ZnPP for 24 h. Cells were harvested to detect HO-1 protein expression via western blotting. PK-15 or ST cells were treated with 0, 20, 40, and 60 µM of ZnPP for 12 h at 37◦C. Then cells were infected with 0.01 MOI of PRV and followed by incubating with 50 µM of CoPP from 1 hpi onwards. Overexpression of HO-1 Suppresses PRV Replication Conversely, the IC50 and CC50 values of CoPP in ST cells were 51.6 and 212.0 µM, respectively, while the SI value of CoPP was 4.11 (Supplementary Figure 3). However, treatment with CoPP was not associated with complete viral inactivation (Figures 6G,H), suggesting that interference with PRV replication occurs partially attribute to downstream mechanism. Taken together, these results suggest that CoPP partially suppresses PRV replication via its direct virucidal effect instead of its cytotoxicity effect. HO-1 Metabolite, CO Mediates the Anti-PRV Activity of HO-1 HO-1 yields three enzymatic downstream metabolites, including CO, which was assessed to determine whether it plays a role in the antiviral activity of HO-1 (Blancou et al., 2011). The intracellular content of CO was assessed following infection of PK-15 and ST cells with PRV. ELISA analysis demonstrated that PRV infection markedly decreased intracellular CO content in both PK-15 and ST cells (Figures 7A,B). Subsequently, intracellular CO content was assessed following HO-1 induction via CoPP. PK-15 cells were incubated with 50 µg/ml of Hb, in the presence or absence of 50 µM of CoPP, supernatants were collected after 36 h and HbCO levels were assessed via ELISA. The results demonstrated that HO-1 induction via CoPP notably increased supernatants HbCO content compared with the control group (Figures 7C,D), March 2020 | Volume 11 | Article 412 Frontiers in Microbiology | www.frontiersin.org 9 Zhang et al. HO-1 Suppresses PRV Replication in vitro FIGURE 4 | Overexpression of HO-1 suppresses PRV replication. PK-15 or ST cells stably expressing HO-1 were verified via (A) RT-qPCR and (B) western blotting. PK-15HO−1 or STHO−1 cells were infected with PRV at a MOI of 0.01. Samples were harvested at 12, 24, 36, 48 hpi. (C,D) Western blotting was performed to assess PRV gB and HO-1 protein expression levels, while (E,F) RT-qPCR was performed to detect supernatant progeny virus copies. Data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. HO-1, heme oxygenase-1; PRV, pseudorabies virus; RT-qPCR, reverse transcription-quantitative PCR; MOI, multiplicity of infection. FIGURE 4 | Overexpression of HO-1 suppresses PRV replication. PK-15 or ST cells stably expressing HO-1 were verified via (A) RT-qPCR and (B) western blotting. PK-15HO−1 or STHO−1 cells were infected with PRV at a MOI of 0.01. Samples were harvested at 12, 24, 36, 48 hpi. (C,D) Western blotting was performed to assess PRV gB and HO-1 protein expression levels, while (E,F) RT-qPCR was performed to detect supernatant progeny virus copies. Data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. HO-1, heme oxygenase-1; PRV, pseudorabies virus; RT-qPCR, reverse transcription-quantitative PCR; MOI, multiplicity of infection. indicating that upregulation of HO-1 can promote endogenous CO production in host cells, although the quantity is small. HO-1 Metabolite, CO Mediates the Anti-PRV Activity of HO-1 indicating that upregulation of HO-1 can promote endogenous CO production in host cells, although the quantity is small. Overall, the results indicate that HO-1 may suppress PRV replication; however, the molecular mechanism underlying HO- 1 inhibition of PRV replication still remains unclear. In order to investigate its antiviral molecular mechanism, PRV-infected PK-15 cells were treated with 50 µM of CoPP in the presence or absence of 50 µg/ml of Hb, or treated only by Hb. Cells and culture supernatants were harvested for detection of PRV gB mRNA and protein expression, and supernatant virus copies at 36 hpi. The results demonstrated that treatment with Hb partially reversed the inhibitory effect of CoPP on PRV gB mRNA (Figure 7E) and protein expression (Figure 7F). Furthermore, treatment with Hb in the presence of CoPP increased supernatant progeny virus copies compared with CoPP treatment alone (Figure 7G). However, treatment with Hb alone Overall, the results indicate that HO-1 may suppress PRV replication; however, the molecular mechanism underlying HO- 1 inhibition of PRV replication still remains unclear. In order to investigate its antiviral molecular mechanism, PRV-infected PK-15 cells were treated with 50 µM of CoPP in the presence or absence of 50 µg/ml of Hb, or treated only by Hb. March 2020 | Volume 11 | Article 412 Frontiers in Microbiology | www.frontiersin.org 10 Zhang et al. HO-1 Suppresses PRV Replication in vitro FIGURE 5 | Knockdown of endogenous HO-1 promotes PRV replication. PK-15shHO−1 or STshHO−1 cells were identified via (A) RT-qPCR and (B) western blotting. PK-15shNC, PK-15shHO−1, STshNC, and STshHO−1 cells were incubated with PRV at a MOI of 0.01. Cells and supernatants were collected at 12, 24, 36, 48 hpi to assess (C,D) PRV gB and HO-1 protein expression levels via western blotting, while (E,F) supernatant virus copies were detected via RT-qPCR (E,F). Data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. HO-1, heme oxygenase-1; PRV, pseudorabies virus; sh, short hairpin; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR; MOI, multiplicity of infection. FIGURE 5 | Knockdown of endogenous HO-1 promotes PRV replication. PK-15shHO−1 or STshHO−1 cells were identified via (A) RT-qPCR and (B) western blotting. PK-15shNC, PK-15shHO−1, STshNC, and STshHO−1 cells were incubated with PRV at a MOI of 0.01. HO-1 Metabolite, CO Mediates the Anti-PRV Activity of HO-1 Cells and supernatants were collected at 12, 24, 36, 48 hpi to assess (C,D) PRV gB and HO-1 protein expression levels via western blotting, while (E,F) supernatant virus copies were detected via RT-qPCR (E,F). Data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. HO-1, heme oxygenase-1; PRV, pseudorabies virus; sh, short hairpin; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR; MOI, multiplicity of infection. had no significant effect on PRV infection (Figures 7E–G). Taken together, these results indicate that endogenous CO produced by HO-1 catalysis partially mediates the antiviral effect of HO-1. CORM-2 was demonstrated to decrease supernatant virus copies in a concentration-dependent manner (Figures 7I–K); however, opposing effects were observed with iCORM-2. Taken together, these results suggest that CO partially mediates the antiviral activity of HO-1. As an effective carrier of exogenous CO, CORM-2 was subsequently assessed to further determine the effect exogenous CO on PRV replication. PK-15 or ST cells were infected with PRV (0.01 MOI), followed by treatment with 0, 10, 25, 50 µM of CORM-2 or 50 µM of iCORM-2. Western blotting analysis demonstrated that CORM-2 suppressed PRV gB protein expression in both PK-15 (Figure 7H) and ST (Figure 7J) cells in a concentration-dependent manner, without inducing cytotoxic effects as observed with CoPP (data not shown). Conversely, iCORM-2 failed to exhibit a significant effect on gB expression (Figures 7H–J). Consistent with intracellular protein expression, Frontiers in Microbiology | www.frontiersin.org HO-1 Metabolite, BV Partially Mediates the Inhibitory Effect of HO-1 on PRV Replication Cells were washed extensively with ice-cold PBS to remove unabsorbed viruses and analyzed for the virus genome via RT-qPCR (A,B) and for the gB protein via western blotting (C,D). For entry assay, following adsorption with PRV (1.0 MOI) for 1 h on ice, CoPP-treated PK-15 cells were transferred to at 37◦C. After 1 h, cells were washed with trypsin to remove virions adsorbed on the cell surface, and subsequently harvested to assess virus genome via RT-qPCR (E) and gB protein expression via western blotting (F). (G,H) PRV virus suspension (0.01 MOI) was co-incubated with CoPP at 37◦C for 1 h and used to inoculate Vero cells, and part of the supernatant is directly used to determine the virus titer. After 24 h, cells were harvested for viral replication analysis, and virus titer was tested using TCID50. Data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. CoPP, cobalt-protoporphyrin; PRV, pseudorabies virus; MOI, multiplicity of infection; RT-qPCR, reverse transcription-quantitative PCR; gB, glycoprotein B. intracellular content of BR, the metabolite of BV, was assessed following PRV infection or treatment with CoPP via ELISA. Progression of PRV infection was associated with a downward trend of BR content in both PK-15 and ST cells compared with the control group (Figures 8A,B). Conversely, treatment with CoPP increased intracellular BR levels in both PK-15 and ST cells compared with the untreated control group (Figures 8C,D). Whether treatment with CoPP increased intracellular BR content by stimulating BVR expression was subsequently investigated. The results demonstrated no significant changes of BVR mRNA and protein expression levels in both PK-15 and ST cells (Supplementary Figure 4). Since the BV-BVR signaling cascade is the only signaling pathway that generates BR (Wegiel et al., 2011), it was assumed that increased BR content was directly due to HO-1 induction. In order to determine whether BV partially mediated the anti-PRV activity of HO-1, PRV-infected PK-15 or ST cells were incubated with 0, 10, 25, 50 µM of BV for 36 h at 37◦C, and samples were subsequently harvested for viral replication analysis. A CCK-8 assay was subsequently performed to determine the cytotoxic effect of BV. Treatment with BV notably decreased PRV gB protein expression in PK- 15 or ST cells (Figures 8E,G), as well as supernatant virus copies (Figures 8F,H), in a dose-dependent manner. HO-1 Metabolite, BV Partially Mediates the Inhibitory Effect of HO-1 on PRV Replication Previous studies have demonstrated the antiviral effect of the HO-1 metabolite, BV (Lehmann et al., 2010; Zhu et al., 2010; Santangelo et al., 2012; Liu et al., 2016). In order to determine the role of BV in mediating the anti-PRV activity of HO-1, March 2020 | Volume 11 | Article 412 Frontiers in Microbiology | www.frontiersin.org 11 Zhang et al. HO-1 Suppresses PRV Replication in vitro FIGURE 6 | CoPP directly inactivates PRV particles without affecting viral attachment or entry into cells. PK-15 or ST cells treated with 0, 10, 25, 50 µM of CoPP for 12 h at 37◦C. After washing three times with PBS to remove residual CoPP, cells were subsequently pre-chilled on ice for 30 min, followed by inoculation with ice-cold PRV (1.0 MOI) for 1 h at 4◦C. Cells were washed extensively with ice-cold PBS to remove unabsorbed viruses and analyzed for the virus genome via RT-qPCR (A,B) and for the gB protein via western blotting (C,D). For entry assay, following adsorption with PRV (1.0 MOI) for 1 h on ice, CoPP-treated PK-15 cells were transferred to at 37◦C. After 1 h, cells were washed with trypsin to remove virions adsorbed on the cell surface, and subsequently harvested to assess virus genome via RT-qPCR (E) and gB protein expression via western blotting (F). (G,H) PRV virus suspension (0.01 MOI) was co-incubated with CoPP at 37◦C for 1 h and used to inoculate Vero cells, and part of the supernatant is directly used to determine the virus titer. After 24 h, cells were harvested for viral replication analysis, and virus titer was tested using TCID50. Data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. CoPP, cobalt-protoporphyrin; PRV, pseudorabies virus; MOI, multiplicity of infection; RT-qPCR, reverse transcription-quantitative PCR; gB, glycoprotein B. FIGURE 6 | CoPP directly inactivates PRV particles without affecting viral attachment or entry into cells. PK-15 or ST cells treated with 0, 10, 25, 50 µM of CoPP for 12 h at 37◦C. After washing three times with PBS to remove residual CoPP, cells were subsequently pre-chilled on ice for 30 min, followed by inoculation with ice-cold PRV (1.0 MOI) for 1 h at 4◦C. Frontiers in Microbiology | www.frontiersin.org HO-1 Metabolite, BV Partially Mediates the Inhibitory Effect of HO-1 on PRV Replication However, no significant cytotoxic effect of BV was observed within the concentrations used in the present study (Supplementary Figures 5A,B). Taken together, these results suggest that like CO, BV also partially mediates the inhibitory effect of HO-1 on PRV replication. March 2020 | Volume 11 | Article 412 Frontiers in Microbiology | www.frontiersin.org 12 HO-1 Suppresses PRV Replication in vitro Zhang et al. hang et al. HO-1 Suppresses PRV Replication in vitro FIGURE 7 | CO mediates the anti-PRV activity of HO-1. (A) PK-15 or (B) ST cells were infected with PRV at a MOI of 0.01, prior to harvesting at 12, 24, 36, and 48 hpi to detect total HbCO levels via ELISA. (C) PK-15 or (D) ST cells were incubated with 50 µM of CoPP for 24 h, prior to harvesting to detect total HbCO levels via ELISA. PRV-infected PK-15 cells were treated with 50 µM of CoPP, in the presence or absence of 50 µg/ml of Hb. After 24 h, cells were harvested to assess PRV gB mRNA and protein expression levels via (E) RT-qPCR and (F) western blotting, respectively, while (G) supernatants were harvested to measure virus copies via RT-qPCR. PRV-infected PK-15 or ST cells were incubated with 3% FBS + DMEM containing 0, 10, 25, and 50 µM of CORM-2 or 50 µM of inactive CORM-2 for 36 h. Cells and supernatants were collected for PRV replication analysis via (I,K) western blotting and (H,J) RT-qPCR. Data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. CO, carbon monoxide; PRV, pseudorabies virus; HO-1, heme oxygenase-1; Hb, hemoglobin; ELISA, enzyme-linked immunosorbent assay; CoPP, cobalt-protoporphyrin; RT-qPCR, reverse transcription-quantitative PCR; CORM, CO-releasing molecules. FIGURE 7 | CO mediates the anti-PRV activity of HO-1. (A) PK-15 or (B) ST cells were infected with PRV at a MOI of 0.01, prior to harvesting at 12, 24, 36, and 48 hpi to detect total HbCO levels via ELISA. (C) PK-15 or (D) ST cells were incubated with 50 µM of CoPP for 24 h, prior to harvesting to detect total HbCO levels via ELISA. PRV-infected PK-15 cells were treated with 50 µM of CoPP, in the presence or absence of 50 µg/ml of Hb. HO-1 Metabolite, BV Partially Mediates the Inhibitory Effect of HO-1 on PRV Replication After 24 h, cells were harvested to assess PRV gB mRNA and protein expression levels via (E) RT-qPCR and (F) western blotting, respectively, while (G) supernatants were harvested to measure virus copies via RT-qPCR. PRV-infected PK-15 or ST cells were incubated with 3% FBS + DMEM containing 0, 10, 25, and 50 µM of CORM-2 or 50 µM of inactive CORM-2 for 36 h. Cells and supernatants were collected for PRV replication analysis via (I,K) western blotting and (H,J) RT-qPCR. Data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. CO, carbon monoxide; PRV, pseudorabies virus; HO-1, heme oxygenase-1; Hb, hemoglobin; ELISA, enzyme-linked immunosorbent assay; CoPP, cobalt-protoporphyrin; RT-qPCR, reverse transcription-quantitative PCR; CORM, CO-releasing molecules. were subsequently infected with PRV (0.01 MOI), followed by treatment with or without 50 µM of BV or CoPP. Cells and culture supernatants were harvested at 36 hpi to detect PRV gB protein expression and progeny virus copies. Western blot analysis demonstrated no significant difference in PRV gB protein expression between PK-15 cells transfected with siBVR In mammalian cells, BV is rapidly converted to BR via BVR catalysis (Wegiel et al., 2011). In order to determine whether BR also exerts anti-PRV effects, PK-15 cells were transfected with BVR-specific siRNA to prevent the conversion of BV to BR, which resulted in the accumulation of BV in PK-15 cells. PK-15 cells transfected with siBVR or siNC March 2020 | Volume 11 | Article 412 Frontiers in Microbiology | www.frontiersin.org 13 HO-1 Suppresses PRV Replication in vitro Zhang et al. FIGURE 8 | BV mediates the anti-PRV activity of HO-1. PK-15 (A) or ST (B) cells were infected with 0.01 MOI of PRV. Samples were collected at 12, 24, 36, 48 hpi for BR detection using ELISA. PK-15 (C) or ST (D) cells were treated with 50 µM CoPP for 24 h, the samples were used for BR content detection by ELISA. PK-15 or ST cells were infected with PRV (0.01 MOI) followed by treating with 0, 10, 25, 50 µM of BV for 36 h. Cells were harvested for gB detection using western blotting (E,G), and supernatants were harvested for progeny virus copies measurement using RT-qPCR (F,H). PK-15 cells were transfected with 100 nM of siBVR or siNC, then cells were inoculated with 0.01 MOI of PRV followed by treatment with 50 µM BV or CoPP. HO-1 Metabolite, BV Partially Mediates the Inhibitory Effect of HO-1 on PRV Replication At 24 hpi, cells and supernatants were harvested, PRV gB was detected using western blotting (I), progeny virus copies was detected using RT-qPCR (J), respectively. (K,L) PRV-infected PK-15 cells were treated with 0, 10, 25, 50 µM of BR for 36 h. Cells and supernatants were collected for PRV replication analysis. Data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. PRV, pseudorabies virus; BR, bilirubin; BV, biliverdin; ELISA, enzyme-linked immunosorbent assay; CoPP, cobalt-protoporphyrin; RT-qPCR, reverse transcription-quantitative PCR; MOI, multiplicity of infection. FIGURE 8 | BV mediates the anti-PRV activity of HO-1. PK-15 (A) or ST (B) cells were infected with 0.01 MOI of PRV. Samples were collected at 12, 24, 36, 48 hpi for BR detection using ELISA. PK-15 (C) or ST (D) cells were treated with 50 µM CoPP for 24 h, the samples were used for BR content detection by ELISA. PK-15 or ST cells were infected with PRV (0.01 MOI) followed by treating with 0, 10, 25, 50 µM of BV for 36 h. Cells were harvested for gB detection using western blotting (E,G), and supernatants were harvested for progeny virus copies measurement using RT-qPCR (F,H). PK-15 cells were transfected with 100 nM of siBVR or siNC, then cells were inoculated with 0.01 MOI of PRV followed by treatment with 50 µM BV or CoPP. At 24 hpi, cells and supernatants were harvested, PRV gB was detected using western blotting (I), progeny virus copies was detected using RT-qPCR (J), respectively. (K,L) PRV-infected PK-15 cells were treated with 0, 10, 25, 50 µM of BR for 36 h. Cells and supernatants were collected for PRV replication analysis. Data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. PRV, pseudorabies virus; BR, bilirubin; BV, biliverdin; ELISA, enzyme-linked immunosorbent assay; CoPP, cobalt-protoporphyrin; RT-qPCR, reverse transcription-quantitative PCR; MOI, multiplicity of infection. or siNC, without BV or CoPP treatment (Figure 8I). Conversely, transfection with siBVR, followed by BV treatment indicated increased PRV gB expression compared with siNC-transfected cells (Figure 8I). Besides, transfection with siBVR, followed by CoPP treatment indicated increased PRV gB expression compared with siNC-transfected cells as well (Figure 8I). Similar results were observed with regards to supernatant virus copies under the same experimental conditions (Figure 8J). DISCUSSION PRV, a member of the α herpesvirus family, is considered infectious to most mammals, which highlights the necessity to develop antiviral strategies as a complementary approach to vaccination. Induction of HO-1 can protect organs or tissues from several types of stresses, including inflammation, oxidative injury and hemorrhage-induced hypoxia (Hashiba et al., 2001; Vallabhaneni et al., 2010). Furthermore, recent studies have reported the antiviral properties of HO-1 and its enzymatic products, emphasizing the key role HO-1 plays in protecting the host against viral infection. However, the molecular mechanism underlying HO-1 function in PRV infection and replication still remains unclear. To the best of our knowledge, the present study was the first to demonstrate that upregulated HO-1 expression inhibits PRV replication in both PK-15 and ST cells, indicating the antiviral potential of this cytoprotective enzyme during PRV infection. Furthermore, the results demonstrated that the downstream metabolites of HO-1, BV and CO, mediate its antiviral effects, elucidating the molecular mechanism by which HO-1 inhibits viral replication. p p p g In order to verify the specificity of upregulated HO- 1 in mediating the antiviral effect of CoPP, HO-1-specific siRNA was used to downregulate CoPP-induced HO-1 expression. Downregulation of HO-1 partially reversed the CoPP-induced inhibitory effect on PRV replication, proving that HO-1 specifically mediates the antiviral activity of CoPP. Overexpression of HO-1 suppressed PRV replication, while knockdown of basal levels of HO-1 promoted PRV replication in both PK-15 and ST cells, indicating that aberrant HO-1 expression may effectively inhibit PRV replication. ZnPP, a HO-1 specific inhibitor that can upregulate HO-1 expression, while inhibiting its enzymatic activity was used to determine whether HO-1 activity was essential to inhibit PRV replication (Sardana and Kappas, 1987). Treatment with ZnPP partially reversed CoPP-induced PRV inhibition, but failed to attenuate PRV replication, suggesting that HO-1 activity is essential for PRV attenuation. The present study confirmed the anti- PRV activity of HO-1; however, this property does not seem to be ubiquitous. For example, HO-1 has been reported to promote KSHV replication in endothelial cells via its downstream metabolite, CO (Botto et al., 2017). Furthermore, HO-1 failed to play a suppressive role in the replication of VSV (Hill-Batorski et al., 2013), indicating different physiological functions of HO-1 against different pathogenic microorganisms. It is hypothesized that these inconsistencies may attribute to the specific molecular mechanisms underlying the antiviral properties of HO-1. HO-1 Metabolite, BV Partially Mediates the Inhibitory Effect of HO-1 on PRV Replication Subsequently, exogenous BR was used to treat PRV-infected PK-15 cells and viral replication was determined, in order to further validate the aforementioned results. BR restrained both gB expression (Figure 8K) and supernatant virus copies (Figure 8L), in a concentration-dependent manner, while no significant cytotoxic effect was observed according to the results March 2020 | Volume 11 | Article 412 Frontiers in Microbiology | www.frontiersin.org 14 HO-1 Suppresses PRV Replication in vitro Zhang et al. PRV with CoPP had obvious, but not complete inhibitory effect on PRV proliferative property (Figures 6G,H), demonstrating certain virucidal activity. The molecular mechanism underlying how porphyrins such as CoPP disrupt the PRV enveloped particles is unknown. However, the evidence that CoPP does not affect viral attachment or internalization into host cells suggest that CoPP directly disrupts the virus as a virucidal agent which may interact with specific structures (glycosylation, phosphorylation, peptide interface, etc.) on the viral particle surface or lipids within the viral envelope, as some studies have suggested that porphyrins abrogate infection by HIV-1 and that this activity appears to be a result of an interaction with the envelope protein. Since all the viral envelopes have a lipid bilayer basal structure similar to that of the cell membrane (Seeger and Mason, 2000) and these compounds are well tolerated by cells, it is unlikely that porphyrin compounds disrupt envelope through lipid depletion. Besides, porphyrins were reported to selectively inactivated a diversity of enveloped viruses, but not non-enveloped viruses (Guo et al., 2011), implies that some common structural features presented on the surfaces of viral envelope proteins are the most possible targets. of the CCK-8 assay (Supplementary Figures 5C,D). Taken together, these results suggest that the secondary metabolite of BV, BR, contributes to the anti-PRV effect of HO-1. of the CCK-8 assay (Supplementary Figures 5C,D). Taken together, these results suggest that the secondary metabolite of BV, BR, contributes to the anti-PRV effect of HO-1. Frontiers in Microbiology | www.frontiersin.org DISCUSSION the only source of intracellular BR (Maines, 2005), the present study set out to determine whether the antiviral activity of BV against PRV attributed to its conversion into BR rather than a direct effect of BV, in PK-15 and ST cells. Knockdown of BVR exerted no significant effect on PRV replication, whereas co-treatment with siBVR and BV or CoPP partially decreased inhibition of PRV replication compared with the siNC and BV or CoPP co-treatment group, suggesting that the conversion of BV to BR may participate in the antiviral effect of BV. As expected, treatment with BR notably suppressed PRV replication in PK-15 cells, further manifesting its antiviral activity. A previous study reported that BR exerts antiviral activity against HSV-1 infection via NO production (Santangelo et al., 2012), and NO synthesized by NOS has been demonstrated to enhance the activity of HO- 1, resulting in an activating positive loop between NO and CO (Zuckerbraun et al., 2003; Wegiel et al., 2010). Thus, the antiviral effect of BV/BR against PRV may be due to the synergistic effect between NO and CO; however, this requires further investigation. is faced with noxious stimuli or infected with certain viruses. For example, BV and CO have been demonstrated to exhibit additive effects in protecting the liver from immune-mediated injury or Huh7.5 cells from HBV infection (Sass et al., 2004; Lehmann et al., 2010). The antiviral effect of HO-1 against PRV may be achieved by administrating either BV or CO, whereby the synergistic effect of the two downstream metabolites is hypothesized to mediate the antiviral effect of HO-1. Despite the novel insights, the present study was unable to determine the downstream molecular mechanisms underlying the antiviral effects of BV/BR and CO; thus, prospective studies will focus on these associations. Taken together, the results of the present study suggest that HO-1 exerts an antiviral effect against PRV, and these functions are at least partially mediated by its downstream products, BV and CO. These findings provide novel insight into the molecular mechanisms underlying HO-1 inhibition on PRV replication, while also indicating potential novel measures for controlling PRV infection. q g HO-1 induction and endogenous CO have been demonstrated to take on a protective role in some animal disease models and in vitro experiments (Otterbein et al., 2003; Knauert et al., 2013). Furthermore, the HO-1/CO axis has favorable properties against inflammatory responses (Chung et al., 2009). DISCUSSION Previous studies indicated that both HBV and HCV infection caused a down-regulation of HO-1 as a means of optimizing viral replication (Abdalla et al., 2004; Protzer et al., 2007). Consistent with previous finding, the results of the present study indicated that PRV infection was associated with decreased HO- 1 expression in both PK-15 and ST cells (Figure 1). Thus it was speculated that the virus develops multiple strategies to evade host antiviral defense responses, by which suppression of HO-1 expression is a manifestation of this strategy. Some previous studies have certificated that HO-1 expression is regulated by transcription factors, including Nrf2, Keap1 and Bach1, which bind to the antioxidant response element in the promoter region of HO-1 (Han, 2011; Fredenburgh et al., 2015). However, although PRV replication is necessary for HO-1 inhibition in the present experimental model, it still remains unclear as to whether viral proteins are involved in suppressing activation of the aforementioned transcription factors, thus abrogating HO-1 expression. Whether viral protein directly interacts with HO-1 requires further investigation, and possesses important implications to understanding the molecular mechanism underlying HO-1 downregulation. Some evidence suggests that host cell oxidative stress status plays an important role in regulating viral replication and infectivity (Cai et al., 2003). Up-regulation of intracellular oxidative stress and down- regulation of HO-1 in PRV-infected cells may indicate that PRV can induce oxidative stress in host cells and antagonizes the HO-1 pathway to facilite self-replication. Certain porphyrins and porphyrin derivatives possess virucidal effect against some other viruses, such as EBOV, HIV and MARV (Vzorov et al., 2002; Chen-Collins et al., 2003; Guo et al., 2011), thus the potential of virucidal effect of CoPP was evaluated. Co-incubation of The roles of HO-1 downstream metabolites were subsequently investigated to determine the precise molecular mechanisms underlying the inhibition of PRV replication by HO-1. Both BV and CO mediated the antiviral activity of HO-1, as demonstrated by inhibition of PRV replication via BV or CORM-2 treatment in cell-based assays. As a downstream metabolite of HO-1, BV has been reported to exert antiviral activity against several domestic animal viruses, including BVDV (Ma et al., 2017). Considering that the majority of organs retain BVR activity to transform BV into BR (Maines, 2005), and the BV-BVR signaling pathway is March 2020 | Volume 11 | Article 412 Frontiers in Microbiology | www.frontiersin.org 15 HO-1 Suppresses PRV Replication in vitro Zhang et al. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2020.00412/full#supplementary-material DATA AVAILABILITY STATEMENT All datasets generated for this study are included in the article/Supplementary Material. DISCUSSION The present study demonstrated that the protective effect of HO-1 against PRV may be achieved via administration of CORM-2, but not iCORM-2, suggesting that besides BV, CO may also exert cytoprotective effects during PRV infection. HO-1-derived CO was also indicated to play a key role in the antiviral activity of HO-1, whereby CO scavenger Hb partially reversed the inhibitory effect of CoPP-induced HO-1 on PRV replication. Previous studies have demonstrated that CO decreases the replication ability of PRRSV by suppressing the activation of the NF- κB signaling pathway, which plays a significant role in PRV replication (Zhang et al., 2017; Zhao et al., 2017). Thus, the NF-κB signaling pathway may act as a potential target for the HO- 1/CO system to exert its antiviral effect. According to previous reports, HO-1 and its metabolite, CO, function as important immune regulatory factors (Remy et al., 2009; Blancou et al., 2011), thus, the HO-1/CO system may be used as an antiviral or innate immune mechanism by host cells infected with PRV. Furthermore, HO-1/CO have been reported to directly inhibit heat shock protein (HSP) 90 activity of host cells, which plays a critical role in nuclear transport of HSV-1 capsid protein (Lee W.Y. et al., 2014; Zhong et al., 2014). Considering that both PRV and HSV-1 belong to the α Herpesviridae family and have similar biological characteristics, CO may interfere with HSP90 activity to suppress PRV replication in host cells; however, this requires further investigation. Previous studies have also reported a synergistic effect between BV and CO when the host FUNDING This study was supported by the National Natural Science Foundation of Major Projects (31490601), National Natural Science Foundation of China (31702217), National Science and Technology Plan (2015BAD12B02-3), and Science and Technology Innovation Fund of Henan Agricultural University (KJCX2019A06). This study was supported by the National Natural Science Foundation of Major Projects (31490601), National Natural Science Foundation of China (31702217), National Science and Technology Plan (2015BAD12B02-3), and Science and Technology Innovation Fund of Henan Agricultural University (KJCX2019A06). AUTHOR CONTRIBUTIONS AZ, GZ, and YD conceived and designed the experiments. AZ and BW performed the experiments. YW, DJ, and PJ analyzed the data. AZ and GZ wrote the manuscript. AZ, GZ, and YD conceived and designed the experiments. AZ and BW performed the experiments. YW, DJ, and PJ analyzed the data. AZ and GZ wrote the manuscript. An, T. Q., Peng, J. M., Tian, Z. J., Zhao, H. Y., Li, N., Liu, Y. M., et al. (2013). Pseudorabies virus variant in Bartha-K61-vaccinated pigs. China, 2012. Emerg. Infect.Dis. 19, 1749–1755. doi: 10.3201/eid1911.13 0177 Bach, F. H. (2006). Heme oxygenase-1 and transplantation tolerance. Hum. Immunol. 67, 430–432. doi: 10.1016/j.humimm.2006.03.006 Abdalla, M. 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Frontiers in Microbiology | www.frontiersin.org REFERENCES Heme oxygenase 1 protects against hepatic hypoxia and injury from hemorrhage via regulation of cellular respiration. Shock 33, 274–281. doi: 10.1097/SHK.0b013e3181b0f566 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. Vzorov, A. N., Dixon, D. W., Trommel, J. S., Marzilli, L. G., and Compans, R. W. (2002). Inactivation of human immunodeficiency virus type 1 by porphyrins. Antimicrob. Agents Chemother. 46, 3917–3925. doi: 10.1128/aac.46.12.3917- 3925.2002 Copyright © 2020 Zhang, Wan, Jiang, Wu, Ji, Du 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. Wegiel, B., Gallo, D., Csizmadia, E., Roger, T., Kaczmarek, E., Harris, C., et al. (2011). Biliverdin inhibits Toll-like receptor-4 (TLR4) expression through nitric oxide-dependent nuclear translocation of biliverdin reductase. Proc. NatL. Acad. Sci. U.S.A. 108, 18849–18854. doi: 10.1073/pnas.1108571108 March 2020 | Volume 11 | Article 412 Frontiers in Microbiology | www.frontiersin.org 18
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Population dynamics, delta vulnerability and environmental change: comparison of the Mekong, Ganges–Brahmaputra and Amazon delta regions
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SPECIAL FEATURE: OVERVIEW ARTICLE Sustainable Deltas: Livelihoods, Ecosystem Services, and Policy Implications Sustain Sci (2016) 11:539–554 DOI 10.1007/s11625-016-0372-6 SPECIAL FEATURE: OVERVIEW ARTICLE Sustainable Deltas: Livelihoods, Ecosystem Services, and Policy Implications Sustain Sci (2016) 11:539–554 DOI 10.1007/s11625-016-0372-6 Sustain Sci (2016) 11:539–554 DOI 10.1007/s11625-016-0372-6 SPECIAL FEATURE: OVERVIEW ARTICLE Sustainable Deltas: Livelihoods, Ecosystem Services, and Policy Implications Population dynamics, delta vulnerability and environmental change: comparison of the Mekong, Ganges–Brahmaputra and Amazon delta regions 1 Division of Social Statistics and Demography, University of Southampton, Southampton, UK 2 Department of Anthropology, The Anthropological Center for Training and Research on Global Environmental Change and the Ostrom Workshop-Indiana University, Bloomington, USA Keywords Population change  Delta vulnerability  Mekong delta  Amazon delta  Ganges–Brahmaputra delta 3 United Nations University, Institute for Environment and Human Security, Bonn, Germany Population dynamics, delta vulnerability and environmental change: comparison of the Mekong, Ganges–Brahmaputra and Amazon delta regions Sylvia Szabo1 • Eduardo Brondizio2 • Fabrice G. Renaud3 • Scott Hetrick2 • Robert J. Nicholls4 • Zoe Matthews1 • Zachary Tessler5 • Alejandro Tejedor6 • Zita Sebesvari3 • EfiFoufoula-Georgiou6 • Sandra da Costa7 • John A. Dearing8 Sylvia Szabo1 • Eduardo Brondizio2 • Fabrice G. Renaud3 • Scott Hetrick2 • Robert J. Nicholls4 • Zoe Matthews1 • Zachary Tessler5 • Alejandro Tejedor6 • Zita Sebesvari3 • EfiFoufoula-Georgiou6 • Sandra da Costa7 • John A. Dearing8 Received: 2 August 2015 / Accepted: 3 May 2016 / Published online: 26 May 2016  The Author(s) 2016. This article is published with open access at Springerlink.com we provide an overview of population trends and dynamics in the Ganges–Brahmaputra, Mekong and Amazon deltas. Using multiple data sources, including census data and Demographic and Health Surveys, a discussion regarding the components of population change is undertaken in the context of environmental factors affecting the demographic landscape of the three delta regions. We find that the demographic trends in all cases are broadly reflective of national trends, although important differences exist within and across the study areas. Moreover, all three delta regions have been experiencing shifts in population struc- tures resulting in aging populations, the latter being most rapid in the Mekong delta. The environmental impacts on the different components of population change are impor- tant, and more extensive research is required to effectively quantify the underlying relationships. The paper concludes by discussing selected policy implications in the context of sustainable development of delta regions and beyond. Abstract Tropical delta regions are at risk of multiple threats including relative sea level rise and human alter- ations, making them more and more vulnerable to extreme floods, storms, surges, salinity intrusion, and other hazards which could also increase in magnitude and frequency with a changing climate. Given the environmental vulnerability of tropical deltas, understanding the interlinkages between population dynamics and environmental change in these regions is crucial for ensuring efficient policy planning and progress toward social and ecological sustainability. Here, Handled by Osamu Saito, United Nations University Institute for the Advanced Study of Sustainability (UNU-IAS), Japan. & Sylvia Szabo s.m.szabo@soton.ac.uk Handled by Osamu Saito, United Nations University Institute for the Advanced Study of Sustainability (UNU-IAS), Japan. Introduction 4 Engineering and the Environment, University of Southampton, Southampton, UK Delta regions constitute dynamic ecological and social environments and are often major contributors to national economies. While overall, deltas account for only 1 % of global land area, they are home to more than a half billion people, or ca. 7 % of world population (Ericson et al. 2006). Given the particular environmental risks faced by tropical deltas and accounting for interlinkages between demographic and environmental factors, it is crucial to analyze population dynamics in delta environments to inform planning and policy making. The Millennium 5 Environmental CrossRoads Initiative, City University of New York, New York, USA 6 Civil, Environmental and Geo-Engineering and National Center for Earth-surface Dynamics, University of Minnesota, Minneapolis, USA 7 Instituto de Pesquisa e Desenvolvimento, Universidade do Vale do Paraı´ba (UNIVAP), Sa˜o Jose´ dos Campos, Brazil 8 Geography and Environment, University of Southampton, Southampton, UK 8 Geography and Environment, University of Southampton, Southampton, UK 12 3 Sustain Sci (2016) 11:539–554 540 Ecosystem Assessment (2005) showed that beyond provi- sioning ecosystem services such as food, water, fiber and fuel, regulating and supporting ecosystem services also influences various aspects of human development, such as health and income, and long-term sustainability of agri- culture and natural resources. Trends in human wellbeing, however, cannot be fully understood without considering the specific demographic context, including evolving population structures by age and sex, and changes in the dominant demographic drivers of fertility, mortality and migration. Neumann et al. 2015). By providing a rigorous demo- graphic overview of three delta regions, the study not only contributes to the literature on delta regions, but also considers policy implications for sustainable development. Ecosystem Assessment (2005) showed that beyond provi- sioning ecosystem services such as food, water, fiber and fuel, regulating and supporting ecosystem services also influences various aspects of human development, such as health and income, and long-term sustainability of agri- culture and natural resources. Trends in human wellbeing, however, cannot be fully understood without considering the specific demographic context, including evolving population structures by age and sex, and changes in the dominant demographic drivers of fertility, mortality and migration. The next section describes the data sources and methods used in this study. Introduction In ‘‘Environmental context for delta vulnerability’’, we discuss the environmental context for vulnerability of tropical deltas using the examples of the three case studies, i.e., the Ganges–Brahmaputra, the Mekong and the Amazon delta. In ‘‘Feedbacks between population dynamics and environmental change’’, we propose an original conceptual framework illustrating the feedbacks between population and environment with a specific focus on delta regions. ‘‘Population growth and structure’’ summarizes the recent trends in population growth across the study areas, while ‘‘Components of population change’’ offers an analysis and discussion of the key components of population change in the three delta regions. The final section summarizes the key arguments and discusses the main policy implications in the context of the projected population dynamics and the current and expected impacts of environment and climate change on the delta regions and delta populations. Understanding population trends and dynamics in del- taic regions is especially important in the context of global environmental change which is expected to exacerbate the existing threats to livelihoods through e.g., sea level rise, land subsidence, increased storminess, flooding, and salinity intrusion (Dun 2011; Nicholls 2011; Szabo et al. 2015a, b, c; Wong et al. 2014; World Bank 2000). In this context, the present study examines population dynamics (population growth, fertility, mortality, and migration) in three selected tropical deltas. It draws on the theory of demographic transition and the literature conceptualizing the interlinkages between population and environment (de Sherbinin et al. 2007; Hummel et al. 2012). According to fundamental demographic theory (Notestein 1945; Dyson 2011; Dyson 1998), it is common for countries to experi- ence concurrent falling mortality and fertility levels as they progress through the ‘demographic transition’. This is a process through which a country evolves from high to low levels of mortality and fertility, usually associated with increasing longevity. With regard to interlinkages between population and environment, specific components of demographic change can also be influenced by the quality of the biophysical environment, environmental hazards and creeping processes, such as salinity intrusion and arsenic contamination of water and soil resources. 123 Study areas The Vietnamese portion of the Mekong delta region (thereafter: the Mekong delta) covers 13 provinces and excludes Ho Chi Min City. As with the GBD, the Mekong delta is highly vulnerable to adverse environmental events, in particular, flooding and salinity intrusion. While it has been recognized that fluvial floods can bring benefits for the economy, as they convey sediment and benefit fisheries (Tri et al. 2013), flooding can also have a disastrous effect on households’ livelihoods. Since 2000, the region expe- rienced three major floods (2000, 2001 and 2002); the first affecting approximately 11 million people. As a result of this flood, 800 9 103 dwellings were inundated, and 55,123 ha of rice crops destroyed (Nguyen and James 2013). Extreme weather events will continue to occur in the region and may occur more frequently (Dun 2011). In addition, climate change is likely to increase not only the risk of flooding, but is also associated with relative sea level rise, salinity intrusion and changes in temperature and rainfall patterns (Dang et al. 2014; Nguyen and James 2013). Finally, for the Amazon delta (Brazil), we combined the parameters provided by Ericson et al. (2006), who used a 5 km buffer zone around the coastline intersecting with the first distributary, and the limits of municipalities intersect- ing this buffer zone to define our study area. In terms of administrative boundaries, the study area comprises 50 municipalities across the Para´ and Amapa´ states in the North region of Brazil (or approximately 6 % of the legal Brazilian Amazon in terms of total territory). In 2010, our study area concentrated approximately 16.5 % of the total population of this legal Amazon and 18 % of its urban population (IBGE 2010). While other parts of the Brazilian Amazon have undergone significant environmental change during the last three decades, the delta region has seen relatively lower levels of environmental degradation. However, the region has experienced rapid urbanization, so there are local hotspots of environmental change and a growing economy based on forest products and agroforestry (Brondizio et al. 2013). Most urban areas in the region lack basic sanitation and other infrastructure and public services, which along with some of the highest poverty rates in Brazil create vulnerable conditions for a significant segment of the population (this issue). On the other hand, farmers in the Fig. 1 Population-environment gradients in the study areas. Study areas The study areas (Fig. 1) encompass three deltaic systems, all located in developing/transition countries. For the pur- poses of data collection, we have defined the spatial extent of each delta primarily by the area downstream of the first distributary as mapped by the Shuttle Radar Topography Mission. For the Ganges–Brahmaputra delta (GBD), the altitude of the first distributary (the Hoogli river) at the Farraka Barrage is about 18–20 m asl. Within Bangladesh, this contour encloses over 45 districts in whole division areas of Khulna, Barisal, Dhaka, Sylhet but most of Chittagong division. Chittagong division excludes the districts of Khagrachari, Rangamati and Bandarban. This is an envi- ronmentally vulnerable region suffering both from the consequences of rapid-onset hazards (e.g. cyclones) (Kay et al. 2015) and creeping processes, such as salinity intrusion (Clarke et al. 2015), arsenic contamination of groundwater (Abedin et al. 2012; Edmunds et al. 2015) and subsidence (Brown and Nicholls 2015). There is extensive evidence that multiple stressors associated with environ- mental change place increasing strains on the livelihoods of populations in the region. In particular, food security has emerged as a key developmental concern (Faisal and Parveen 2004; MEF 2009). The present study focuses on three specific delta regions, i.e., the Ganges–Brahmaputra delta in Bangladesh (GBD), Mekong delta in Vietnam and the Amazon delta in Brazil. These deltas were selected as they are each locally and globally significant and encompass a range of biophysical and social conditions. Population size, the rate of popula- tion growth and population distribution constitute crucial factors which are affecting and affected by natural habitat. While a relatively large body of literature examined the interlinkages between population growth and environment (de Sherbinin et al. 2007; Hummel et al. 2012; Lutz et al. 2002), there is limited consideration of the dynamics of population change in delta regions. Yet, approximately 10 % of the world’s population live in areas lower than 10 m above sea level, and the population in these low-lying coastal areas is projected to grow in all continents, espe- cially in the developing world (McGranahan et al. 2007; 123 541 Sustain Sci (2016) 11:539–554 For the Mekong, the first distributary point at Phnom Penh lies at about 7–9 m asl which delineates a delta mainly in Vietnam with a small part in Cambodia. Environmental context for delta vulnerability Sea level rise associated with global climate change is rightly considered a major factor in setting the vulnerability and sustainability of coastal systems (Brakenridge et al. 2013). Rising seas increase the risk of flooding due to coastal storms (Balica et al. 2012) and increase rates of coastal wetland loss (Nicholls 2004; Spencer et al. 2016). In deltas, sea level rise is exacerbated by land subsidence, resulting in relative sea level rise (RSLR) rates several orders of magnitude greater than eustatic sea level rise rates alone (Ericson et al. 2006; Syvitski et al. 2009). As envi- ronments of active sedimentation, deltas rely on delivery and deposition of new sediment from the upstream catch- ment area to offset natural sediment compaction (Syvitski and Saito 2007). Human activity on the delta can accelerate these natural rates through urban development (Mazzotti et al. 2009), groundwater extraction (Higgins et al. 2013) and hydrocarbon extraction (Morton et al. 2002). While deltas require increased sediment delivery to keep pace with accelerated compaction and sea level rise, the sediment fluxes reaching most major deltas have decreased due to construction of dams and reservoirs in the upstream river network (Vo¨ro¨smarty et al. 2003). The increased rates of RSLR associated with these anthropogenic factors lead to increased coastal flood risk in deltas (Tessler et al. 2015a, b). Following the method of Tessler et al. (2015a, b), we have estimated the relative magnitude of eight anthro- pogenic drivers associated with relative sea level rise in deltas (Fig. 2). From the coastal delta domain, the anthropogenic drivers include hydrocarbon extraction, groundwater extraction, impervious surface area, and wetland disconnectivity. From the upstream contributing watershed, we include impervious surface area, wetland disconnectivity and sediment trapping in artificial reser- voirs. We also include estimates of sea level rise trends Sea level rise associated with global climate change is rightly considered a major factor in setting the vulnerability and sustainability of coastal systems (Brakenridge et al. 2013). Rising seas increase the risk of flooding due to coastal storms (Balica et al. 2012) and increase rates of coastal wetland loss (Nicholls 2004; Spencer et al. 2016). In deltas, sea level rise is exacerbated by land subsidence, resulting in relative sea level rise (RSLR) rates several orders of magnitude greater than eustatic sea level rise rates alone (Ericson et al. 2006; Syvitski et al. 2009). Study areas Note 1 Bangladesh Population and Housing Census (2001, 2001 and 1991); 2 Brazilian Institute of Geography and Statistics (IBGE 2010); 3 2009 Vietnam Population and Housing Census; 4 Ericson et al. (2006); 5 World Development Indicators (WDI), World Bank; 6 Parry et al. (2007); 7 Giri et al. (2011); 8 Syvitski et al. (2009); 9 Syvitski and Saito (2007) Fig. 1 Population-environment gradients in the study areas. Note 1 Bangladesh Population and Housing Census (2001, 2001 and 1991); 2 Brazilian Institute of Geography and Statistics (IBGE 2010); 3 2009 Vietnam Population and Housing Census; 4 Ericson et al. (2006); 5 World Development Indicators (WDI), World Bank; 6 Parry et al. (2007); 7 Giri et al. (2011); 8 Syvitski et al. (2009); 9 Syvitski and Saito (2007) 12 3 542 Sustain Sci (2016) 11:539–554 IPEA 2010). Data are aggregated at the municipal and census sector levels for 1991, 2000 and 2010 in all three delta regions. Table 1 summarizes the demographic data sources used in the analysis. region are reporting increasing tidal flooding and changing salinity in coastal ecosystems, but these changes have not been systematically documented. Figure 1 illustrates the key delta relevant population- environment gradients and socio-environmental character- istics of the three study areas. Gradient bars, associated with variables are shaded to represent the respective ‘‘values’’ of each variable for each study area. Darker shades represent higher values. Population size and popu- lation density are greatest in the GBD where the total population exceeded 108 million, and the population den- sity in the study area is approximately 1280 people per km2 (Ericson et al. 2006). The proportion of delta population at risk is highest in the Mekong delta region, and so is the proportion of delta area potentially lost by 2050 (Ericson et al. 2006). It should, however, be noted that, because of the population size of the GDB, in terms of absolute numbers, the greatest impact on population loss is expected to take place in this delta region (Ericson et al. 2006). Among the three delta regions, the Amazon delta has the highest proportion of urban population, while the Mekong delta is least urbanized. Environmental context for delta vulnerability As envi- ronments of active sedimentation, deltas rely on delivery and deposition of new sediment from the upstream catch- ment area to offset natural sediment compaction (Syvitski and Saito 2007). Human activity on the delta can accelerate these natural rates through urban development (Mazzotti et al. 2009), groundwater extraction (Higgins et al. 2013) and hydrocarbon extraction (Morton et al. 2002). While deltas require increased sediment delivery to keep pace with accelerated compaction and sea level rise, the sediment fluxes reaching most major deltas have decreased due to construction of dams and reservoirs in the upstream river network (Vo¨ro¨smarty et al. 2003). The increased rates of RSLR associated with these anthropogenic factors lead to increased coastal flood risk in deltas (Tessler et al. 2015a, b). Table 1 Sources of demographic data Delta region Data source Ganges– Brahmaputra Bangladesh Demographic and Health Surveys (DHS), 2010 household income and expenditure survey (HIES), Bangladesh Population and Housing Census (2001, 2001 and 1991) Mekong Vietnamese Living Standards Survey (VLSS), Vietnam Demographic and Health Surveys (DHS), 2009 Vietnam Population and Housing Census, online data repository developed by the General Statistics Office of Vietnam Amazon Brazilian Institute of Geography and Statistics (IBGE 2010, 2014) Institute of Applied Economic Research (IPEA 2010) Cross-cutting World Population Prospects (United Nations, 2012) Data 2 Selected rank-normalized anthropogenic impacts on deltas associated with sea level rise and land subsidence The three focus The gray region indicates the uncertainty of each delta’s aggreg anthropogenic impact following Tessler et al (2015a b) Fig. 2 Selected rank-normalized anthropogenic impacts on deltas associated with sea level rise and land subsidence. The three focus deltas of this study are highlighted in the context of 48 global deltas. The gray region indicates the uncertainty of each delta’s aggregate anthropogenic impact following Tessler et al. (2015a, b) River is likely to be important in the near future (Kuenzer et al. 2015). There appears to be very low anthropogenic impact on the Amazon delta, relative to other deltas, with the largest stress associated with sea level rise. from the oceanic domain. For global consistency, these indicators are derived from global-scale remote-sensing and numerical modeling. Each indicator is rank-normalized across 48 globally-distributed deltas, then aggregated to provide a relative scale for comparing the environmental state of river deltas and each delta’s susceptibility to RSLR. Constructed at the global scale, these estimates do not consider spatial patterns of environmental change within deltas, or important anthropogenic drivers of change other than those associated with RSLR, such as soil salinization, wetland loss and coastal eutrophication. Data Our paper draws on a number of secondary macro and micro level data sources to overview and analyze the three deltas discussed above. More specifically, for Bangladesh, the data used include the 2010 household income and expenditure survey (HIES) conducted by the Bangladesh Bureau of Statistics (BBS 2011a), Demographic and Health Surveys (DHS) (Mitra et al. 1994; NIPORT, Mitra and Associates and ICF International 2013; NIPORT, Mitra and Associates and Macro International 2009; NIPORT, Mitra and Associates and ORCM 2001) as well as census data. Similarly, for Vietnam, we used the Vietnamese Living Standards Survey (VLSS), census data and Demo- graphic and Health Surveys. Finally, for the Amazon delta, we used census data from the Brazilian Institute of Geog- raphy and Statistics (IBGE) and demographic data and human development indices compiled by The Institute of Applied Economic Research (IPEA) of Brazil (IBGE 2010; Following the method of Tessler et al. (2015a, b), we have estimated the relative magnitude of eight anthro- pogenic drivers associated with relative sea level rise in deltas (Fig. 2). From the coastal delta domain, the anthropogenic drivers include hydrocarbon extraction, groundwater extraction, impervious surface area, and wetland disconnectivity. From the upstream contributing watershed, we include impervious surface area, wetland disconnectivity and sediment trapping in artificial reser- voirs. We also include estimates of sea level rise trends Table 1 Sources of demographic data Delta region Data source Ganges– Brahmaputra Bangladesh Demographic and Health Surveys (DHS), 2010 household income and expenditure survey (HIES), Bangladesh Population and Housing Census (2001, 2001 and 1991) Mekong Vietnamese Living Standards Survey (VLSS), Vietnam Demographic and Health Surveys (DHS), 2009 Vietnam Population and Housing Census, online data repository developed by the General Statistics Office of Vietnam Amazon Brazilian Institute of Geography and Statistics (IBGE 2010, 2014) Institute of Applied Economic Research (IPEA 2010) Cross-cutting World Population Prospects (United Nations, 2012) Table 1 Sources of demographic data 12 Sustain Sci (2016) 11:539–554 543 Fig. 2 Selected rank-normalized anthropogenic impacts on deltas associated with sea level rise and land subsidence. The three focus deltas of this study are highlighted in the context of 48 global deltas. The gray region indicates the uncertainty of each delta’s aggregate anthropogenic impact following Tessler et al. (2015a, b) Fig. international development debate (de Sherbinin et al. 2007; Hummel et al. 2012). Rapid population growth and resulting population density put pressure on provisioning ecosystem services in coastal areas, and the interactions between these factors are likely to intensify in the context of population growth and climate change (Kirwan and Megonigal 2013). fertility rates, although this relationship is more complex and not fully established. There is, however, some evidence that environmental pollutants can negatively affect fertility, although research findings are not consistent (Fisch et al. 2003; Foster et al. 2008). Feedbacks between population dynamics and environmental change A delta-specific conceptual framework was developed (Fig. 3), which will serve for analyzing distinct compo- nents of population change in the three study deltas and how these demographic factors relate to environmental context in each delta. The framework will also serve to draw conclusions regarding the key demographic and environmental factors in each delta, and implications for development of sustainable management policies. All delta regions, as will be shown in the next sections of this paper, experienced relatively rapid population growth accompa- nied by decline in fertility and high out-migration. Demographic change can be explained at least partially, by the environmental context in which populations find themselves. The associations between population and environment have become key factors contributing to the The Ganges–Brahmaputra, Mekong and Amazon deltas represent three different regions of the environmental stress space (Tessler et al. 2016). By these measures, the Ganges– Brahmaputra delta is the most environmentally stressed delta system in the study, with high levels for nearly all the environmental indicators. The only moderate-valued indi- cator is sediment retention in upstream reservoirs, though this may change in the near future with several new dams on the Brahmaputra River under construction or planned in India, Nepal and China (Zarflet al. 2015). The Mekong delta, though affected by similar drivers as the Ganges– Brahmaputra delta, has an overall moderate level of envi- ronmental impact. New dam construction on the Mekong 12 3 3 544 Sustain Sci (2016) 11:539–554 Fig. 3 Feedbacks between population dynamics and environment Fig. 3 Feedbacks between population dynamics and environment Fig. 3 Feedbacks between population dynamics and environment Sustain Sci (2016) 11:539–554 Fig. 4 Recent population growth in the a Ganges– Brahmaputra, b Mekong and c Amazon deltas Data sources: Bangladesh Bureau of Statistics (BBS), Brazilian Institute of Geography and Statistics (IBGE), and General Statistics Office (GSO), Vietnam population in the Mekong delta increased from around 15.5 million in 1995 to over 17.5 million in 2013 (Fig. 4b). However, the Mekong delta is one of the regions with the lowest population growth rate in Vietnam with large spatial differences within the delta (Garschagen et al. 2012). When analysing population dynamics of delta regions, it is important to consider their population structures. The current population structure of the GBD (Fig. 5a) is the youngest, although it is visible that the youngest age groups (0–4 and 5–9) are disproportionally small, which reflects recent trends in fertility decline. In the Mekong delta region, the population aged 15 to 35 constitutes the greatest percentage of the total population, while the bottom of the pyramid is relatively narrow indicating an aging population structure (Fig. 5b). This population structure is reflected in the region’s dependency ratios, which is around 42.3 % as compared to the national average of 44.7 % (General Statistics Office 2011a). Compared to the Mekong delta region, the population structure in the Amazon delta (Fig. 5c) has not yet reached the rapid ageing pattern; however, its overall population is older than that of the GBD. Likewise, the Amazon delta region has experienced continuous population growth since 1990 (Fig. 4c), most of it being in urban areas of the delta. As highlighted previ- ously, the study area for the Amazon delta is shared by two states in the Northern region of Brazil. To the north, the state of Amapa´ includes nine municipalities, while to the south, the state of Para´ includes 41 municipalities within the Amazon delta region. With approximately 4 million people, the population size of the Amazon delta is the smallest of the three delta regions. Since 1990, the region has experienced over 56 % increase in total population growth reaching a total of ca. 4 million people in 2010 (IBGE 2010). The majority of the population in 2010 (78.5 %) declared urban residency. However, there exists considerable variation within the region (IBGE 2010). The Amazon delta experienced slight decrease in rural popu- lation from 1990 to 2000, followed by a slight increase from 2000 to 2010. Sustain Sci (2016) 11:539–554 The increase in rural population reflects the increasing economic importance of forest and agro- forestry products in the region, opening economic oppor- tunities in rural areas and motivating strong connections between rural and urban areas (Brondizio et al. 2013). Population growth and structure Specific components of demographic change can also be influenced by the quality of the natural environment, environmental hazards and creeping processes, such as salinity intrusion and arsenic contamination of water and soil resources. Environmental migration contributes to population loss in an area and might even lead to popula- tion collapse in certain areas of delta regions, as aban- donment of settlements becomes the main coping strategy of vulnerable households (McLeman 2011). Mortality rates can be affected by the quality of provisioning ecosystem services, natural hazards and extreme weather events. For example, in Bangladesh, 3406 people died as a conse- quence of Cyclone Sidr in 2007 (Paul 2007). Deteriorating ecosystem services, such as poor water quality was found to be positively associated with health outcomes and child and maternal mortality (Brown et al. 2013; Cheng et al. 2012; Silva 2011). Salinization of soil and water has neg- ative effects of quality of water and food security (Szabo et al. 2015a, b, c), which can lead to ill health. Finally, the quality of the environment can also have an impact on Figure 4 illustrates the trends in population growth in the three delta regions. In the GBD, the population size in 2011 increased by approximately 19 million people compared to 1991, which represents a 17.5 % increase in population size over the last two decades (Fig. 4a). Based on the 2011 census data, the total population of the study area was about 108 million, comprising approximately 75 % Ban- gladesh’s population. Out-migration combined with below replacement level fertility rates in some districts con- tributes to changing population structure and negative rates of population growth. For example, during the last two decades, in Pirojpur district in south-western Bangladesh, the population decreased by around 18.4 %, while in the nearby Barisal district, the population declined by approximately 14.3 % (BBS 2012). Similarly to the GBD, in the past two decades, the Mekong delta continued to experience population growth despite falling fertility and relatively high out-migration (Dun 2011). The overall 123 123 545 Sustain Sci (2016) 11:539–554 Fig. 5 Population structure in the a Ganges–Brahmaputra (2011), b Mekong (2009) and c Amazon (2010) deltas Data sources: BBS, IBGE and GSO Mortality The IMR in the Mekong delta is estimated at 12.0 per 1000 live births, while the national average is 15.3 per 1000 live births (General Statistics Office 2013). It is dif- ficult to examine to what extent mortality trends have been affected by environmental factors. It is, however, reason- able to assume that gains in life expectancy could have been greater in the absence of environmental and climate change. The negative effects on population can occur through at least two sets of pathways: first, through direct impact of natural hazards on human life, and second, through the effects of the quality of environment, such as poor water quality linked to salinity ingression and arsenic contamination. As discussed in detail by the 2015 Lancet Commission on Health and Climate Change, the environ- mental impacts on human health, and thus, mortality rates are likely to be exacerbated by climate change. These will involve not only a potentially greater magnitude of natural hazard and diseases and creeping processes, such as soil and water salinization, but also the negative effects of higher temperatures (Watts et al. 2015). Also, it should be remembered that traditional socio-economic factors, such as access to quality healthcare and availability of and access to nutritious food have been found to be important determinants and mitigating factors of child, adolescent and adult health (Braveman et al. 2011). Similarly, in the GBD, environmental factors and cli- mate change can have an important direct and indirect effect on human mortality. It should be stressed that Ban- gladesh as a country has achieved a significant progress in a number of health indicators, despite its relatively poor economic situation (Chowdhury et al. 2013). Current life expectancy in Bangladesh is estimated at 69 years for males and 71 years for females, an increase by 9 and 12 years, respectively since 1990 (WHO 2014) (Fig. 6). At the same time, however, maternal and child mortality rates are worryingly high. Maternal mortality ratio is 170 per 1,000,000 live births, which is similar to that of Pakistan and Cambodia and considerably higher than the maternal mortality rate (MMR) in Vietnam and Brazil (World Bank 2015). The population of the GBD is particularly vulner- able to cyclones, especially in coastal areas. It has been estimated that during the last 50 years, approximately 718 9 103 people died due to cyclones (Haque et al. 2012). Mortality Mortality is one of the components of population change. In Vietnam, life expectancy was 59.1 years in 1960 (World Bank 2015), and increased to 72.2 years in 2005 and 73.1 years in 2013 (General Statistics Office 2014). In the Mekong delta region, life expectancy increased from 12 3 3 546 Sustain Sci (2016) 11:539–554 Fig. 5 Population structure in the a Ganges–Brahmaputra (2011), b Mekong (2009) and c Amazon (2010) deltas Data sources: BBS, IBGE and GSO 5 6 Susta Sc ( 0 6) :539 55 123 123 Sustain Sci (2016) 11:539–554 547 Fig. 6 Recent trends in life expectancy at birth in the Mekong, Ganges–Brahmaputra and Amazon delta regions Data sources: World Development Indicators (WDI), IBGE and GSO, Vietnam. For GBD national data were used to approximate trends (Government of Vietnam 2011). Between 2000 and 2002, 1144 people were killed due to floods in the Mekong delta region (Central Committee for Flood and Storm Control 2015). Children were found to be particularly vulnerable to floods, especially in poorer households where parents worked outside often leaving children without supervision (Nguyen and James 2013). Secondly, indirect environ- mental effects on mortality rates include the quality of water and sanitation, which are associated with water- borne diseases, such as cholera and typhoid and paraty- phoid fevers. Water quality in the frequently populated smaller waterways of the Mekong delta is relatively poor, regardless of the sources exploited (surface-, ground-water, rain water, piped water), with contamination by pesticides (Chau et al. 2015; Toan et al. 2013), nutrients, metals, salinity and microbial organisms (Wilbers et al. 2014a, b; Wilbers et al. 2013; Wilbers et al. 2014a, b). In peri-urban and rural regions of the Mekong delta, large portions of the population are directly exposed to polluted water and consume polluted water with treatments that do not elim- inate all contaminants (Wilbers et al. 2014a, b; Wilbers et al. 2013; Wilbers et al. 2014a, b). Fig. 6 Recent trends in life expectancy at birth in the Mekong, Ganges–Brahmaputra and Amazon delta regions Data sources: World Development Indicators (WDI), IBGE and GSO, Vietnam. For GBD national data were used to approximate trends 73.4 years in 2005 to 74.4 years in 2013 (General Statistics Office 2014) (Fig. 6). Concerning child mortality, infant mortality rate (IMR) in the Mekong delta region is the second lowest regionally in Vietnam (after the Southeast region). Mortality However, the death toll has fallen dramatically, and in 2007, 4234 people died as a result of cyclones compared to 500 9 103 deaths in 1970 (Haque et al. 2012). This reflects improved early warnings and provision of a network of cyclone shelters. In addition to their direct effect, i.e., the loss of human life, cyclones affect the quality of water and increase the risk of disease transmission, in particular, in resource-poor areas. They can also have a post-disaster impact on mental health by increasing the risk of stress and depression (Haque et al. 2012; Shultz et al. 2005). With regards to the direct interlinkages between envi- ronmental factors and mortality (as conceptualized in Fig. 3), between 2001 and 2010, natural disasters affecting the country were responsible for death of 9.5 9 103 people Finally, the population of the Amazon delta region experienced a rapid increase in life expectancy reaching 71.8 years in 2010, an increase by 7.2 years since 1991 12 3 Sustain Sci (2016) 11:539–554 548 (IPEA 2010) (Fig. 6). Overall, life expectancy is slightly higher in the Amapa´ state, although trends between both states (Para´ and Amapa´) are quite similar (IPEA 2010). Infant mortality rate remains high (21 per 1000) although declined more than twofold from 49.7 in 1991. This regional IMR is considerably higher when compared to the national IMR, which has been estimated at 15 in 2010 (World Bank 2015). According to a recent study by the International Institute for Environment and Development (IIED) (Viana et al. 2014), the infant mortality rate in Amapa´ state is the highest in Brazil. In the Brazilian Amazon region, natural disasters, in particular, floods, are associated with environmental and health impacts, includ- ing loss of life, however, disaggregated numbers are dif- ficult to obtain (de Resende Londe et al. 2014). In this region, health problems have also been associated with the fast pace of urbanization accompanied by poor infrastruc- ture. Health challenges included infectious diseases, such as malaria, in particular, in peripheral areas bordering forests (OPA 2010). Perhaps, the largest impact of envi- ronmental change can be noted in the case of migration. same for males and females. Within the Mekong delta region, Bac Lieˆu Province reported the highest out-mi- gration rates (-14.2 for males and -13.5 for females) (General Statistics Office 2013). Mortality Similar to migration trends in the Mekong delta region, internal migration in the Bangladeshi GBD is an important demographic and social phenomenon. Environmental shocks combined with the economic vulnerability of large strata of the society are the key push factors affecting relatively high out-migration rates in coastal districts. A recent report by UNDP pointed out that 40 out of 64 dis- tricts have been identified to be environmentally at risk (Marshall and Rahman 2013), and the delta as a whole is the most environmentally stressed of those we analyzed (Fig. 1). High out-migration, in particular, from rural locations contributes to creation of large slum areas and informal settlements in cities. Because of limited or absent income generating opportunities, migration to cities is often perceived as a coping strategy for the rural poor. Also, crop losses or damage caused by natural hazards further exacerbate the existing social vulnerabilities. In Bangladesh, the number of life time migrants increased from 950 9 103 in 1950 to 12,773 9 103 in 2004, and out- migration from rural to urban areas grew from 7.3 per 1000 in 1984 to 25.9 per 1000 in 2010 (BBS 2011b). As is the case widely in other delta regions, climate change, and in particular, sea level rise, are projected to have negative impact on households’ livelihoods, which is likely to increase the volume of out-migration in the future (De Souza et al. 2015; Mallick and Vogt 2012). Migration Migration is a key element of population change in all three study deltas, with the high environmental vulnera- bility of tropical deltas potentially being an important factor. At the same time, due to its unpredictability, migration is also the most difficult part of demographic modeling. Nonetheless, out-migration from three deltas as well as internal migration to the deltas’ megacities are likely to continue given unequal spatial development, biophysical transformations and environmental vulnera- bility (Seto 2011).The Mekong delta region is exposed to environmental hazards with extreme weather events lead- ing to frequent flooding which affects people’s livelihoods (Dun 2011; Nguyen and James 2013). Also, slow onset hazards such as salinity intrusion continue to pose a severe risk to water and soil quality, and thus, to water supply and agriculture. Climate change is likely to exacerbate the existing risks, and thus, further affect future population distribution. Out-migration from the environmentally vul- nerable areas is a widely recognized coping strategy (Rayhan 2008). Seasonal migration to the cities can pro- vide income during the times of distress. For example, in the Thfnh My¨ Taˆy commune in An Giang province, there were 5000 seasonal migrants reported in 2009 (Nguyen and James 2013). Between April 2012 and April 2013, there were a total of 121,443 out migrants from the Mekong delta region, with most of them moving to the Southeast region where Ho Chi Minh City is located (General Statistics Office 2013). As per the above-mentioned report, the net migration rates in the Mekong delta region are the Migration dynamics in the Amazon delta show inter- esting patterns. Based on the analysis of the data for the period 2001–2007 originating from the National Household Survey (PNAD), the state of Para´ has consistently experi- enced high levels of out-migration. In contrast, migration patterns in Amapa´ have been highly volatile with only some years showing net out-migration flows (Ferreira- Filho and Horridge 2010). Nevertheless, recent research reports that over 28 % of the current population in Amapa´ originates from outside of this state which may explain the rising trends in population growth (Viana et al. 2014). Another body of research highlights that in the Amazon delta, similar to other delta regions, migration dynamics are largely intertwined with urbanization. The difficulty of categorizing and quantifying these migration trends is related to the fact that a large proportion of migration includes circular movements (Padoch et al. 2008). Fertility As highlighted previously, during the past half-century, Vietnam has undergone rapid demographic transition with the TFR declining from 6.4 children per woman in 1960 to 123 123 549 Sustain Sci (2016) 11:539–554 1.8 children per woman in 2013 (World Bank 2015). Today, the unmet need for contraception is estimated at 4.3 % at the national level and 3.6 % for the Mekong delta region (General Statistics Office 2011b). Based on DHS data, it can be observed that since the 1990s, the TFR in the Mekong delta region has been continuously decreasing and is currently estimated at 1.92 (Fig. 7). In terms of early childbearing, the percentage of women aged 20–27 who had their first birth before the age of 18 is 5.8 % in the Mekong delta region, while the equivalent national pro- portion is 3 % (General Statistics Office 2011b). sterilization. In 1996, in North-eastern states, 51 % of married women between 15 and 49 reported having undergone sterilization (Caetano 2001), a trend confirmed for areas of the Northern region by Siqueira et al. (2007). These past trends indicate that future delta populations are likely to continue to significantly change and see smaller households with a greater proportion of elderly dependents. This predicted change is expected to be most rapid in the Mekong delta. Vietnam saw a decrease from an average household size of 4.8 in 1989 to 3.8 in 2009 (General Statistics Office 2011a), and in 2009, household size in the Mekong delta was estimated at 3.9 (Fig. 8). The trends toward smaller households are accompanied by a rising age at marriage and rising divorce rates. The ratios of divorce/separation to marriage are the highest in the Mekong delta and Southeast regions, which is likely to be associated with high rates of out-migration. Comparatively, in the GBD, there has been a trend toward smaller house- hold size, although there exists considerable inter divi- sional differences. In our study area, an average household size varies from 4.3 in Khulna to 5.4 in Sylhet (BBS 2011b). It should be highlighted that during the last decade, all five divisions which fall under the study area have seen a decrease in household size. In 2001, the average family size in the delta region was 5.1, while in 2010 in was 4.8 (BBS 2011b). Fertility Finally, in the Amazon delta region, this decline was even more rapid, although an average house- hold is larger than in the Mekong delta region. These changing population structures should be considered when assessing the consequences and implications of environ- mental change. Similar to the Mekong delta, analyzing trends in the total fertility rate in the GBD reveals a decline from 3.5 children per woman in 1993 to roughly below 2.5 in 2011 (Fig. 7). Fertility decline has been more pronounced for women from wealthier households than for women from poorer households. The average total fertility rates in the coastal divisions of Khulna and Barisal are below the national average except for Barisal district. The lowest fertility rates among the coastal districts are observed in Satkhira and Barguna districts with TFR 1.56 and 1.59, respectively (BBS 2012). Finally, since the early 1990s, the Amazon delta region has experienced the most rapid decline in TFR with a drop in TFR from over 6.1 children per women in 1991 to 3.3 children per woman in 2010. This fast decline in fertility mirrors the general trends in Brazil which is explained by a combination of government programs, migration from rural to urban areas, and social- cultural change regarding women’s rights and household roles (Siqueira et al. 2007). This trend started first in the urban areas of South and South East regions and then spread gradually to the rest of the country (Siqueira et al. 2007). A key feature of the process was the reliance on irreversible methods of contraception, such as female Conceptualizing direct links between environmental factors and fertility is more complex, albeit not impossible. As highlighted previously, an emerging body of evidence suggests that environmental pollutants may negatively affect fertility (Fisch et al. 2003; Foster 1998) and that the perceived risks related to climate change may be linked to Fig. 7 Recent trends in total fertility rate (TFR) in the Mekong, Ganges–Brahmaputra and Amazon delta regions Data sources: BBS, IBGE and General Statistics Office (GSO), Vietnam affect fertility (Fisch et al. 2003; Foster 1998) and that the perceived risks related to climate change may be linked to Fig. 7 Recent trends in total fertility rate (TFR) in the Mekong, Ganges–Brahmaputra and Amazon delta regions Data sources: BBS, IBGE and General Statistics Office (GSO), Vietnam Fig. Discussion and conclusions The increased population pressure on delta regions, toge- ther with the acceleration of urbanization and more intensive agricultural use, have magnified risks and exposure to relative sea level rise, flooding, and loss of ecosystem services. Furthermore, environmental factors exert a direct influence on population dynamics both over short (e.g. hurricanes and tropical storms) and mid/long (e.g. coastal retreat) term horizons. The complexity of the problem is compounded by the feedbacks between popu- lation and environmental factors in different socio-eco- nomic scenarios. O’Neill et al. (2015) described five basic Shared Socioeconomic Pathways (SSPs) to explore the possible outcomes in terms of mitigation and adaptation challenges related to climate change scenarios. The dif- ferent narratives are based on alternative plausible futures of societal development at global scale and provide a set of qualitative descriptors of projected changes, including changes in demographic futures and environmental sus- tainability. However, extending the application of that general framework to specific areas, such as deltas, necessitates the identification of the distinctive factors (demographic, environmental, etc.) that are relevant regionally. Thus, this study aimed at investigating the dynamics of population change and identifying the key environmental factors expected to affect population dynamics across deltas, in general, and the three delta regions, in particular: the Ganges–Brahmaputra, the Mekong and the Amazon deltas (as defined in ‘‘Materials and methods’’). Secondly, our findings suggest that all delta regions have been experiencing shifts in population structure resulting in aging populations, with the most rapid changes occurring in the Mekong delta. These expected shifts will comple- ment the changes in population distribution. Although at different rates, rural–urban population distribution is likely to continue to change across the three deltas as rural families seek employment opportunities and better services in urban areas. Migration to urban areas, however, do not represent a rupture with the rural, but tend to be marked by intense circulation of people and resources, and thus, are likely to continue to shape the economy and governance of rural sectors in the three deltas. Finally, while environ- mental impacts on population trends and dynamics in the delta regions are hardly contestable, they remain difficult to quantify. Future research should therefore consider ways in which these integrated systems and associations could be analyzed and modeled (Lazar et al. 2015; Nicholls et al. 2013). The results of the analysis presented in this study nev- ertheless have important policy implications. Fertility 8 Change in mean household size across the three study areas Data sources: BBS, IBGE and GSO, Vietnam 1 3 Fig. 8 Change in mean household size across the three study areas Data sources: BBS, IBGE and GSO, Vietnam Fig. 7 Recent trends in total fertility rate (TFR) in the Mekong, Ganges–Brahmaputra and Amazon delta regions Data sources: BBS, IBGE and General Statistics Office (GSO), Vietnam Fig. 8 Change in mean household size across the three study areas Data sources: BBS, IBGE and GSO, Vietnam 12 3 Sustain Sci (2016) 11:539–554 550 couples’ fertility intensions (Arnocky et al. 2012). Also, fertility and family size can have direct and indirect effects on natural habitat and environmental and climate change at macro, meso and possibly micro levels. At the macro level, the most intuitive link is that through population growth, increased consumption and hence greater pollution and CO2 emissions. This link can operate at the local and regional level. For example, in the urban areas of GBD, including the megacity of Dhaka, rapid population growth has been associated with environmental degradation; including wetland vegetable cover reduction and frag- mentation of landscape (Dewan et al. 2012). Given the changing fertility rates across the three study areas, shifts in population structure and projected impacts of climate change, it is crucial that further research is undertaken on the interlinkages between fertility and environmental fac- tors in climate hotspots, such as delta regions. of population change: fertility, mortality, and migration. The results of the analysis are summarized in Table 2, which provides an overview of key population dynamics, environmental factors and policy implications for each delta region. This summary draws from the conceptual frame- work presented in ‘‘Feedbacks between population dynamics and environmental change’’. While there are considerable differences in terms of environmental and population change across the three deltas, several conclu- sions applicable to all three delta regions can be drawn. Firstly, our results suggest that, consistent with national averages, fertility rates in the delta regions have been steadily declining, falling below replacement levels in some geographical areas, such the Khulna and Barisal districts in south-western Bangladesh. This trend, combined with increasing life expectancy, improving child mortality rates and migration dynamics imply that in the longer term population in coastal delta regions are likely to stabilize or even decline, as recently projected for several districts in south-west Bangladesh (Szabo et al. 2015a, b, c). Fertility This differs from general expectations of expanding coastal populations and coastal cities (Neumann et al. 2015), and hence, is worthy of investigation in other deltas. Discussion and conclusions Coupled with out-migration, these trends will require the development of specific policies to tackle potential shifting from labor surplus to labor shortages in To examine the dynamics of population change across three delta regions, we analyzed the trends in population growth, population structure and three specific components 123 123 551 Sustain Sci (2016) 11:539–554 Table 2 Overview of important population change and environmental factors in each delta, and implications for development of sustainable management policies Environmental threats that could affect demographic change Timing/duration of impact Population dynamics FloodingGB,M,A Sea level riseGB,M,A Land subsidenceGB,M Cyclones and storm surgeGB,A(M) Soil salinizationGB Water salinizationGB,M,A Upstream impacts of climate change and dams/catchment management on river hydrologyGB,M,A Immediate—MT–LT LT MT–LT Immediate LT LT MT–LT Population growth and size Population growthGB,M,A Shifts in population structure (including population aging)GB,M,A Fertility Declining (sometimes below replacement) fertilityGB,M,A Mortality Gains in life expectancyGB,M,A Improvements in maternal and child healthGB,M,A Migration High out-migrationGB,M Socio-demographic change Declining household sizeGB,M,A Rapid urbanisationGB,M,A Policy implications Investments in rapid emergency response programmes and policies Social protection schemes in view of changing population structures, including population aging Policies aiming at tackling the drivers and consequences of changing and unequal population distribution Region specific rural development and urban planning policies tailored to local contexts Policies aimed at improving sanitation infrastructure and treatment particularly in urban areas Addressing potential labor shortages in the farming sector Investments in collection and analysis of desegregated demographic, socio-economic and environmental data Delta development plans MT medium term, LT long term, GB Ganges–Brahmaputra, M Mekong, A Amazon Table 2 Overview of important population change and environmental factors in each delta, and implications for management policies Policy implications Policy implications y p Investments in rapid emergency response programmes and policies Social protection schemes in view of changing population structures, including population aging Policies aiming at tackling the drivers and consequences of changing and unequal population distribution Region specific rural development and urban planning policies tailored to local contexts Policies aimed at improving sanitation infrastructure and treatment particularly in urban areas Addressing potential labor shortages in the farming sector Investments in collection and analysis of desegregated demographic, socio-economic and environmental data Delta development plans MT medium term, LT long term, GB Ganges–Brahmaputra, M Mekong, A Amazon narratives, enriched by the specific characteristics of del- tas, would improve the framework to assess mitigation and adaptation challenges. certain sectors and geographical areas. 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Discussion and conclusions doi:10.1016/S0140-6736(13)62148-0 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://crea tivecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://crea tivecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Clarke D, Williams S, Jahiruddin M, Parks K, Salehin M (2015) Projections of on-farm salinity in coastal Bangladesh. Environ Sci Process Impacts 17(6):1127–1136. doi:10.1039/c4em00682h Dang LH, Li E, Nuberg I, Bruwer J (2014) Perceived risks of climate change and influencing factors: a study in the Mekong Delta Vietnam. Environ Manag. doi:10.1007/s00267-014-0299-6 de Resende Londe L, Coutinho MP, Di Grego´rio LT, Lima Santos LB, Soriano E (2014) Water-related disasters in Brazil: perspec- tives and recommendations. Ambient Soc 17(4):133–152. doi:10. 1590/1809-4422ASOC1082V1742014 Discussion and conclusions Additionally, envi- ronmental stressors, such as relative sea level rise, could entail large population displacements, including across national borders (Smajgl and Ward 2013). With an aging population, disaster preparedness programs will need to be appropriately adjusted to reflect the changing population needs during disasters. The study presented here is unusual in looking at pop- ulation dynamics across a set of deltas. More studies of this type are required to better characterize deltas and their populations around the world. In particular, focused research is urgently needed to further disentangle popula- tion–environment associations, through stakeholder engagement initiatives, targeted data collection, and dynamic/statistical modeling. Changes in occupational structure are also likely to be linked to internal migration and resulting urban growth. Addressing potential labor shortages in the farming sector is likely to be a challenge given that delta regions are food baskets for many nations (Foufoula-Georgiou et al. 2013), thus potentially contributing to reducing global food in security. Given the key role of demographic and envi- ronmental issues for sustainability of tropical deltas, as well as their wider interlinkages with other factors affecting sustainable development, such as public health and good governance, it is critical to undertake thorough assessments of specific population-environment dynamics on a case-by-case basis. Thus, locally informed SSP Acknowledgments The research forms part of the international Belmont Forum project BF-DELTAS ‘‘Catalyzing action toward sustainability of deltaic systems with an integrated modeling frame- work for risk assessment’’. Sylvia Szabo, Zoe Matthews, Robert Nicholls and John Dearing also acknowledge support from the project ‘Assessing health, livelihoods, ecosystem services and poverty alle- viation in populous deltas’ (NE/J002755/1) funded by the Ecosystem Services for Poverty Alleviation Programme (ESPA). Eduardo S. Brondizio acknowledge support from the United States National Science Foundation to Indiana University (NSF # 1342898). The UK 12 3 552 Sustain Sci (2016) 11:539–554 ESPA programme is funded by the Department for International Development, Economic and Social Research Council (ESRC) and the Natural Environment Research Council (NERC). water, sanitation and infant, child, and maternal mortality. Environ Health 11:1–8. doi:10.1186/1476-069x-11-4 Chowdhury AMR, Bhuiya A, Chowdhury ME, Rasheed S, Hussain Z, Chen LC (2013) The Bangladesh paradox: exceptional health achievement despite economic poverty. Lancet 382(9906): 1734–1745. References Retrieved on 19 Feb 2015 Haque U, Hashizume M, Kolivras KN, Overgaard HJ, Das B, Yamamoto T (2012) Reduced death rates from cyclones in Bangladesh: what more needs to be done? 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https://openalex.org/W4214817856
https://vestnik.guu.ru/jour/article/download/3319/2264
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Tax policy impact on shadow economy
Vestnik universiteta
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cc-by
5,285
Экономика: проблемы, решения и перспективы Экономика: проблемы, решения и перспективы TAX POLICY IMPACT ON SHADOW ECONOMY Avgustina S. Yakovleva Assistant, Chuvash State University named after I.N. Ulyanov, Cheboksary, Russia ORCID: 0000-0003-4879-0091 e-mail: avgserg@yandex.ru Abstract. The article examines the relationship between tax policy and the shadow economy. The tax burden was chosen as a tax policy indicator; the shadow economy is assessed using the Rosstat methodology. When comparing the tax burden in the country as a whole and the shadow economy volumes, a decrease in the shadow economy indicators was noted with a rela­ tively high tax burden. When comparing the shadow economy and the tax burden by econom- ic activity types, an insignificant relationship between the studied indicators was revealed, however, in some sectors of the economy, a feedback was noted. The introduction of a tax on professional income has led to an increase in the number of self-employed. However, the share of the self-employed in the number of those employed in the informal sector in the Volga Federal District showed a high heterogeneity across regions. The conclusion was drawn about the influence of many factors on the shadow economy, as a result of which the use of only fis- cal policy instruments to reduce the shadow sector seems insufficient. Elena G. Lyubovtseva Cand. Sci. (Econ.), Chuvash State University named after I.N. Ulyanov, Cheboksary, Russia ORCID: 0000-0003-2408-1442 e-mail: lyubovceva_elena@mail.ru Elena G. Lyubovtseva Cand. Sci. (Econ.), Chuvash State University named after I.N. Ulyanov, Cheboksary, Russia ORCID: 0000-0003-2408-1442 e-mail: lyubovceva_elena@mail.ru Keywords: tax policy, shadow economy, legal entities’ shadow transactions, informal sector, informal sector operations, tax burden, self-employed workers For citation: Yakovleva A.S., Lyubovtseva E.G. (2022) Tax policy impact on shadow economy. Vestnik universiteta, no. 1, pp. 109–116. DOI: 10.26425/1816-4277-2022-1-109-116 DOI 10.26425/1816-4277-2022-1-109-116 ВЛИЯНИЕ НАЛОГОВОЙ ПОЛИТИКИ НА ТЕНЕВУЮ ЭКОНОМИКУ УДК 336.02 JEL H26, H30, O17 Яковлева Августина Сергеевна ассистент, ФГБОУ ВО «Чувашский государственный университет им. И.Н. Ульянова», г. Чебоксары, Российская Федерация ORCID: 0000-0003-4879-0091 e-mail: avgserg@yandex.ru Любовцева Елена Геннадьевна канд. экон. наук, ФГБОУ ВО  «Чувашский государственный универ- ситет им. И.Н. Ульянова», г. Чебоксары, Российская Федерация ORCID: 0000-0003-2408-1442 e-mail: lyubovceva_elena@mail.ru УДК 336.02 JEL H26, H30, O17 Яковлева Августина Сергеевна ассистент, ФГБОУ ВО «Чувашский государственный университет им. И.Н. Ульянова», г. Чебоксары, Российская Федерация ORCID: 0000-0003-4879-0091 e-mail: avgserg@yandex.ru УДК 336.02 JEL H26, H30, O17 Яковлева Августина Сергеевна ассистент, ФГБОУ ВО «Чувашский государственный университет им. И.Н. Ульянова», г. Чебоксары, Российская Федерация ORCID: 0000-0003-4879-0091 e-mail: avgserg@yandex.ru Аннотация. В статье изучается связь между налоговой политикой и теневой экономи- кой. В качестве показателя налоговой политики выбрана налоговая нагрузка, теневая экономика оценивается по методике Росстата. При сопоставлении налоговой нагрузки в целом по стране и объемов теневой экономики отмечено снижение показателей те- невой экономики при относительно высокой налоговой нагрузке. При сравнении теневой экономики и налоговой нагрузки по видам экономической деятельности обнаружена не- значимая связь между изучаемыми показателями, однако по некоторым отраслям эко- номики отмечена обратная связь. Введение налога на профессиональный доход приве- ло к увеличению числа самозанятых. Однако доля самозанятых в численности занятых в неформальном секторе по Приволжскому федеральному округу показала высокую не- однородность по регионам. Сделан вывод о влиянии на теневую экономику множест- ва факторов, вследствие чего применение только инструментов фискальной политики для сокращения теневого сектора видится недостаточным. Любовцева Елена Геннадьевна канд. экон. наук, ФГБОУ ВО  «Чувашский государственный универ- ситет им. И.Н. Ульянова», г. Чебоксары, Российская Федерация ORCID: 0000-0003-2408-1442 e-mail: lyubovceva_elena@mail.ru Ключевые слова: налоговая политика, теневая экономика, теневые операции юридических лиц, неформальный сектор, операции неформального сектора, налоговая нагрузка, самозанятые работники Ключевые слова: налоговая политика, теневая экономика, теневые операции юридических лиц, неформальный сектор, операции неформального сектора, налоговая нагрузка, самозанятые работники Для цитирования: Яковлева А.С., Любовцева Е.Г. Влияние налоговой политики на теневую экономику//Вестник университета. 2022. № 1. С. 109–116. Avgustina S. Yakovleva Assistant, Chuvash State University named after I.N. Ulyanov, Cheboksary, Russia ORCID: 0000-0003-4879-0091 e-mail: avgserg@yandex.ru Avgustina S. Yakovleva Assistant, Chuvash State University named after I.N. Ulyanov, Cheboksary, Russia ORCID: 0000-0003-4879-0091 e-mail: avgserg@yandex.ru Elena G. Lyubovtseva Cand. Sci. (Econ.), Chuvash State University named after I.N. Ulyanov, Cheboksary, Russia ORCID: 0000-0003-2408-1442 e-mail: lyubovceva_elena@mail.ru © Яковлева А.С., Любовцева Е.Г., 2022. Статья доступна по лицензии Creative Commons «Attribution» («Атрибуция») 4.0. всемирная (http://creativecommons.org/licenses/by/4.0/). © Yakovleva A.S., Lyubovtseva E.G., 2022. This is an open access article under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/). © Яковлева А.С., Любовцева Е.Г., 2022. Статья доступна по лицензии Creative Commons «Attribution» («Атрибуция») 4.0. всемирная (http://creativecommons.org/licenses/by/4.0/). © Yakovleva A.S., Lyubovtseva E.G., 2022. This is an open access article under the CC BY 4 0 license (http://creativecommons org/licenses/by/4 0/) © Яковлева А.С., Любовцева Е.Г., 2022. на по лицензии Creative Commons «Attribution» («Атрибуция») 4.0. всемирная (http://creativecommons.org/licenses/by/4.0/). S., Lyubovtseva E.G., 2022. access article under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/). Avgustina S. Yakovleva Assistant, Chuvash State University named after I.N. Ulyanov, Cheboksary, Russia ORCID: 0000-0003-4879-0091 e-mail: avgserg@yandex.ru Введение Теневая экономика оказывает в большей части негативное влияние на экономическую систему в целом. Скрытая, неформальная и нелегальная экономические деятельности приводят к получению доходов узким кругом лиц без их участия в формировании доходов бюджетов бюджетной системы. Вследствие этого участ- ники теневой деятельности пользуются общественными благами безвозмездно. Нелегальная деятельность нарушает экономическую безопасность страны. Поэтому для контроля масштабов теневой экономики необходимо выявить факторы, оказывающие влияние на ее объемы. Одним из таких факторов выступает налоговая политика. «Налоговая политика 109 Вестник университета № 1, 2022 призвана: обеспечивать формирование финансовых ресурсов, необходимых государству для выполнения взятых на себя функций; оказывать регулирующее воздействие на процессы социально-экономического развития страны» [3, c. 29]. призвана: обеспечивать формирование финансовых ресурсов, необходимых государству для выполнения взятых на себя функций; оказывать регулирующее воздействие на процессы социально-экономического развития страны» [3, c. 29]. Цель статьи – выявление связи между налоговой политикой и теневой экономикой. Для этого проанализирова- но влияние налоговой нагрузки на теневую экономику России в целом и по отраслям народного хозяйства, рассмо- трены результаты внедрения налога на профессиональный доход и его влияние на масштабы теневой экономики. Литературный обзор и на пла- новый период 2023 и 2024 годов» предусмотрены следующие мероприятия по сокращению теневого сектора: – «развитие национальной системы прослеживаемости товаров, обеспечивающей контроль за оборо- том товаров на всех этапах от ввоза до реализации в рознице; – «развитие национальной системы прослеживаемости товаров, обеспечивающей контроль за оборо- том товаров на всех этапах от ввоза до реализации в рознице; – формирование правовых основ для внедрения таможенного мониторинга; – запуск нового налогового режима упрощенной система налогообложения (УСН); – онлайн для микропредприятий с численностью сотрудников до 5 человек; – введение института «единого налогового платежа»; формирование правовых основ для внедрения таможенного мониторинга; – запуск нового налогового режима упрощенной система налогообложения (УСН); – онлайн для микропредприятий с численностью сотрудников до 5 человек; – введение института «единого налогового платежа»; – запуск нового налогового режима упрощенной система налогообложения (УСН); – онлайн для микропредприятий с численностью сотрудников до 5 человек; – введение института «единого налогового платежа»; – повышение качества администрирования акцизов на табак» [2]. Литературный обзор Большинство экономистов считает, что высокая налоговая нагрузка способствует росту теневой эконо- мики [5–7]. Ф. Шнайдер и Д. Энст полагают, что низкие налоговые ставки, меньшее количество норматив- ных документов, требующих соблюдения, развитые институты способствуют формированию теневой эко- номики в значительно меньших масштабах [15]. Однако есть и прямо противоположные исследования. Так, Д. Ю. Федотов провел корреляционный анализ между объемами теневой экономики и показателями налого- вой системы 120 стран на основе данных Всемирного банка и выявил, что между теневой экономикой и на- логовой нагрузкой наблюдается обратная зависимость [8, с. 13]. В. Г. Пансков предполагает, что величина налоговой нагрузки занижается в странах с высоким уровнем теневизации экономики вследствие недопо- ступления налогов от теневого сектора [6, с. 83]. Сторонники снижения налоговой нагрузки в целях уменьшения теневой экономики допускают, что данное мероприятие сподвигнет предпринимателей выйти из теневой экономики, но «вовсе не вынуждает их к этому». С другой стороны, наличие чрезмерной налоговой нагрузки, значительного количества нор- мативных документов способствует развитию коррупции, нелегальной деятельности чиновников, налого- вых и правоохранительных органов, из-за чего все государственные меры по снижению теневого сектора являются бессмысленными [4, с. 36]. В «Основных направлениях бюджетной, налоговой и таможенно-тарифной политики на 2022 г. и на пла- новый период 2023 и 2024 годов» предусмотрены следующие мероприятия по сокращению теневого сектора: В «Основных направлениях бюджетной, налоговой и таможенно-тарифной политики на 2022 г. и на пла- новый период 2023 и 2024 годов» предусмотрены следующие мероприятия по сокращению теневого сектора: – «развитие национальной системы прослеживаемости товаров, обеспечивающей контроль за оборо- том товаров на всех этапах от ввоза до реализации в рознице; В «Основных направлениях бюджетной, налоговой и таможенно-тарифной политики на 2022 г. Основные результаты Проанализируем, существует ли связь между налоговой нагрузкой и теневой экономикой (табл. 1). ц Динамика налоговой нагрузки и объемов теневой экономики России в 2002–2019 гг., % к валовому внутреннему продукту в рыночных ценах Год Теневые операции юридических лиц Операции неформального сектора экономики и производ- ство домашних хозяйств для собственного конечного использования Экономические опера- ции, не наблюдаемые прямыми статистиче- скими методами Налоговая нагрузка 2002 13,0 11,7 24,7 30,0 2003 13,7 9,9 23,7 28,7 2004 7,5 9,7 17,2 29,9 2005 7,5 9,6 17,1 30,0 110 Экономика: проблемы, решения и перспективы Окончание табл. 1 Год Теневые операции юридических лиц Операции неформального сектора экономики и производ- ство домашних хозяйств для собственного конечного использования Экономические опера- ции, не наблюдаемые прямыми статистиче- скими методами Налоговая нагрузка 2006 7,4 9,4 16,8 28,9 2007 7,4 8,9 16,4 30,8 2008 6,0 8,0 14,1 28,2 2009 6,6 9,4 16,0 25,5 2010 5,6 5,5 11,1 25,7 2011 6,6 4,7 11,3 27,2 2012 6,1 4,5 10,6 28,0 2013 4,4 9,6 14,0 25,1 2014 4,4 9,2 13,6 25,4 2015 4,2 8,8 13,0 26,7 2016 4,4 8,7 13,1 27,4 2017 4,3 8,3 12,6 29,4 2018 4,0 7,6 11,7 30,9 2019 3,9 7,6 11,6 31,6 Источники: [11; 13] Окончание табл. 1 В качестве измерителя теневой экономики будем использовать экономические операции, не наблюдае- мые прямыми статистическими методами, оцениваемые Росстатом в ходе уточнения объемов валового вну- треннего продукта (далее – ВВП). Они включают в себя теневые операции юридических лиц и операции неформального сектора экономики и производство домашних хозяйств для собственного конечного исполь- зования. К теневым операциям юридических лиц относится осуществление законных видов деятельности, но скрытых «от органов государственной власти в целях уклонения от уплаты налогов или следования за- конодательным нормам». Производство неформального сектора – это производственная деятельность отно- сительно небольших предприятий сектора домашних хозяйств, которая в силу недостаточного количества занятых официально не зарегистрирована и имеет «какое-либо рыночное производство» [11, с. 89]. Неле- гальная экономическая деятельность не оценивается при подсчете валового внутреннего продукта. Налоговая нагрузка будет определена как процентное отношение налоговых доходов консолидирован- ного бюджета Российской Федерации к валовому внутреннему продукту в рыночных ценах. Налоговая нагрузка будет определена как процентное отношение налоговых доходов консолидирован- ного бюджета Российской Федерации к валовому внутреннему продукту в рыночных ценах. Налоговые доходы бюджета имеют прямую линейную связь с ВВП, коэффициент корреляции по дан- ным с 2002 г. по 2019 г. составляет 0,99. Что свидетельствует о росте налоговых поступлений в периоды ро- ста деловой активности и уменьшении при спаде экономики. Основные результаты В целом налоговые поступления меняются бо- лее плавно, чем ВВП, что может повлиять на колебания налоговой нагрузки. Налоговые доходы бюджета имеют прямую линейную связь с ВВП, коэффициент корреляции по дан- ным с 2002 г. по 2019 г. составляет 0,99. Что свидетельствует о росте налоговых поступлений в периоды ро- ста деловой активности и уменьшении при спаде экономики. В целом налоговые поступления меняются бо- лее плавно, чем ВВП, что может повлиять на колебания налоговой нагрузки. В течение изучаемого периода налоговая нагрузка составляет в среднем 28,3 %. В 2002–2008 гг. налого- вая нагрузка колебалась от 28,2 до 30,8 %. В 2009–2010 гг. и 2013–2014 гг. наблюдаются наименьшие значе- ния налоговой нагрузки от 25,1 до 25,7 %. С 2015 г. по 2019 г. фиксируется рост налоговой нагрузки. В течение изучаемого периода налоговая нагрузка составляет в среднем 28,3 %. В 2002–2008 гг. налого- вая нагрузка колебалась от 28,2 до 30,8 %. В 2009–2010 гг. и 2013–2014 гг. наблюдаются наименьшие значе- ния налоговой нагрузки от 25,1 до 25,7 %. С 2015 г. по 2019 г. фиксируется рост налоговой нагрузки. Объемы теневой экономики, в отличие от налоговой нагрузки, практически с каждым годом снижают- ся. Причем теневые операции юридических лиц за 2002–2019 гг. сократились на 9,1 п. п., операции нефор- мального сектора экономики и производство домашних хозяйств для собственного конечного использова- ния – на 4,1 п. п., теневая экономика – на 13,1 п. п. Объемы теневой экономики, в отличие от налоговой нагрузки, практически с каждым годом снижают- ся. Причем теневые операции юридических лиц за 2002–2019 гг. сократились на 9,1 п. п., операции нефор- мального сектора экономики и производство домашних хозяйств для собственного конечного использова- ния – на 4,1 п. п., теневая экономика – на 13,1 п. п. Значительное снижение теневых операций юридических лиц могло быть связано либо с более эффек- тивной работой налоговых и правоохранительных органов, либо усовершенствованием применяемых мето- дик для сокрытия теневой деятельности. При сохранении высокой налоговой нагрузки наблюдается сниже- ние масштабов теневых операций, регистрируемых Росстатом. 111 Вестник университета № 1, 2022 Таблица 2 Таблица 2 Налоговая нагрузка и объемы теневой экономики по видам экономической деятельности за 2019 г. Основные результаты Вид экономической деятельности Удельный вес теневых опе- раций юриди- ческих лиц, % к ВДС от- расли Удельный вес операций неформального сектора экономики и производ- ства домашних хозяйств для собственного конеч- ного использования, % к ВДС отрасли Удельный вес экономиче- ских операций, не наблюдае- мых прямыми статистически- ми методами, % к ВДС отрасли Налоговая нагрузка, % Отрасли с низким уровнем теневой экономики Обеспечение электрической энерги- ей, газом и паром; кондиционирова- ние воздуха 0,0 0,0 0,0 7,1 Добыча полезных ископаемых 0,6 0,0 0,6 41,4 Водоснабжение, водоотведение, ор- ганизация сбора и утилизации отхо- дов, деятельность и ликвидация за- грязнений 0,6 0,2 0,8 9,9 Деятельность в области информа- ции и связи 1,7 0,0 1,7 17,2 Транспортировка и хранение 3,0 1,3 4,2 6,4 Обрабатывающие производства 4,5 0,3 4,8 7,6 Отрасли с высоким уровнем теневой экономики Торговля оптовая и розничная; ре- монт автотранспортных средств и мо- тоциклов 9,4 0,8 10,2 2,8 Деятельность административная и со- путствующие дополнительные услуги 13,4 0,7 14,1 17,9 Деятельность гостиниц и предприя- тий общественного питания 15,8 0,2 15,9 10,5 Строительство 9,3 7,0 16,4 11,9 Сельское, лесное хозяйство, охота, рыболовство, рыбоводство 1,9 31,8 33,7 4,5 Деятельность по операциям с недви- жимым имуществом 8,7 60,1 68,8 25,0 Источник: [1; 11] Налоговая нагрузка и объемы теневой экономики по видам экономической деятельности за 2019 г. Проанализируем изменение налоговой нагрузки и теневой экономики по видам экономической деятель- ности (табл. 2). К отраслям с низким уровнем теневой экономики отнесем те виды экономической деятель- ности, уровень теневизации которых не превышает 5 % валовой добавленной стоимости данной отрасли. К отраслям с высоким уровнем теневой экономики включим все остальные виды экономической деятель- ности. Налоговая нагрузка по видам экономической деятельности определена как результат деления собран- ных усилиями Федеральной налоговой службы налогов и сборов на оборот организаций. Налоговая нагрузка по видам экономической деятельности достаточно неоднородна. Сильная дифферен- циация видов экономической деятельности по налоговой нагрузке увеличивает зависимость бюджета от кон- кретных отраслей народного хозяйства. Среди отраслей с низким уровнем теневой экономики присутствуют виды деятельности с высокой налоговой нагрузкой, среди отраслей с высоким уровнем теневой экономики Налоговая нагрузка по видам экономической деятельности достаточно неоднородна. Сильная дифферен- циация видов экономической деятельности по налоговой нагрузке увеличивает зависимость бюджета от кон- кретных отраслей народного хозяйства. Основные результаты Среди отраслей с низким уровнем теневой экономики присутствуют виды деятельности с высокой налоговой нагрузкой, среди отраслей с высоким уровнем теневой экономики 112 Экономика: проблемы, решения и перспективы есть отрасли с низкой налоговой нагрузкой, также есть виды деятельности, сочетающие в себе значительный уровень теневой экономики и налоговой нагрузки и наоборот. Коэффициенты корреляции налоговой нагруз- ки с показателями теневой экономики по видам экономической деятельности имеют следующие значения: с теневыми операциями юридических лиц – -0,08; с операциями неформального сектора экономики – 0,17; с экономическими операциями, не наблюдаемыми прямыми статистическими методами – 0,14. Однако по- лученные значения оказались незначимыми по критерию Стьюдента. Это объяснимо тем, что налоговая по- литика является не единственным фактором, оказывающим влияние на масштабы теневого сектора. Сфор- мировавшиеся политические и экономические институты определяют, какой траекторией будет происходить развитие страны, создают разные стимулы для субъектов экономики. И не всегда курс, выбранный высши- ми элитами, ведет к процветанию всего общества. В отраслях с низким уровнем теневой экономики существуют большие барьеры для входа на рынок в виде первоначальных затрат на приобретение факторов производства, вследствие чего здесь имеют место низкие объемы экономических операций, не наблюдаемых прямыми статистическими методами. В добыче полезных ископаемых наблюдается обратная связь между уровнем теневизации и налоговой нагрузкой, максимальная налоговая нагрузка 41,4% сочетается с низким уровнем теневой экономики в размере 0,6 %. Факт присутствия теневой деятельности в данной отрасли можно объяснить тем, что чрезмерное налоговое давление стимулирует искать пути для оптимизации налоговых платежей. По дан- ным формы 1-НОМ Федеральной налоговой службы России, в структуре уплачиваемых налогов, сбо- ров и страховых взносов по рассматриваемому виду экономической деятельности в 2019 г. преоблада- ют налоги и сборы за пользование природными ресурсами и составляют 77 % [12]. В остальных видах деятельности, характеризующихся низким уровнем теневой экономики, доминируют в основном на- лог на добавленную стоимость, налог на прибыль организаций и страховые взносы. В обрабатываю- щих производствах, помимо перечисленных налогов, высока роль акцизов. Страховые взносы во всех видах экономической деятельности, за исключением добычи полезных ископаемых, составляют от 19 до 54 % фискальных платежей. В отраслях с высоким уровнем теневой экономики структура уплачиваемых налогов, сборов и стра- ховых взносов имеет отличия. Здесь преобладают НДС, налог на доходы физических лиц, страховые взносы, но есть отрасли, где также высока роль налога на прибыль организаций. К тому же в отраслях с высоким уровнем теневой экономики в 2019 г. было сосредоточено почти 43 % занятых. Основные результаты Проблема высоких налогов на труд поднимается и на высших уровнях управления, однако, она до сих пор не на- шла разрешения. Низкая налоговая нагрузка в сельскохозяйственной и торговой отраслях привлекает не только добросо- вестных экономических субъектов, но и субъектов теневой экономики, которые наблюдая низкие барьеры для входа на рынок, стремятся организовать свою деятельность и заработать прибыль, применяя незакон- ные способы уклонения от уплаты налогов и экономя на НДФЛ и страховых взносах. Самый высокий уровень теневой экономики в размере 68,8 % зафиксирован в деятельности по опе- рациям с недвижимым имуществом несмотря на то, что по налоговой нагрузке данный вид деятельнос- ти занимает второе место после добычи полезных ископаемых. Эта отрасль опровергает предположение о том, что высокая налоговая нагрузка в отрасли сопровождается меньшим объемом теневой экономики. Разрастание теневой экономики в этой отрасли связано с большими затратами на контроль операций сдачи имущества в аренду со стороны налоговых органов. Хотя в последнее время формирующее граждан- ское поведение со стороны соседей, управляющих компаний приводит к выявлению случаев сдачи имуще- ства в аренду. Кроме того, создание института самозанятых позволяет арендодателям платить налог с полу- ченного дохода в размере 4 % вместо 13 % и стимулирует выйти из теневого сектора. Несмотря на льготную ставку, отсутствие отчетов и деклараций, простую регистрацию и другие упро- щения по налогу на профессиональный доход доля самозанятых в численности занятых в неформальном секторе по регионам Приволжского федерального округа достаточно неравномерна (табл. 3). Наиболее вы- дающиеся результаты достигнуты в Республике Татарстан, где доля самозанятых в численности занятых в неформальном секторе составила 25,7 %. 113 Вестник университета № 1, 2022 Таблица 3 Динамика численности самозанятых и занятых в неформальном секторе в Приволжском федеральном округе в 2020 г. Регионы Численность самозанятых на 31.12.2020 Численность занятых в неформальном секторе за 2020 г. Основные результаты Доля самозаня- тых в численно- сти занятых в неформальном секторе, % всего, человек удельный вес, % всего, человек удельный вес, % Республика Башкортостан 31 824 11,3 432 001 14,4 7,4 Республика Марий Эл 2 024 0,7 63 255 2,1 3,2 Республика Мордовия 2 413 0,9 70 044 2,3 3,4 Республика Татарстан 102 624 36,5 399 153 13,3 25,7 Удмуртская Республика 8 099 2,9 138 932 4,6 5,8 Чувашская Республика 6 324 2,3 157 749 5,2 4,0 Кировская область 5 175 1,8 152 028 5,1 3,4 Нижегородская область 29 483 10,5 311 115 10,3 9,5 Оренбургская область 8 367 3,0 225 407 7,5 3,7 Пензенская область 4 995 1,8 161 570 5,4 3,1 Пермский край 23 734 8,5 197 211 6,6 12,0 Самарская область 39 998 14,2 279 457 9,3 14,3 Саратовская область 10 078 3,6 325 189 10,8 3,1 Ульяновская область 5 734 2,0 94 528 3,1 6,1 Приволжский федеральный округ 280 872 100,0 3 007 638 100,0 9,3 Источники: [9; 14] Динамика численности самозанятых и занятых в неформальном секторе в Приволжском федеральном округе в 2020 г. В состав занятых в неформальном секторе включаются «граждане, занимающиеся предпринимательской деятельностью без образования юридического лица или на индивидуальной основе, независимо от того, име- ют ли они или не имеют государственную регистрацию в качестве предпринимателя; лица, занятые оказанием платных услуг по дому (горничные, сторожа, водители, гувернантки, няни, домашние повара, домашние се- кретари и т. п.), независимо от того, рассматриваются ли они как наемные работники или как самостоятель- но занятые; лица, работающие по найму у физических лиц, индивидуальных предпринимателей и т. д.» [10]. Хотя Росстат не относит занятых в неформальном секторе к теневой экономике, но в деятельности пред- приятий неформального сектора может присутствовать и теневая составляющая. Наличие занятых в нефор- мальном секторе свидетельствует о том, что население в силу недостаточности знаний, умений и навыков или отсутствия в достаточном количестве рабочих мест не может реализовать себя в формальном секторе. Если удельный вес занятых в неформальном секторе по регионам колеблется от 2,1 % до 14,4 %, то удель- ный вес самозанятых – от 0,7 % до 36,5 %. Это свидетельствует о том, что население в регионах в разной степени готово выйти из теневого сектора. Заключение 7, № 4 (25). – С. 194–202. 7. Трусова, Н. С. Мировой опыт выявления и противодействия уклонению от уплаты налогов // Известия Юго-Западного государственного университета. Серия: Экономика. Социология. Менеджмент. – 2017. – Т. 7, № 4 (25). – С. 194–202. 8. Федотов, Д. Ю. Влияние налоговой системы на теневую экономику // Финансы и кредит. – 2015. – № 41 (665). – С. 10–21. государственного университета. Серия: Экономика. Социология. Менеджмент. – 2017. – Т. 7, № 4 (25). – С. 194–202. 8. Федотов, Д. Ю. Влияние налоговой системы на теневую экономику // Финансы и кредит. – 2015. – № 41 (665). – С. 10–21. 9. Итоги выборочного обследования рабочей силы // Федеральная служба государственной статистики [Электронный ресурс]. – Режим доступа: https://rosstat.gov.ru/compendium/document/13265 (дата обращения: 03.11.2021). 8. Федотов, Д. Ю. Влияние налоговой системы на теневую экономику // Финансы и кредит. – 2015. – № 41 (665). – С. 10–21. 9. Итоги выборочного обследования рабочей силы // Федеральная служба государственной статистики [Электронный ресурс] Режим доступа: https://rosstat gov ru/compendium/document/13265 (дата обращения: 03 11 2021) 8. Федотов, Д. Ю. Влияние налоговой системы на теневую экономику // Финансы и кредит. – 2015. – № 41 (665). – С. 10–21. 9. Итоги выборочного обследования рабочей силы // Федеральная служба государственной статистики [Электронный ресурс]. – Режим доступа: https://rosstat.gov.ru/compendium/document/13265 (дата обращения: 03.11.2021). 10. Методологические положения по измерению занятости в неформальном секторе экономики // Федеральная служба государственной статистики [Электронный ресурс]. – Режим доступа: https://www.gks.ru/bgd/free/b99_10/isswww.exe/ stg/d030/i030150r.htm (дата обращения: 03.11.2021). 10. Методологические положения по измерению занятости в неформальном секторе экономики // Федеральная служба государственной статистики [Электронный ресурс]. – Режим доступа: https://www.gks.ru/bgd/free/b99_10/isswww.exe/ stg/d030/i030150r.htm (дата обращения: 03.11.2021). 11. Национальные счета России в 2013–2020 годах: Стат. сб. – M.: Росстат, 2021. – 429 [Электронный ресурс]. – Режим доступа: https://rosstat.gov.ru/storage/mediabank/Nac-sch_2013-2020.pdf (дата обращения: 03.11.2021). 11. Национальные счета России в 2013–2020 годах: Стат. сб. – M.: Росстат, 2021. – 429 [Электронный ресурс]. – Режим доступа: https://rosstat.gov.ru/storage/mediabank/Nac-sch_2013-2020.pdf (дата обращения: 03.11.2021). 12. Отчет по форме 1-НОМ по состоянию на 01.01.2020 // Федеральная налоговая служба [Электронный ресурс]. – Режим до- ступа: https://www.nalog.gov.ru/rn21/related_activities/statistics_and_analytics/forms/8826515/ (дата обращения: 03.11.2021). 13. Российский статистический ежегодник // Федеральная служба государственной статистики [Электронный ресурс]. – Режим доступа: https://rosstat.gov.ru/folder/210/document/12994 (дата обращения: 03.11.2021). 12. Отчет по форме 1-НОМ по состоянию на 01.01.2020 // Федеральная налоговая служба [Электронный ресурс]. – Режим до- ступа: https://www.nalog.gov.ru/rn21/related_activities/statistics_and_analytics/forms/8826515/ (дата обращения: 03.11.2021). 13. Российский статистический ежегодник // Федеральная служба государственной статистики [Электронный ресурс]. – Режим доступа: https://rosstat.gov.ru/folder/210/document/12994 (дата обращения: 03.11.2021). 14. Заключение 114 Исходя из проведенного анализа можем констатировать, что для снижения размеров теневой экономи- ки нужно найти оптимальный уровень налоговой нагрузки для каждой отрасли народного хозяйства. Пото- му что связь между налоговой политикой и теневой экономикой существует, но в силу большего влияния прочих факторов направление влияния может отличаться. Поэтому борьба с теневой экономикой должна быть комплексной, включающей и воспитание более ответственного гражданского поведения, гибкость на- селения в части использования налогового законодательства, сервисов налогоплательщика, более тщатель- ные проверки со стороны налоговых органов, влекущие значительные санкции для налогоплательщиков 114 Экономика: проблемы, решения и перспективы за нарушения налогового законодательства, государственную политику по созданию новых рабочих мест и подготовке кадров согласно спросу на них. Должно быть сформировано коллективное сознание о необ- ходимости уплаты налогов и контроля расходования собранных средств. 1. Приказ ФНС России от 30.05.2007 № ММ-3-06/333 (ред. от 10.05.2012) «Об утверждении Концепции системы пла- нирования выездных налоговых проверок» // СПС «КонсультантПлюс» [Электронный ресурс]. – Режим доступа: http://www.consultant.ru/document/cons_doc_LAW_55729/ (дата обращения: 03.11.2021). 1. Приказ ФНС России от 30.05.2007 № ММ-3-06/333 (ред. от 10.05.2012) «Об утверждении Концепции системы пла- нирования выездных налоговых проверок» // СПС «КонсультантПлюс» [Электронный ресурс]. – Режим доступа: http://www.consultant.ru/document/cons_doc_LAW_55729/ (дата обращения: 03.11.2021). 2. Основные направления бюджетной, налоговой и таможенно-тарифной политики на 2022 год и на плановый период 2023 и 2024 годов // Министерство финансов Российской Федерации [Электронный ресурс]. – Режим доступа: https://minfin. gov.ru/common/upload/library/2021/09/main/ONBNiTTP_2022-2024.pdf (дата обращения: 03.11.2021). 3. Алиев, Б. Х., Эльдарушева, М. Д. Налоговая политика государства: сущность, механизм реализации и перспективы // Финансы и кредит. – 2014. – № 40 (616). – С. 27–36. 3. Алиев, Б. Х., Эльдарушева, М. Д. Налоговая политика государства: сущность, механизм реализации и перспективы // Финансы и кредит. – 2014. – № 40 (616). – С. 27–36. 4. Арипов, М. Г. Налоговые механизмы снижения уровня теневой экономики и стимулирования инвестиционной деятель- ности // Финансы и кредит. – 2012. – № 42 (522). – С. 35–41. 5. Мазур, Л. В., Батяев, А. В. Налоговая нагрузка и теневая экономика // Территория науки. – 2018. – № 5. – С. 98–102. 6. Пансков, В. Г. Теневая экономика и налогообложение // ЭТАП: экономическая теория, анализ, практика. – 2017. – № 5. – С. 77–91. 6. Пансков, В. Г. Теневая экономика и налогообложение // ЭТАП: экономическая теория, анализ, практика. – 2017. – № 5. – С. 77–91. 7. Трусова, Н. С. Мировой опыт выявления и противодействия уклонению от уплаты налогов // Известия Юго-Западного государственного университета. Серия: Экономика. Социология. Менеджмент. – 2017. – Т. Заключение Статистика для национального проекта «Малое и среднее предпринимательство и поддержка индивидуальной пред- принимательской инициативы» // Федеральная налоговая служба [Электронный ресурс]. – Режим доступа: https://rmsp. nalog.ru/statistics2.html (дата обращения: 03.11.2021). 15. Шнайдер, Ф., Энст, Д. Скрываясь в тени. Рост подпольной экономики // Международный валютный фонд [Электронный ресурс]. – Режим доступа: https://www.imf.org/external/pubs/ft/issues/issues30/rus/issue30r.pdf (дата обращения: 03.11.2021). 15. Шнайдер, Ф., Энст, Д. 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Shadow economy and taxation, ETAP: economic theory, analysis, practice, 2017, no. 5, pp. 77–91. (In Russian). 7. Trusova N. S. World experience of identification and counteraction by the damage from the payment of taxes, Izvestiya Yugo-Za- padnogo gosudarstvennogo universiteta. Seriya Ekonomika, sotsiologiya, menedzhment, 2017, vol. 7, no. 4 (25), pp. 194–202. References The impact of the tax system on shadow economy, Finance and credit, 2015, no. 41 (665), pp. 10–21. (In Russian). 9. The results of a sample survey of the labor force, Federal State Statistics Service. Available at: https://rosstat.gov.ru/compendium/ 8. Fedotov D. Yu. The impact of the tax system on shadow economy, Finance and credit, 2015, no. 41 (665), pp. 10–21. (In Russian). 9. The results of a sample survey of the labor force, Federal State Statistics Service. Available at: https://rosstat.gov.ru/compendium/ document/13265 (accessed 03.11.2021). 9. 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Marx jornalista: a empiria a serviço da crítica da economia política
Nova Economia
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DOI: http://dx.doi.org/10.1590/0103-6351/4632 DOI: http://dx.doi.org/10.1590/0103-6351/4632 Marx jornalista: a empiria a serviço da crítica da economia política* Adriano Lopes Almeida Teixeira (1) (1) Universidade Federal do Espírito Santo Resumo This article seeks to highlight the impor- tance of the journalistic activities developed by Marx between the years 1848 and 1857 for the process of elaborating his critique of political economy. Without abandoning his theoretical work, the journalism and his involvement with the apprehension of the practical aspects of social reality made pos- sible to Marx the refi nement of two essential aspects for the elaboration of his critique: the form and content of the categories of political economy. Therefore, in addition to theoretical developments of that period con- tained both in London Notebooks, as in epis- tolary Marx-Engels, the empirical elements appear as a fundamental moment in Marx’s theoretical-methodological trajectory, which would culminate in the elaboration of the Grundrisse in the years 1857-1858. Este artigo busca evidenciar a importância das atividades jornalísticas desenvolvidas por Marx entre os anos de 1848 e 1857 para o processo de elaboração de sua crítica da economia política. Sem abandonar seus trabalhos teóricos, o jorna- lismo e seu envolvimento com a apreensão dos aspectos práticos da realidade social possibilita- ram a Marx o refi namento de dois aspectos es- senciais para a elaboração de sua crítica: a forma e o conteúdo das categorias da economia política. Por isso, para além dos desenvolvimentos teóricos daquele período contidos tanto nos Cadernos de Londres, quanto no epistolário Marx-Engels, a empiria aparece como momento fundamental da trajetória teórico-metodológica de Marx, que cul- minaria na elaboração dos Grundrisse nos anos de 1857-1858. 1 Introdução De forma não intencional, jornalismo e teoria se imbricaram na trajetória intelectual de Marx, num processo de retroalimentação constante, inter- rompido em alguns momentos pelas vicissitudes de uma vida constante- mente atribulada por questões fi nanceiras, familiares e políticas. Se é bas- tante conhecida a proposição de que não se pode fatiar Marx, e que seu pensamento só pode ser relativamente compreendido por uma perspectiva totalizante que englobe fi losofi a, economia e prática política, suas ativida- des jornalísticas foram uma espécie de amálgama desses três elementos. Como bem destaca Heinrich (2018, p. 32) em recente biografi a de Marx, As ideias científi cas desenvolvidas infl uenciavam as atividades jornalísticas e políticas; por outro lado, essas atividades muitas vezes exigiam uma interrupção dos trabalhos científi cos, levantando novos temas e problemáticas, levando à for- mação de novos conceitos e, assim, infl uenciando a própria pesquisa científi ca. Com efeito, era no calor de suas atividades profi ssionais como jornalista que Marx reunia elementos para seu processo de elaboração teórica ao mesmo tempo que buscava incendiar o cenário político com artigos que denunciavam a opressão sofrida pela classe trabalhadora e divulgavam a iminência da revolução. Com efeito, era no calor de suas atividades profi ssionais como jornalista que Marx reunia elementos para seu processo de elaboração teórica ao mesmo tempo que buscava incendiar o cenário político com artigos que denunciavam a opressão sofrida pela classe trabalhadora e divulgavam a iminência da revolução. As atividades jornalísticas de Marx começam no ano de 1842 na Gazeta Renana. Não tendo conseguido ocupar uma cátedra na Universidade de Berlim, restou-lhe buscar o jornalismo como meio de sobrevivência. Para autores como Krätke (2008a, p. 162), aquele ano representou o início de uma longa relação com a economia política. O presente artigo vai se concentrar nas atividades jornalísticas desempe- nhadas por ele a partir de 1848, ano da eclosão de diversas revoluções pela Europa. Foi somente a partir de A miséria da fi losofi a, de 1847, que Marx es- boça uma crítica esteada não somente no seu amplo conhecimento fi losó- fi co e jurídico, mas também na teoria econômica legada pelos economistas clássicos, objeto de estudo intensivo desde sua chegada a Paris, em 1844. Esse procedimento não signifi ca concordar com uma suposta cisão en- tre o Marx jovem e o maduro. Keywords journalism, empirical elements, critique of political economy, dialectical method. jornalismo, empiria, crítica da economia política, método dialético. jornalismo, empiria, crítica da economia política, método dialético. JEL Codes B14, B41, B51. Códigos JEL B14, B41, B51. Códigos JEL B14, B41, B51. Códigos JEL B14, B41, B51. * Este artigo é uma versão modifi cada de um dos capítulos da tese de doutorado do autor, A genealogia da mais-valia: fi losofi a, economia e crítica da economia política (2014). 939 v.29 n.3 p.939-962 2019 Nova Economia Teixeira 940 Nova Economia v.29 n.3 2019 1 Introdução Apenas se reconhece que, entre 1842 e 1847, a vida profi ssional de Marx foi entrecortada por diversos reveses no campo político, que o impediram de atuar de forma contínua pelos jornais por onde passou, o que somente iria acontecer a partir de sua chegada a Londres, no fi nal de 1849. 940 Nova Economia v.29 n.3 2019 940 Marx jornalista O ano de 1848 é, pois, emblemático na confi guração do Marx jornalista. Ano de publicação de O Manifesto Comunista, ele – que já tinha feito seu ajuste de contas com a fi losofi a alemã através de obras escritas sozinho e com Engels, ajuste consumado em A ideologia alemã – reconhecia que, embora tivesse acumulado um estoque de conhecimento sufi ciente para confrontar Proudhon na Miséria da fi losofi a, seu conhecimento teórico em economia precisaria sofrer considerável evolução. Por isso, o Marx que chega ao fi nal da década de 1840 é o Marx que reconhecia tanto a derro- ta temporária do projeto político quanto a necessidade de dedicar mais tempo ao estudo da economia, o que signifi caria, na mesma medida, se abastecer de maior conteúdo empírico. No seu embate com Proudhon, em 1847, Marx, já conhecedor da fi loso- fi a hegeliana, critica o uso inadequado que o autor de A fi losofi a da miséria faz de Hegel. Embora esse autor tenha recebido os estilhaços dos ataques proferidos por Marx e Engels à fi losofi a especulativa em obras como A sagrada família e A ideologia alemã, não parece que Marx tenha em algum momento pretendido uma ruptura completa com Hegel. A dialética legada por Hegel, transformada e aportada no projeto de compreensão de uma sociedade reconhecidamente mistifi cada, necessitava que suas estruturas essenciais estivessem em relação direta com as condições concretas da rea- lidade. Por isso, o jornalismo iria se apresentar como atividade capaz de arramar as pontas que estavam soltas no seu projeto, fato que alcançaria plenitude no período londrino. A partir de 1949, já em Londres, Marx entra em ritmo alucinante de tra- balho para garantir o próprio sustento material. Sem abandonar totalmente os estudos teóricos, dedica-se a intensas pesquisas de cunho empírico para os artigos jornalísticos que tinha que produzir. Essa etapa da vida de Marx é o momento em que ele amplia seus conhecimentos de economia visando à realização da crítica da economia política. Na NGR [Nova Gazeta Renana] podemos seguir o passo a passo como in- terpretam Marx e Engels o processo revolucionário, suas opções táticas e estra- tégicas e suas primeiras generalizações teóricas da prática que estão vivendo; vemos nascer muitas das refl exões e teses que encontraram logo expressão mais 1 Introdução Se, em Miséria da fi losofi a, de 1847, ele deu um passo à frente na análise teórica da totalidade da repro- dução capitalista, tratava-se agora, em meio às atividades de apropriação da realidade concreta, de preparar as condições para a realização da crítica. A importância, portanto, do período entre 1848 e 1857 para o enrique- cimento teórico do projeto marxiano contrasta com certo descaso da tra- dição marxista com os trabalhos jornalísticos de Marx. Pino (2013, p. 22) lembra que a indiferença quanto à importância do jornalismo para os ob- jetivos teóricos de Marx é decorrente da primazia concedida pela tradição 941 v.29 n.3 2019 Nova Economia Teixeira marxista ao Marx fi lósofo e ao Marx economista. Este artigo, ao contrário, busca situar o jornalismo de Marx como etapa fundamental de constituição dos elementos teóricos e metodológicos da crítica da economia política. A forma como o presente artigo foi dividido refl ete a difi culdade de sepa- rar o jornalista do fi lósofo, do ativista político ou do economista, pois, como já mencionado, eles se interseccionam. Na próxima seção, abordaremos a trajetória de Marx desde a fundação da Nova Gazeta Renana em Colônia, em 1848, até o ano de 1851, quando, já em Londres, recebe convite de Charles Dana, editor do jornal norte-americano New York Tribune para ser correspon- dente na Europa e escrever uma série de artigos sobre os eventos políticos ocorridos na Alemanha. Na seção seguinte, a terceira, o foco estará sobre o conjunto das atividades desempenhadas por Marx desde o convite de Char- les Dana até a elaboração dos Grundrisse. Por fi m, na última seção buscare- mos evidenciar a relação umbilical entre atividades jornalísticas e elaboração teórica na evolução do pensamento de Marx durante aquele período, com foco nos Cadernos de Londres, escritos entre 1850 e 1853, e no epistolário Marx-Engels, seguindo-se, então, brevíssimas considerações fi nais. 2 Marx (1848/1851): da Nova Gazeta Renana ao New York Tribune A rigor, não há razões para contestar o depoimento dado por Marx quanto a ter interrompido seus estudos formais de economia durante os anos de 1848 e 1849. Ele, até então, tinha se debruçado no estudo dos teóricos mais proeminentes no campo do socialismo e da economia política, através de análise cuidadosa, quase sempre parágrafo a parágrafo, de suas principais obras. De fato, essa prática é interrompida com a ida de Marx em 1848 para Colônia, onde, no calor da revolução que infl amava os alemães, ele se dedicaria à publicação diária de um jornal, a Nova Gazeta Renana (NGR), que em algumas edições era acompanhado por volumosos suplementos, demandando quase o seu tempo total disponível. Sobre a importância des- se jornal na trajetória intelectual de Marx, Claudín (1985, p. 93) diz que: 942 Nova Economia v.29 n.3 2019 942 Marx jornalista desenvolvida – ou seriam retifi cadas – nos textos clássicos sobre a revolução de 48: Revolução e Contrarrevolução na Alemanha, A luta de classes na França, O 18 Brumário de Luís Bonaparte. desenvolvida – ou seriam retifi cadas – nos textos clássicos sobre a revolução de 48: Revolução e Contrarrevolução na Alemanha, A luta de classes na França, O 18 Brumário de Luís Bonaparte. Nesse período, Marx teve que dividir o seu tempo com questões paralelas a sua atividade principal, como os processos judiciais e a militância políti- ca. Se, por um lado, Marx na Nova Gazeta Renana se afastava dos tradicio- nais compêndios de economia política, por outro, adquiria paulatinamente a capacidade de estar em fi na sintonia com a ordem do dia. Tornava-se um exímio analista de conjunturas. Sua ida para a Alemanha signifi cou um afastamento das atividades po- líticas que desempenhava no Comitê Central em Bruxelas, mas também uma aproximação dos fatos políticos que norteavam a causa proletária na Alemanha. Percebe-se, assim, que o perfi l de sua atuação, bem como o perfi l do órgão que comandava, compelia Marx a se calçar de ampla base empírica. Afastava-se dos estudos da economia política, mas não da aná- lise dos fatos econômicos do dia a dia, uma prática que atenderia aos seus propósitos de dar à economia política uma base científi ca, que signifi cava a recusa às leis abstratas como ponto de partida. 2 Marx (1848/1851): da Nova Gazeta Renana ao New York Tribune É essa nova confi guração das suas investigações que permite relativizar a afi rmação de Marx sobre a interrupção dos seus estudos econômicos no período da Nova Gazeta Renana. Como alerta Chasin (1993, p. 33), “cabe atentar bem para o signifi cado intrínseco da afi rmação, pois, não se há de supor, evidentemente, que Marx interromperia seus estudos econômicos por tão largo período em troca de alguma bagatela”. Os olhos de Marx permaneciam, pois, atentos aos fatos econômicos da época, como mostram alguns dos seus textos publicados naquele jornal. Os artigos citados a seguir são apenas alguns, entre outros, que abordam a temática econômica e que são elencados prioritariamente, e em ordem cronológica, devido à ênfase própria dos seus títulos: “O projeto de lei so- bre o empréstimo compulsório e sua exposição de motivos” (Marx, 2010, p. 167-170), de 26 de julho de 1848, artigo no qual, de forma sucinta, Marx menciona categorias como circulação, dinheiro, capital e juros; “A Gazeta de Colônia sobre o empréstimo compulsório” (Marx, 2010, p. 191-192), de 04 de agosto de 1848, que contém elementos sobre a questão fi scal da Prússia; “O discurso de Proudhon contra Thiers” (Marx, 2010, p. 195-198), de 05 de agosto de 1848, em que Marx critica a ideia de criar um Banco Nacional com consequente redução dos juros a zero; “A Bélgica, ‘Estado- -Modelo’” (Marx, 2010, p. 199-202), de 07 de agosto de 1848, com o uso 943 v.29 n.3 2019 Nova Economia 94 v.29 n.3 2019 Nova Economia 943 Teixeira de dados demográfi cos, comércio exterior, produção da indústria nacional etc.; “O orçamento dos Estados Unidos e o germano-cristão” (Marx, 2010, p. 373-376), de 07 de janeiro de 1849; “A situação fi nanceira prussiana sob Bodelschwingh e consortes” (Marx, 2010, p. 440-449), de 17 de fevereiro de 1849; “Outra contribuição sobre a administração fi nanceira velho-prus- siana” (Marx, 2010, p. 454-456), de 23 de fevereiro de 1849; “A situação do comércio” (Marx, 2010, p. 484-488), de 07 de março de 1849 e “Os bilhões” (Marx, 2010, p. 513-517), de 16 de março de 1849, com elementos da política monetária do Governo Provisório na França. No dia 05 de abril de 1849, Marx publica o primeiro editorial, de um total de cinco, do que fi cou conhecido como Trabalho Assalariado e Capital. 944 Nova Economia v.29 n.3 2019 1 Queixa semelhante fora feita anos antes por Arnold Ruge (1981, p. 8), que, com Marx, fundou no ano de 1844, em Paris, os Anais Franco-Alemães: “Ele tem uma personalidade pecu- liar – perfeito como erudito e escritor, mas um completo desastre como jornalista. Lê muito; trabalha com uma intensidade incomum e tem um talento crítico que de vez em quando degenera numa dialética temerária. Mas não termina nada, interrompe tudo e volta a mergu- lhar num oceano de livros. (…) É irritadiço e exaltado, particularmente quando trabalha até adoecer e não vai para a cama por três, até quatro noites seguidas.” 2 Stephan Born foi um tipógrafo e revolucionário alemão, membro da Liga Comunista, com participação ativa na revolução de 1848. A citação faz parte de suas memórias, publicadas em 1898, disponíveis em <https://www.gutenberg.org/fi les/55895/55895-h/55895-h.htm>, p. 196, acesso em 14/08/2018. 2 Marx (1848/1851): da Nova Gazeta Renana ao New York Tribune Engels menciona os textos da Nova Gazeta Renana como um dos mo- mentos, além de O Manifesto Comunista, em que Marx consegue, com sucesso, aplicar a concepção materialista da história “para explicar acon- tecimentos políticos contemporâneos. (...) Tratava-se, pois, de reduzir, seguindo a concepção do autor, os acontecimentos políticos a efeitos de causas que, em última instância, eram econômicas” (Engels, 1953, p. 93). Na década seguinte, e sobretudo até a publicação de O Capital, a feição empírica dos trabalhos de Marx permanece. Em Londres, a partir de 1850, volta aos estudos de economia política, em alguns momentos de forma simultânea às atividades jornalísticas, buscando construir uma teoria do valor própria, num movimento que não poderia prescindir dos dados eco- nômicos e estatísticos constantes das mais diversas fontes, como jornais, relatórios de agências ofi ciais, cadernos dos inspetores de fábrica, os Blue Books (cadernos azuis, publicados pelo Parlamento Britânico com informa- ções sobre a história econômica e diplomática do país) etc. O objeto de estudo de Marx, a sociedade burguesa, não poderia ser compreendido sem o apelo ao empírico. De certa forma, o que aqui é apre- sentado como resultado do entendimento de Marx de que o método cor- reto exigia submeter as categorias da economia política à sua concepção materialista da história – o que requeria pesquisas mais profundas sobre elementos da vida material que até já tinham sido tratados pelos clássi- cos – tem também o seu lado fortuito, pois Marx foi obrigado a se apegar ao jornalismo como única fonte de sobrevivência. Se a adoção da carreira jornalística esteve longe de ser uma escolha planejada e espontânea, não se diga o mesmo da forma como essa contingência foi direcionada para seus propósitos de construir uma crítica da economia política. É razoável Marx jornalista entender que a publicação dos textos, especialmente na Nova Gazeta Rena- na, se tinha objetivos políticos, tinha também certa funcionalidade teórica, muitas vezes com a proeminência desta última sobre aqueles, como se pode extrair da queixa1 de Engels, testemunhada por Born2 (1981, p. 16) ao visitar Marx como editor do referido jornal em janeiro de 1849: “Ele não é e nunca será um jornalista”, dizia ele. “Passa o dia inteiro estudando minuciosamente um artigo de fundo que outra pessoa levaria umas duas horas para fazer, como se ele concernisse ao manejo de um problema fi losófi co pro- fundo. 2 Marx (1848/1851): da Nova Gazeta Renana ao New York Tribune Altera e aprimora, e depois modifi ca as alterações, e, graças a seu rigor incansável, nunca consegue terminar na hora certa.” Marx chega a Londres no fi nal de agosto de 1849 decidido a dar prosse- guimento às suas atividades políticas e teóricas. Envolve-se com ativida- des de apoio a refugiados políticos alemães, com a reorganização da Liga Comunista e com a criação de um jornal alemão, de publicação mensal, cujo objetivo mais uma vez, além do próprio nome escolhido, demonstra o processo de retroalimentação entre aquelas duas atividades: “investigar de modo detalhado e científi co as relações econômicas que constituem a base de todo o movimento político.” (Claudín, 1985, p. 228). Assim como o seu jornal da Renânia, o nome escolhido foi Neue Rheinische Zeitung (Nova Gazeta Renana), distinguindo-se apenas quanto ao acréscimo do termo Po- litisch-ökonomische Revue (Revista Econômico-Política). Segundo Engels, Marx tinha acompanhado os principais acontecimentos dos anos 1848-1849 de tal forma, que estava apto a escrever sobre a situação econômica e política das principais nações europeias, sem as perturbações que a proximidade dos eventos pode causar a um analista que tenha participado dos fatos ocorridos (Engels, 1953, p. 94). De fato, política e economia se imbricam nos três únicos números do jornal, publicados em forma de revista. Os temas versam sobre conjuntu- ra econômica e política internacional tanto no primeiro número (janeiro- 945 v.29 n.3 2019 Nova Economia 945 Teixeira -fevereiro de 1850) quanto nos dois outros (março-abril e maio-outubro de 1850). Este último número antes da falência do jornal foi precedido por um aprofundamento da análise econômica por parte de Marx, que chega à conclusão de que “a crise comercial contribuiu infi nitamente mais para as revoluções de 1848 do que a revolução para a crise comercial” (Musto, 2011, p. 42). Essa constatação recrudesce a visão que Marx já defendia desde a década anterior, especialmente na Nova Gazeta Renana, de recusar o método pelo qual a revolução seria fruto de algum plano habilmente preparado, o que gerava forte oposição de quadros políticos aliados.3 Antes de entrar em completo isolamento político, “uma grande quanti- dade de artigos econômicos nas Notes to the People e no People’s Paper foram escritos com a colaboração direta de Marx” (Krätke, 2008a, p. 163). 3 O Comitê Central da Liga dos Comunistas de Londres envia uma carta à Direção Geral, em princípios de 1851, contendo ataques a Marx e Engels e explicando os motivos da diver- gência: “(...) A única diferença entre eles e nós com referência aos princípios – quando ainda trabalhávamos juntos – era que esses homens afi rmavam a necessidade de prosseguir por pelo menos durante 50 anos mais em oposição, isto é: de atuar de forma meramente crítica, enquanto nós afi rmávamos que com uma adequada organização de nosso partido seríamos capazes de impor já na próxima revolução as medidas tendentes à instauração da sociedade proletária” (Enzensberger, 2009, p. 163-164). 2 Marx (1848/1851): da Nova Gazeta Renana ao New York Tribune Com o isolamento imposto pelas circunstâncias, Marx permanece em Londres, enquanto Engels abandona o jornalismo e volta para Manchester, onde se empregaria na empresa têxtil de seu pai, a Ermen & Engels. Por alguns meses, Marx ministra cursos de economia para os amigos em sua própria casa, o que o mantém em contato com a teoria, conforme depoimento de Wilhelm Liebknecht, amigo e discípulo de Marx, prestado em 1896 (En- zensberger, 2009, p. 130-131). 3 Marx (1851/1857): o nascedouro dos Grundrisse Com efeito, a resistência de Marx em participar dos projetos de prepa- ração para a revolução guardava relação com a sua convicção de que era necessário antes compreender cientifi camente a natureza e a estrutura de funcionamento da sociedade capitalista. A dissolução da Liga Comunista acontece em 17 de dezembro de 1852, mas, desde 1851, o afastamento das atividades políticas dá a Marx a oportunidade de se voltar para os estudos de economia política. Ele não seria membro de nenhum partido político pelos dez anos seguintes. 946 Nova Economia v.29 n.3 2019 Marx jornalista Esses acontecimentos impactariam positivamente o ritmo dos estudos de Marx que, em busca de respostas, empreende uma acelerada troca de cartas com Engels sobre temas da economia política. O ritmo na troca de cartas entre os dois seria tão intenso que às vezes uma carta era enviada antes que chegasse a resposta referente à carta anterior. Por isso, nos in- tervalos entre as “cartas teóricas”,4 Marx e Engels se correspondiam sobre os mais variados assuntos. Sem saber ainda que voltaria a se ocupar com tarefas jornalísticas, Marx escreve a Engels em 11 de fevereiro de 1851: “Eu gosto muito do isolamento do público no qual nos encontramos. Tem a ver com nossa atitude e nossos princípios” (Marx; Engels, 1982, p. 286). Em janeiro de 1851, Wilhelm Pieper, um jovem fi lólogo alemão que foi secretário de Marx, escreve a Engels, dizendo: “Marx tem estado muito isolado. Seus únicos amigos são John Stuart Mill e Lloyd. Quando alguém chega em sua casa, em lugar de cumprimentos é saudado com categorias econômicas” (Enzensberger, 2009, p. 168). Engels passaria aproximadamente os próximos 20 anos em Manchester, naquilo que ele considerou seu próprio exílio pessoal. O epistolário Marx- Engels, tema que será retomado mais adiante, torna-se, desde então, um repo- sitório valioso do desenvolvimento teórico conjunto desses dois autores. Eles, que escreveram diversas obras juntos, tinham nas cartas a única possibilidade de interação. Em 07 de janeiro de 1851, Marx escreve: “Te escrevo somente para submeter a seu exame uma pequena questão teórica de natureza políti- co-econômica. Comecemos desde o princípio: sabes que, segundo a teoria de Ricardo, a renda não é mais do que a diferença entre custo de produção e o preço do produto agrícola (...)” (Marx; Engels, 1982, p. 258). Engels responde somente em 29 de janeiro de 1851. 4 As cartas aqui recuperadas são as que fornecem pistas do desenvolvimento teórico d Marx. Por isso denominá-las “cartas teóricas”. 3 Marx (1851/1857): o nascedouro dos Grundrisse Em 03 de fevereiro, Marx escreve longa carta a Engels, com grande quantidade de dados referentes à circulação mo- netária na Inglaterra. Engels envia seu parecer a Marx em 25 de fevereiro de 1851. Em 02 de abril, Marx informa a Engels que teve que interromper seus estudos devido aos problemas fi nanceiros e de saúde de Jenny, sua esposa, mas que, dado o estado adiantado de suas pesquisas sobre economia, em cinco semanas concluiria a sua Economia Política e se dedicaria a outro campo de aprendizagem no Museu Britânico (Marx; Engels, 1982, p. 325). Em agosto de 1851, Marx recebe convite de Charles Dana, editor do jornal norte-americano New York Tribune, para ser correspondente na Eu- 947 v.29 n.3 2019 Nova Economia Teixeira ropa e escrever uma série de artigos sobre os eventos políticos ocorridos na Alemanha. Esses artigos seriam escritos por Engels, a pedido de Marx, que ainda não dominava o idioma inglês. Além dessa difi culdade, que seria logo superada, Marx não queria parar seus estudos de economia política. Em carta de 14 de agosto de 1851, escreve a Engels: “Tens que me ajudar, agora que estou ocupadíssimo com a economia política” (Marx; Engels, 1982, p. 425). Durante 11 anos, de agosto de 1851 e fevereiro de 1862, Marx, com a participação de Engels, enviou artigos semanais para aquele jornal, que era publicado em três versões diferentes: o New York Daily Tri- bune, o New York Semi-Weekly Tribune e o New York Weekly Tribune. Segun- do Musto (2011, p. 47), “o primeiro publicou 487 artigos, dos quais mais da metade reapareceram no New York Semi-Weekly Tribune e mais de um quarto no New York Weekly Tribune”, além dos que foram recusados por Charles Dana. Daquele total, 350 foram de autoria de Marx, 125 de Engels (dos quais a maioria foi sobre assuntos militares) e 12 escritos em conjunto (McLellan, 1990, p. 307). Aproximadamente um terço dos artigos de Marx destinava-se à aná- lise de assuntos fi nanceiros e econômicos da atualidade, principalmente da Grã-Bretanha, mas também de outros países e da economia mundial como um todo (Krätke, 2008a, p. 163). Nesses artigos, Marx se expressa de maneira aprofundada sobre temas que reaparecem em O capital sem o mesmo nível de profundidade, como o mercado monetário e a legis- lação bancária, com exceção dos temas relacionados à legislação fabril, expostos detalhadamente por Marx naquela obra (Krätke, 2007, p. 3 Marx (1851/1857): o nascedouro dos Grundrisse 129). Certamente, este é mais um exemplo de como a exposição dos resulta- dos da pesquisa, em Marx, situa-se de forma aparentemente desvinculada da investigação, pois, em O capital, as digressões mais específi cas sobre jornada de trabalho e legislação fabril aparecem a partir do capítulo oito, enquanto a exposição da mais-valia acontece no capítulo quatro. O víncu- lo entre investigação e exposição subsequente fi ca claro quando se nota, por exemplo, que, para a elaboração posterior da categoria mais-valia re- lativa, a complexa questão da relação entre produtividade e intensidade do trabalho teve que ser analisada cuidadosamente por Marx, “que argu- menta estritamente em termos da teoria do valor: o que os proprietários da fábrica perdiam pela limitação legal da jornada de trabalho, em tempo de trabalho absoluto, recuperavam através da intensifi cação do tempo de trabalho” (Krätke, 2007, p. 131). 948 Nova Economia v.29 n.3 2019 948 Marx jornalista Krätke menciona, ainda, a seção quinta do livro III de O capital como consistindo, em grande parte, de matérias daquele período. Ademais, te- mas como (...) crises, mercado mundial, estruturas internacionais do comércio, por meio das quais umas nações exploram outras, colônias, colonialismo e sua importância para o desenvolvimento do capitalismo, e diferentes formas de fi nanças públicas e economia pública – somente foram tratadas por Marx em seus artigos de jornal. Algumas das mais sofi sticadas refl exões sobre o estado moderno, seu desenvol- vimento histórico dentro do contexto do sistema europeu de estados nacionais, e o desenvolvimento das principais formas da política nas sociedades burguesas modernas são somente encontradas numa série de artigos escritos por Marx em diversas ocasiões (Krätke, 2008a, p. 163-164). É necessário compreender a teoria de Marx como resultado de experi- mentos, tentativas, correções, descobertas, recuos e avanços dentro de um processo que signifi cava acompanhar um objeto histórico no seu próprio ritmo. Obviamente, Marx não sabia de antemão algo que ele ainda te- ria que descobrir. A evolução dos seus conhecimentos empíricos e do seu instrumental metodológico precisava ser acompanhada em igual medida pela evolução dos seus conhecimentos teóricos, no mesmo sentido em que ontologia e epistemologia caminham juntas. A premência da busca pelo sustento, aliada a outros projetos que surgiam nesse meio tempo, adiava o esperado livro sobre economia. De certa maneira pode-se dizer que nem os trabalhos jornalísticos interromperam os estudos de economia, nem es- tes interromperam aqueles. 3 Marx (1851/1857): o nascedouro dos Grundrisse Marx era “o jornalista e economista crítico, consciente de viver um período turbulento do capitalismo, e que compa- rou a rápida expansão do mercado global durante a década 1849-1858 aos mais brilhantes períodos do século XVI” (Krätke, 2007, p. 134-135). De fato, a consolidação do modo de produção capitalista é apreendida por Marx. A realidade era analisada por ele nos marcos de sua concepção materialista da história, num momento em que se generalizavam tanto as forças produtivas especifi camente capitalistas quanto as relações sociais ca- pitalistas de produção. No prefácio de Contribuição à crítica da economia polí- tica, de 1859, menciona “o novo estágio de desenvolvimento em que esta [a sociedade burguesa] parecia entrar com a descoberta do ouro na Califórnia e Austrália” (Marx, 1986, p. 27) como fator que o levou a recomeçar os estu- dos. Krätke (2007, p. 135) acrescenta outras circunstâncias históricas, como a revogação da lei de cereais, a enorme onda de imigração europeia etc. Os trabalhos jornalísticos de Marx perduram até 1862, intercalado por diversos momentos em que a prioridade se volta para os estudos teóricos v.29 n.3 2019 Nova Economia 949 Teixeira e até políticos. O 18 Brumário de Luís Bonaparte, considerada por muitos como sua obra histórica mais profunda, foi escrito entre dezembro de 1851 e março de 1852, para uma revista mensal norte-americana, a Die Revolution. Nessa obra, Marx analisa os efeitos do golpe de 02 de dezem- bro de 1851 no contexto do confl ito de classes existente na França. Ainda no mês de dezembro, escreve Revelações sobre o processo de comunistas de Colônia, e, entre maio e junho de 1852, escreve Os grandes homens do exí- lio, em que analisa os problemas internos entre os exilados no interior da Liga Comunista. Se, entre 1850 e 1853, Marx esteve ocupado com os Cadernos de Londres, seus estudos teóricos seriam retomados em algum momento entre o fi m de 1854 e o início de 1855 (Musto, 2011, p. 54). Simultaneamente a essas iniciativas, continuava produzindo artigos jornalísticos, embora suas pu- blicações no New York Tribune decrescessem. Em 1853, 80 artigos de Marx foram impressos naquele jornal. Em 1854 o número se repete, mas, no ano seguinte, cai para 40, e, em 1856, para 24 (Mcllelan, 1990, p. 306). Os artigos de Marx produzidos em 1856 focavam principalmente as crises monetárias na Europa (Krätke, 2008a, p. 950 Nova Economia v.29 n.3 2019 3 Marx (1851/1857): o nascedouro dos Grundrisse 163), tendo escrito, em junho de 1856, três artigos sobre o Crédit Mobilier (Marx; Engels, 1986, p. 8-24), banco francês fundado pelos irmãos Péreire, principal instrumen- to da política fi nanceira de Napoleão III. Os irmãos Isaac e Jacob Émile Péreire criavam sociedades por ações, através das quais lançavam títulos de curto prazo na Bolsa e captavam recursos de pequenos investidores para o banco. Marx o considerou “como um dos maiores fenômenos de nossa época”. Nos dois anos seguintes, Marx e Engels escreveriam 117 artigos, sendo 98 de autoria de Marx e 19 de Engels. Dividindo esses artigos em blocos temáticos, vê-se que as crises econômicas são o segundo tema mais abor- dado, com 17 artigos, logo atrás da Revolta dos Sipaios, com 19. Outros seis artigos tratam do sistema bancário europeu, 11 da situação política da Grã-Bretanha, e o restante sobre questões políticas, sociais e bélicas diver- sas (De Paula, 2011, p. 133-134). “Nestes artigos, Marx fazia uso frequente do material que estava coletando diligentemente para o seu ‘livro sobre a crise’” (Krätke, 2008a, p. 165). Em abril de 1857, Marx recebe outro convite de Charles Dana, desta vez para enviar artigos para o New American Cyclopaedia, em forma de verbe- tes que, em sua maioria, tratariam de temas biográfi cos e militares. Nesse 950 Marx jornalista período, a maior parte dos trabalhos fi ca por conta de Engels, para que Marx possa dedicar mais tempo a sua pesquisa. Um total de 67 artigos é publicado até o fi nal de 1858, sendo 51 de autoria de Engels e 16 de Marx (Mcllelan, 1990, p. 307). Com efeito, mais do que qualquer obrigação contratual, as crises eco- nômicas foram o acicate que conduziu Marx a iniciar a elaboração dos Grundrisse.5 A crise, que na visão dele fi nalmente chegaria, ou, conforme suas próprias palavras, o dilúvio, seria a conjuntura perfeita para a defl a- gração dos ideais revolucionários. Por isso, dizia ele a Engels, em carta de 08 de dezembro de 1857: “Eu trabalho como um louco, noites inteiras adentro, para organizar meus estudos econômicos, para por às claras pelo menos os elementos fundamentais antes do dilúvio” (Marx; Engels, 1983b, p. 217). Em outra carta a Engels, de 18 de dezembro, descreve mais uma vez o ritmo alucinante dos seus trabalhos: “Eu estou trabalhando muito, como regra até às 4 horas da manhã. 5 Como assinala Krätke (2008b, p. 169), “Sem a crise mundial de 1857-8, Marx provavelmen- te não teria escrito os Grundrisse”. de prejuízos para o esforço de elaboração da crítica da economia política o tempo e o trabalho dedicados ao jornalismo. De fato, é preciso ver a atividade jornalística como fornecendo base empírica, que informará e subsidiará a elaboração dos Grundrisse, que sendo um discurso do capital em geral, sobre as formas mais ge- rais de presentifi cação do capital, não prescinde de referências histórico-concretas, que são trazidas ao texto tanto para exemplifi cações, quanto como materializa- 3 Marx (1851/1857): o nascedouro dos Grundrisse Estou envolvido em uma, tarefa du- pla: 1. Elaborar os contornos da economia política (...); 2. A atual crise (...)” (Marx; Engels, 1983b, p. 224). Por ocasião dessa carta, Marx realizava uma dupla empreitada, ilustrando mais uma vez a vinculação entre jornalismo e teoria: ao mesmo tempo que escrevia sua obra teórica, os Grundrisse, compilava os Cadernos da Crise (outubro de 1857 a fevereiro de 1858), que consistia de notas variadas extraídas de jornais sobre a evolução da crise, movimentação da bolsa de valores, falências, etc. (Musto, 2011, p. 58). Esse material totalizaria mais de quinhentas páginas impressas para publi- cação no volume IV/14 da MEGA2 (Krätke, 2008b, p. 175). Sobre os efeitos que as intensas pesquisas desenvolvidas por Marx na fundamentação de seus artigos jornalísticos teve no seu desenvolvimento teórico, os seus depoimentos parecem ser contraditórios. Por um lado, diz, em carta ao amigo Cluss, de 15 de setembro de 1853: “A contínua porca- ria jornalística me aborrece. Toma muito tempo, dispersa meus esforços e, na análise fi nal, não é nada. Por mais independente que se queira ser, ainda se é dependente do jornal (...) Obras puramente científi cas são algo completamente diferente” (Marx; Engels, 1983a, p. 367). Por outro, sugere uma relação positiva entre suas atividades no jornalismo e seus estudos de economia política, no prefácio de Contribuição à crítica da economia política, em 1859: v.29 n.3 2019 Nova Economia 951 Teixeira Teixeira Para Krätke (2007, p. 129), “como um pensador da economia política, Marx aprendeu com seu trabalho jornalístico mais do que ele estava disposto a admitir”. Pino (2013, p. 20) é ainda mais enfático ao dizer que a lista de linhas temáticas trabalhadas por Marx, especialmente no New York Tribune “constitui, desde uma perspectiva teórica, um trabalho de investigação, análise de dados e teste dos conceitos crucial na sua formação, através do qual poderá estabelecer no ano de 1858 – na frenética escritura dos Grun- drisse (1857-1858) – a noção vertebral de sua crítica à econômica política: o conceito de mais-valia”. Para Krätke (2007, p. 129), “como um pensador da economia política, Marx aprendeu com seu trabalho jornalístico mais do que ele estava disposto a admitir”. Pino (2013, p. de prejuízos para o esforço de elaboração da crítica da economia política o tempo e o trabalho dedicados ao jornalismo. De fato, é preciso ver a atividade jornalística como fornecendo base empírica, que informará e subsidiará a elaboração dos Grundrisse, que sendo um discurso do capital em geral, sobre as formas mais ge- rais de presentifi cação do capital, não prescinde de referências histórico-concretas, que são trazidas ao texto tanto para exemplifi cações, quanto como materializa- 3 Marx (1851/1857): o nascedouro dos Grundrisse 20) é ainda mais enfático ao dizer que a lista de linhas temáticas trabalhadas por Marx, especialmente no New York Tribune “constitui, desde uma perspectiva teórica, um trabalho de investigação, análise de dados e teste dos conceitos crucial na sua formação, através do qual poderá estabelecer no ano de 1858 – na frenética escritura dos Grun- drisse (1857-1858) – a noção vertebral de sua crítica à econômica política: o conceito de mais-valia”. A conexão quase instantânea que Marx possuía com os últimos aconte- cimentos lhe permitia diminuir o hiato entre a ocorrência do fato e a sua análise. Se ele não era essencialmente um jornalista, como disse Engels referindo-se ao ritmo da fabricação de suas matérias, é necessário reco- nhecer que ele também tinha adquirido hábitos próprios de um jornalista quanto aos meios usados para ter acesso às informações. Seus defeitos como jornalista eram a razão da alta qualidade dos seus artigos, valoriza- dos nos Estados Unidos por conta da profundidade de suas análises. O uso de suas fontes, relacionamento pessoal e de todos os recursos disponíveis que viabilizasse o acesso a dados de variados tipos, desde relatórios a li- vros de tiragem limitada, era prática típica do jornalismo que desautoriza uma construção caricata de Marx como teórico de gabinete. Ao mesmo tempo que se dedicava à análise da realidade capitalista con- creta e à luta política, esta última como contraface do uso de sua pena, Marx se empenhava em continuar os seus estudos de economia política, abandonados desde sua ida para Colônia. O jornalismo, pois, não consti- tuiu uma “pausa teórica”, um intervalo na trajetória intelectual de Marx. Nas palavras de De Paula (2011, p. 133), não se deve falar 952 Nova Economia v.29 n.3 2019 952 Nova Economia v.29 n.3 2019 952 Marx jornalista ções do movimento geral do capital como “particularidade” e como “singularida- de”, isto é, pela explicitação de suas formas fenomênicas de existência. ções do movimento geral do capital como “particularidade” e como “singularida- de”, isto é, pela explicitação de suas formas fenomênicas de existência. Retome-se, mais uma vez, a discussão sobre o papel fundamental da em- piria para o método dialético de Marx. Enquanto em Miséria da fi losofi a ele hostiliza o método das abstrações de Proudhon, em Introdução de 1857, lon- ge do contexto que impulsionou aquela obra, as abstrações são apresenta- das como instrumento analítico fundamental. 6 “A produção em geral é uma abstração, mas uma abstração razoável, na medida em que, efetivamente sublinhando e precisando os traços comuns, poupa-nos a repetição. Esse caráter geral, contudo, ou esse elemento comum, que se destaca através da comparação, é ele pró- prio um conjunto complexo, um conjunto de determinações diferentes e divergentes. Alguns desses elementos comuns pertencem a todas as épocas, outros apenas são comuns a poucas” (Marx, 1986, p. 4). 7 Por aplicação não se entenda tratar-se de um procedimento específi co, executado externa- mente ao objeto. 3 Marx (1851/1857): o nascedouro dos Grundrisse Nesta, Marx discorre sobre as “abstrações razoáveis”,6 necessárias no processo de elaboração teórica que, diferentemente dos clássicos, teria que incorporar no âmbito de uma totalidade orgânica tanto os aspectos gerais referentes a toda e qualquer sociedade quanto os aspectos específi cos de uma determinada formação social. Ele, portanto, reconhece que há determinações comuns a todos os modos de produção e que elas precisam ser integradas nos diversos ele- mentos do desenvolvimento histórico. Importa, pois, reconhecer também as especifi cidades de cada etapa histórica. Por isso, as determinações mais gerais, juntamente com as específi cas de uma determinada época, se arti- culam dentro das abstrações gerais como totalidades orgânicas. Deve-se ressaltar que as categorias econômicas que vão se constituindo ao longo da análise não são resultado de uma construção idealizada; elas são a ex- pressão de relações reais de produção. Nesse sentido, pode-se dizer que, especialmente quanto à especifi cidade de cada etapa histórica, o conheci- mento fornecido pelos trabalhos jornalísticos seria fundamental para que Marx evidenciasse as determinações próprias e contraditórias do modo de produção capitalista. De fato, a crítica da economia política seria o resultado da aplicação7 de seu método às categorias da economia política analisadas no âmbito de relações históricas determinadas, para a qual concorreu tanto seu intenso trabalho de prospecção de dados empíricos quanto o estudo de inumerá- veis livros nos mais variados terrenos, como economia política, história, fi losofi a, ciências naturais, matemática e, de forma original, também a lite- 953 953 v.29 n.3 2019 Nova Economia 953 Teixeira Teixeira ratura, dado que Marx usa, a todo instante, citações de Shakespeare, Bal- zac, Cervantes, Goethe, e outros, para ilustrar seus argumentos teóricos. Segundo Pino (2013, p. 22), “os artigos do New York Tribune ensinariam a Marx que as contradições geradas pelo capital eram defi nitivamente in- solúveis”. Os Grundrisse são tributários desse amplo conhecimento empíri- co. Com efeito, o caminho teórico em direção à essência da mais-valia, por exemplo, inicia-se depois da análise de suas formas de manifestação. Sem dúvida, Marx havia avançado bastante no conhecimento sobre o funciona- mento concreto do capitalismo. Havia estudado questões complexas e avan- çadas, como as relações entre os mecanismos de circulação monetária e os momentos de crises econômicas e de prosperidade, mas não se sentia ainda capaz de desvendar por completo a natureza interna da sociedade burguesa. A tarefa específi ca do teórico que parte de toda esta informação sobre a realidade não consiste, por assim dizer, em dar a esta expressão “abstrata” uma forma ain- da mais abstrata. Ao contrário, começa sempre procedendo a uma análise crítica de abstrações do nível empírico do conhecimento e as refaz para seguidamente avançar, criticando a estreiteza e o subjetivismo de tais abstrações e destruindo as ilusões que elas contêm desde o ponto de vista da realidade em seu conjunto 8 A perspectiva de Ilienkov é semelhante à de Vygodsky, descrita por Fineschi (2009, p. 58) da seguinte forma: “Temos, então: a) uma íntima ligação entre o modo de exposição e a ele- vação do abstrato ao concreto, b) uma íntima ligação entre o modo de pesquisa e a elevação do concreto para o abstrato, c) a separação, no espaço e no tempo – embora não absoluta – de exposição e pesquisa.” 954 Nova Economia v.29 n.3 2019 3 Marx (1851/1857): o nascedouro dos Grundrisse Nos Grundrisse, Marx busca reunir tudo o que havia apreendido nos seus anos de estudo teórico e de pesquisas empíricas. As experimentações na forma dialética de apresentação dos conceitos da economia política têm a empiria como um recurso imprescindível. Se no caminho que vai do concreto ao abstrato, a realidade mesma é o ponto de partida da teoria científi ca, no caminho de volta, do abstrato ao concreto, embora lógico, encontra correspondência também no próprio processo real da história. Entretanto, é preciso diferenciá-los, pois a exposição, como momento que sucede a investigação, busca ordenar, fazer o encadeamento lógico dos fatos já pesquisados. Embora o presente trabalho não tenha a pretensão de analisar a “ope- racionalização” do método de Marx, a maneira diferenciada como a em- piria é absorvida na exposição, vale recuperar indicações feitas por Ilien- kov (1978, p. 34) em obra clássica, ao apontar para uma distinção entre o método dialético de tratamento dos fatos/dados empíricos e o método de elevar-se do abstrato ao concreto,8 considerando apenas uma exceção: 8 A perspectiva de Ilienkov é semelhante à de Vygodsky, descrita por Fineschi (2009, p. 58) da seguinte forma: “Temos, então: a) uma íntima ligação entre o modo de exposição e a ele- vação do abstrato ao concreto, b) uma íntima ligação entre o modo de pesquisa e a elevação do concreto para o abstrato, c) a separação, no espaço e no tempo – embora não absoluta – de exposição e pesquisa.” 954 Nova Economia v.29 n.3 2019 954 Marx jornalista concreto. Neste sentido (e somente neste sentido), o passo do estado empírico ao estado racional do conhecimento aparece também como um passo “do abstrato ao concreto”. Em suma, o tratamento dos dados empíricos que subsidiam a análise de Marx é, em si, um procedimento dialético. Como diz Ilienkov (1978, p. 34), “este procedimento de análise permanece o mesmo, isto é, dialético, tanto no tratamento preliminar dos dados, como no curso de sua elabora- ção defi nitiva, ainda que, naturalmente, vá se aperfeiçoando na medida em que avança em direção a O capital”. 3 Marx (1851/1857): o nascedouro dos Grundrisse A título de ilustração, a descoberta da mais-valia foi resultado desse método, pois Marx partiu da circulação, do mundo visível, para chegar à conclusão de que a geração de valor novo só poderia ser encontrada em outra esfera, a produção, uma descoberta que aparece inicialmente em Miséria da fi losofi a, mas que só se potencializa metodologicamente com os seus primeiros artigos jornalísticos da década de 1850. Ressalte-se, to- davia, que, ao longo daqueles anos, a profundidade e o acurado espírito crítico dos seus textos jornalísticos eram refl exos do aprimoramento para- lelo dos seus conhecimentos teóricos. Como projeto nunca abandonado, a crítica da economia política requeria, especialmente a partir de Miséria da fi losofi a, que Marx elaborasse uma teoria do valor própria. Por isso, o estu- do dos economistas políticos, notadamente aquele que foi sua referência maior naquela obra, David Ricardo, era um exercício obrigatório desde sua chegada a Londres. 4 Jornalismo e teoria: os Cadernos de Londres (1850- 1853) e o epistolário Marx-Engels O período de elaboração dos Cadernos de Londres é entrecortado por diver- sas outras atividades e acontecimentos de cunho político e pessoal. Marx volta-se para os estudos teóricos, em relação aos quais suas cartas refl etem todo um conjunto de questões, dúvidas e descobertas que acompanhavam a própria evolução de suas pesquisas. Como já mencionado, o período de isolamento, entre os anos de 1850 e 1851, permitiu que Marx retornasse às suas pesquisas sobre economia política. Nesse período, há um incremento acentuado no número de cartas trocadas por Marx e Engels, que era soli- citado continuamente por aquele para dar sua opinião sobre as diversas questões teóricas que estava analisando. A partir de 1853 a quantidade 955 v.29 n.3 2019 Nova Economia Teixeira de correspondências entre Marx e Engels avoluma-se novamente. Assim, não tendo Marx publicado obras econômicas no período entre a Miséria da fi losofi a e os Grundrisse, sua evolução teórico-metodológica entre 1850 e 1856 encontra registros tanto em suas anotações nos Cadernos de Londres9 quanto nas diversas cartas escritas. De setembro de 1850 a fevereiro de 1862, os estudos teóricos serão feitos, em maior ou menor intensidade, simultaneamente às atividades jornalísticas. Dessa forma, na esteira do reconhecimento da importância dos traba- lhos jornalísticos de Marx para sua elaboração teórica, cabe recuperar al- gumas das suas aquisições teóricas dessa fase. Foi com o iminente fecha- mento da Nova Gazeta Renana – Revista Político-Econômica que Marx voltou a estudar economia política. O último número da revista foi publicado em novembro, já sob a nuvem negra da falência. Antes disso, em setembro de 1850, Marx dá início aos seus estudos teóricos, aproveitando-se do rico acervo do Museu Britânico. O jejum teórico de três anos é, pois, rompido com uma incessante pesquisa, os chamados Cadernos de Londres, compila- dos em 24 cadernos de notas durante os três anos seguintes, de setembro de 1850 a agosto de 1853. De acordo com a esquematização proposta por Musto (2011, p. 44-49), os Cadernos de Londres10 podem ser divididos em três grupos, conforme Tabela 1, adiante. O estudo sobre as crises econômicas feito nos primeiros cadernos, e que redundou no último número da Nova Gazeta Renana – Revista Político- -Econômica, deu a Marx a percepção de que a revolução só aconteceria no ambiente de uma crise econômica. 9 Quanto aos Cadernos de Londres, há autores que mencionam sua publicação tardia na MEGA2 (1983-1991) como um dos fatores fundamentais que impossibilitou melhor com- preensão do método de Marx (Fineschi, 2009, p. 58). 10 Obviamente, esta seção não pretende percorrer os Cadernos de Londres em todos os seus detalhes, mas apenas extrair alguns elementos essenciais da evolução intelectual de Marx. Por isso, além da esquematização e comentários de Musto (2011a), esta seção se vale da análise pormenorizada sobre esse tema feita por Leonardo de Deus (2010), e, em menor medida, do texto de Rubel (1974, p. 312-325). 9 Quanto aos Cadernos de Londres, há autores que mencionam sua publicação tardia na MEGA2 (1983-1991) como um dos fatores fundamentais que impossibilitou melhor com preensão do método de Marx (Fineschi, 2009, p. 58). 4 Jornalismo e teoria: os Cadernos de Londres (1850- 1853) e o epistolário Marx-Engels A análise das crises econômicas tor- nava-se, assim, objeto múltiplo do interesse de Marx, por constituir tema vital tanto para suas investigações teóricas quanto para suas atividades jornalísticas, e também para os projetos políticos posteriores. Marx estuda incessantemente todos aqueles autores do primeiro grupo, que, excluído o Caderno VII, totalizam cerca de 600 páginas impressas da seção IV, volume V da MEGA2. Ainda não havia iniciado o Caderno VIII 956 Nova Economia v.29 n.3 2019 Marx jornalista quando escreve a Engels, em carta de 02 de abril de 1851, informando o estado adiantado de suas pesquisas (Marx; Engels, 1982, p. 325). Tabela 1 Cadernos de Londres (1850-1853) Grupo Período Cadernos Principais temas e autores analisados 1º Set./1850 - mar./1851 I-VII História e teorias das crises econômicas, dinheiro e crédito / Thomas Tooke, James Taylor, Henry Thornton, Adam Smith, David Ricardo 2º Abr./1851 - nov./1851 VIII Salário, renda da terra / David Ricardo, James Steuart IX-X Obras críticas sobre Ricardo / John Tuckett, Thomas Hodgskin, Thomas Chalmers, Richard Jones, Henri Carey XI Condição da classe trabalhadora – salários, padrões de vida dos trabalhadores, greves, trabalho infantil / J. Fielden, P. Gaskell, Thomas Hodgskin (1) XII-XIII Química agrária – renda da terra / Justus Liebig, James F. W. Johnston XIV Debate sobre a teoria da população, modos pré-capitalistas de produção, colonialismo / Thomas Malthus, Archibald Alison, Adolphe D. de La Malle, William H. Prescott. XV História da tecnologia / Johann Poppe, J. Gray XVI Diversas questões de economia política / Bastiat, Proudhon (2) 3º Abr./1852 - ago./1853 XVII-XXIV Controvérsias históricas sobre a Idade Média, história da litera- tura, da cultura e dos costumes. Fonte: Musto (2011). (1) Rubel (1974, p. 315); (2) Rubel (1974, p. 318). Caderno escrito entre outubro e novembro de 1851. Tabela 1 Cadernos de Londres (1850-1853) (1) Rubel (1974, p. 315); (2) Rubel (1974, p. 318). Caderno escrito entre outubro e novembro de 1851. Ricardo era o alvo principal de Marx, que reserva a ele os Cadernos IV, VII e VIII, considerados “a parte mais importante dos [Cadernos de Londres] de- vido aos inúmeros comentários e refl exões pessoais que as acompanham” (Musto, 2011, p. 44).11 Quando termina o Caderno VII, Marx escreve dois outros cadernos de notas, sob o título Ouro: o sistema monetário perfeito, con- siderada por Musto (2011, p. 1 Parte dos extratos sobre Ricardo, constantes dos Cadernos IV e VIII, aparecem no 3º volu- me da edição dos Grundrisse, publicada pela Siglo Veintiuno Editores (2006). 4 Jornalismo e teoria: os Cadernos de Londres (1850- 1853) e o epistolário Marx-Engels 45) como, possivelmente, a “primeira formu- lação autônoma de Marx sobre a teoria do dinheiro e da circulação”. Marx retorna a Ricardo no Caderno VIII. Sua investigação tinha avança- do anteriormente pelas concepções ricardianas sobre a renda da terra, con- forme carta enviada a Engels, em 07 de janeiro de 1851, que lhe responde 11 Parte dos extratos sobre Ricardo, constantes dos Cadernos IV e VIII, aparecem no 3º volu- me da edição dos Grundrisse, publicada pela Siglo Veintiuno Editores (2006). v.29 n.3 2019 Nova Economia 957 Teixeira em 29 de janeiro, em tom aprobatório, sugerindo que Marx se apressasse para publicar sua economia (Marx; Engels, 1982, p. 271). Na carta seguinte, de 03 de fevereiro de 1851, Marx pretende discutir com Engels a currency- theory, e escreve a ele resumindo conclusões que já apareciam no seu Ca- derno IV. Na referida carta, além de resumir parte do conteúdo do Caderno IV, fi ca explícito o recurso aos dados empíricos: Tooke faz a mesma afi rmação; mas não encontrei nenhuma prova em sua his- tória dos preços para 1843-1847. Note a importância do assunto. Em primeiro lugar, toda a teoria da circulação se encontra comprometida em sua própria base. Em segundo lugar, fi ca demonstrado como o desenvolvimento das crises, em que pese o sistema de crédito ser uma de suas condições, não tem relação com a cur- rency, e que as insensatas regulamentações estatais podem ainda mais agravar (como em 1847) a crise. (Marx; Engels, 1982, p. 275). A evolução nos conhecimentos de economia, notadamente quanto ao di- nheiro, renda da terra, crédito, crises etc., não eram ainda sufi cientes para ensejar a construção de sua crítica. Com efeito, Marx não tinha ainda, nes- sa época, compreendido a dupla natureza da mercadoria e do trabalho, lacunas que obstaculizavam a progressão dos seus conhecimentos sobre os assuntos que já vinha estudando, ao mesmo tempo que o impulsionava a investigar mais. No Caderno VIII, escrito entre abril e maio de 1851, Marx prossegue com a análise de Ricardo, que se estende, pelos Cadernos IX e X, escrito entre maio e julho de 1851, nas leituras que faz sobre os críticos daquele autor. O Caderno VIII se notabiliza, entre outros motivos, porque é nele que reaparece a intuição12 sobre a mais-valia, anteriormente esboçada em Trabalho assalariado e capital. 12 “Ele troca seu produto por outro cujo valor está determinado pelo tempo de trabalho nele empregado (...) O excedente não surge da troca, ainda que tão somente nela se realize. Surge de que deste produto que consome 20 dias de trabalho, o obreiro só obtém o produto de 10 etc., dias de trabalho. Na mesma medida em que cresce a força produtiva do trabalho, decresce o valor do salário” (Marx, 2006, p. 77). 4 Jornalismo e teoria: os Cadernos de Londres (1850- 1853) e o epistolário Marx-Engels Tendo aceitado o convite para trabalhar como jornalista na New York Tribune, em agosto de 1851, Marx, paralelamente, continua suas pesquisas. Apesar de Engels ter insinuado, em carta de 29 de janeiro, que a “teoria da renda da terra” de Marx poderia até mesmo ser publicada (Marx; Engels, 1982, p. 271), ele continuava a buscar no mundo real elementos para as so- luções teóricas que ainda necessitava. Assim, entre setembro e novembro de 1851, escreve o Caderno XV, com temas sobre tecnologia, e o Caderno XVI, sobre temas diversos da economia política. Nova Economia v.29 n.3 2019 958 Marx jornalista Apesar das múltiplas tarefas, já que, em março de 1852 – paralelamente à elaboração de O 18 Brumário – começa, de fato, a escrever para a New York Tribune (Krätke, 2007, p. 150), Marx inicia, em abril de 1852, o ter- ceiro grupo de cadernos, que consubstanciam uma grande dispersão de temas (Musto, 2011, p. 49). Esses cadernos são fi nalizados em agosto de 1853, quando, então, Marx interrompe novamente seus estudos teóricos. No mês seguinte, escreve a Cluss, dizendo que tinha esperança de voltar a se isolar para trabalhar novamente na sua Economia (Marx; Engels, 1983a, p. 367). Isso acontece, como indica Musto (2011, p. 54), entre o fi m de 1854 e o início de 1855, prosseguindo até meados desse ano. Com a nova interrupção de suas pesquisas ocorrida em meados de 1855, Marx só iria retornar aos estudos teóricos em junho de 1856, com os artigos para o The People’s Paper sobre o Crédit Mobilier, em grande medida por causa de suas obrigações como jornalista. A crise ainda não havia chegado e Marx intensifi ca seus estudos, que culminam com o início de elaboração dos Grundrisse, em agosto de 1857. A busca por dados empíricos e por todo tipo de informação útil para a construção da teoria se acelera. Como já mencionado, entre outubro de 1857 e fevereiro de 1858, Marx escreve os Cadernos da Crise. 4 Jornalismo e teoria: os Cadernos de Londres (1850- 1853) e o epistolário Marx-Engels Também nes- se período, as “cartas teóricas” entre Marx e Engels adquirem um perfi l mais próximo de “cartas empíricas”, pois, estando Engels mergulhado no dia a dia da indústria em Manchester, mantinha-se em condições de enviar a Marx dados sobre a crise, além de uma série de informações de cará- ter prático, relacionadas ao funcionamento das máquinas, ciclo de vida, disponibilidade de determinadas matérias-primas, situação de mercado de algumas indústrias, nível salarial etc. Entre os dias 07 e 17 de dezembro de 1857, Engels escreve quatro cartas a Marx com as informações referidas. Em 29 de janeiro de 1858, Marx es- pecifi ca ainda mais o pedido: “acabo de chegar a um determinado ponto, em meus trabalhos econômicos, sobre o qual desejo algumas explicações práticas, porque não tenho encontrado nada sobre isto nas obras teóricas” (Marx; Engels, 1983b, p. 256). Em outra carta, de 02 de março de 1858, Marx pede a Engels informações sobre o período médio de tempo para a renovação das máquinas (Marx; Engels, 1983b, p. 278). Engels responde com carta de 04 de março, que é seguida por outra de Marx, de 05 de mar- ço, agradecendo as respostas e enviando outras perguntas (Marx; Engels, 1983b, p. 279-284). v.29 n.3 2019 Nova Economia 959 Teixeira Teixeira Categorias econômicas como capital variável, capital constante, com- posição orgânica, força de trabalho, e sobretudo a mais-valia, são apreen- didas por Marx não como construções arbitrárias de um pesquisador, mas como traços constitutivos do objeto analisado. A descoberta dessas ca- tegorias nos Grundrisse seria legatária tanto da trajetória teórica iniciada com a Crítica de 43 quanto das vicissitudes do laboratório da vida real na Londres dos anos 1850. 960 Nova Economia v.29 n.3 2019 5 Considerações fi nais Em certo sentido, é possível dizer que os Grundrisse representaram um recomeço para Marx, o que explica o fato de muitos analistas não leva- rem em conta os primeiros anos da década de 1850, ou simplesmente os caracterizarem como meros “antecedentes dos Grundrisse”, dando, assim, um salto de Miséria da fi losofi a para os Grundrisse. Esta obra aparece, então, em algumas análises, como um momento mágico de Marx, quando toda a sua inspiração teórica fl oresce, em questão de meses, e se consubstan- cia numa obra magistral. Esquece-se, como já apontado na seção anterior, que os anos desde 1848 foram, de fato, o nascedouro dos Grundrisse, uma espécie de período de gestação da obra, fi lho temporão, que só veio à luz sob intensas dores de parto. Diga-se que, se os Grundrisse são um ponto de partida da crítica, são também um ponto de chegada de um longo período de investigação, o que invoca uma reconfi guração no peso dos anos de intermitentes estudos teóricos entre 1850 e 1856 para a compreensão do pensamento de Marx, pois realçam a mutualidade entre empiria e teoria na elaboração da crítica da economia política, um dos traços característicos do seu método dialético. Os anos de Marx em Londres, especialmente os que antecederam os Grundrisse, precisam ser compreendidos como integrantes do itinerário teórico por ele percorrido desde a Crítica de 43 até a realização da crítica da economia política, consumada naquela obra. Naquela metrópole, li- dou com imensa quantidade de informações relativas a aspectos práticos do funcionamento das máquinas, legislação fabril, tecnologia e diversos outros elementos que lhe seriam úteis na construção de categorias eco- nômicas dentro dos Grundrisse. Por isso, a caracterização de Marx nessa época como, primordialmente, um economista crítico. Não era, a rigor, 960 Nova Economia v.29 n.3 2019 960 Nova E 960 Marx jornalista um economista, no sentido tradicional do termo. Sempre que entrava na seara dos economistas políticos, era para sair dela mais à frente, para exer- citar a crítica, o método pelo qual aqueles conhecimentos eram analisados, incorporados ou descartados de forma parcial ou integral, e depois supra- -assumidos. Com efeito, Marx só se tornou Marx a partir do momento em que ela- borou sua crítica da economia política, quando, enriquecido pelas determi- nações empíricas da sociedade capitalista, conseguiu, fi nalmente, submeter o objeto da economia política a uma operação específi ca, embora de cariz hegeliano. 5 Considerações fi nais Mas isso não signifi ca que tenha havido um Marx fi lósofo que depois se tornou um Marx economista. Como demonstrado, Marx nunca deixou de ser fi lósofo. A reconstrução de sua trajetória intelectual, notada- mente a etapa referente ao laboratório londrino, permitiu apreender uma linha de continuidade, um fi o condutor, que norteou os seus estudos desde A crítica de 43: a busca pela compreensão da forma de ser da sociedade burguesa. E a partir daí, o caminho que leva Marx de uma etapa exclusiva- mente fi losófi ca à crítica da economia política é permeado de sinuosidades, capazes de confundir os observadores não dialéticos, levando-os a enxergar os períodos da evolução teórica de Marx como compartimentos estanques. Referências BORN, Stephan. Stephan Born. In: MCLELLAN, David (Org.). Karl Marx: Interviews and Recollections. Londres: Macmillan, 1981. p. 16. CHASIN, José. Marx no tempo da Nova Gazeta Renana. Prefácio. In: MARX, Karl. A burguesia e a contra-revolução. 3. ed. São Paulo: Ensaio, 1993. p. 15-40. CLAUDÍN, Fernando. Marx, Engels y la Revolución de 1848. 4. ed. Madri: Siglo XXI, 1985. DE DEUS, Leonardo. Reconstrução categorial de O Capital à luz de seus esboços. A instauração da crítica da economia política (1857,1863). 259 f. Tese (Doutorado em Economia) – Fa- culdade de Ciências Econômicas, Cedeplar, Universidade Federal de Minas Gerais. Belo Horizonte, 2010. DE PAULA, João. A. Marx 1857/1858: além dos Grundrisse. Revista da Sociedade Brasileira de Economia Política. São Paulo, n. 30, p. 123-142, out. 2011. ENGELS, Friedrich. Introdução. Lutas de classes na França de 1848 a 1850. In: MARX, Karl; Engels Friedrich. Obras escolhidas. São Paulo: Alfa-Ômega, 1953. v. 1. p. 93-198. ENZENSBERGER, Hans. M. Conversaciones con Marx y Engels. Barcelona: Editorial Anagrama, 2009. 961 v.29 n.3 2019 Nova Economia Teixeira Teixeira FINESCHI, Roberto. Dialectic of the Commodity and Its Exposition: The German Debate in the 1970s – a Personal Survey. In: BELLOFIORE, R.; FINESCHI, R. Re-reading Marx: New Perspectives after the Critical Edition. Hampshire: Palgrave Macmillan, 2009. cap. 4, p. 50-70. HEINRICH, Michael. Karl Marx e o nascimento da sociedade moderna. São Paulo: Boitempo, 2018. v. 1. ILIENKOV, Evald V. Elevar-se de lo abstrato a lo concreto. In: DIAS, P. L. (Org.). El capital, teoria, estructura y método. México: Ediciones de Cultura Popular, 1978. V. 1. p. 27-83. KRÄTKE, Michael. R. Journalisme et science. L’importance des travaux journalistiques de Marx pour la critique de l’économie politique. Actuel Marx, n. 42, p. 128-163, 2007. KRÄTKE, Michael. R. The First World Economic Crisis: Marx as an Economic Journalist. In: MUSTO, Marcello. Karl Marx’s Grundrisse: Foundations of the Critique of Political Economy 150 Years Later. New York: Routledge, 2008a. cap. 10. p. 162-168. KRÄTKE, Michael. R. Marx’s ‘Books of CRISIS’ of 1857-8. In: MUSTO, Marcello. Karl Marx’s Grundrisse: Foundations of the Critique of Political Economy 150 Years Later. New York: Routledge, 2008b. cap. 10. p. 169-175. MCLELLAN, David. Karl Marx. Vida e pensamento. Petrópolis: Vozes, 1990. MARX, Karl. Para a crítica da economia política. Salário, preço e lucro. O rendimento e suas fontes. 2. ed. São Paulo: Abril Cultural, 1986. MARX, Karl. Referências Elementos Fundamentales para la crítica de la economía política (Grundrisse) 1857- 1858. 13. ed. Madri: Siglo Veintiuno Editores, 2006.v. 3. MARX, Karl. Nova Gazeta Renana. Tradução de Lívia Cotrim. São Paulo: Educ, 2010. MARX, Karl; ENGELS, Friedrich. Collected Works. Moscou: Progress, 1982. v. 38. MARX, Karl; ENGELS, Friedrich. Collected Works. Moscou: Progress, 1983a. v. 39. MARX, Karl; ENGELS, Friedrich. Collected Works. Moscou: Progress, 1983b. v. 40. MARX, Karl; ENGELS, Friedrich. Collected Works. Moscou: Progress, 1986. v. 15. MUSTO, Marcello. A formação da crítica de Marx à economia política: dos estudos de 1843 aos Grundrisse. Revista Crítica Marxista. São Paulo, n. 33, p. 31-65, 2011. PINO, Mario Espinosa. Introducción. In: MARX, Karl. Artículos periodísticos. Barcelona: Alba Clásica, 2013. p. 8-27. RUBEL, Maximilien. Les cahiers d’études de Marx. In: RUBEL, Maximilien. Marx: critique du marxisme. Paris: Payot, 1974. p. 301-359. RUGE, Arnold. Arnold Ruge. In: MCLELLAN, David (Org.). Karl Marx: Interviews and Recol- lections. Londres: Macmillan, 1981. p. 8-9. Sobre o autor Adriano Lopes Almeida Teixeira – adriano.teixeira@ufes.br Universidade Federal do Espírito Santo, Vitória, Espírito Santo, Brasil. ORCID: https://orcid.org/0000-0001-9881-5483. Adriano Lopes Almeida Teixeira – adriano.teixeira@ufes.br Universidade Federal do Espírito Santo, Vitória, Espírito Santo, Brasil. ORCID: https://orcid.org/0000-0001-9881-5483. Adriano Lopes Almeida Teixeira – adriano.teixeira@ufes.br Universidade Federal do Espírito Santo, Vitória, Espírito Santo, Brasil. ORCID: https://orcid.org/0000-0001-9881-5483. Sobre o artigo Recebido em 23 de janeiro de 2018. Aprovado em 18 de dezembro de 2018. 962 Nova Economia v.29 n.3 2019 962
https://openalex.org/W2125222922
https://europepmc.org/articles/pmc3480903?pdf=render
English
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Oral health-related quality of life in an aging Canadian population
Health and quality of life outcomes
2,012
cc-by
9,676
Abstract Background: The purpose of the study is to describe the impact of oral health-related quality of life (OHRQoL) on the lives of pre-seniors and seniors living in Nova Scotia, Canada. Methods: This cross-sectional study involved 1461 participants, grouped by age (pre-seniors [45–64] and seniors [65+]) and residential status (long-term care facility [LTC] or community). OHRQoL was measured using the 14-item Oral Health Impact Profile questionnaire (OHIP-14) in a random digit dialing telephone survey (for community residents) or a face-to-face interview (for LTC residents). Intra-oral examinations were performed by one of six dentists calibrated to W.H.O. standards. Results: Approximately one in four pre-seniors and seniors reported at least one OHRQoL impact ‘fairly/very often’. The most commonly reported impacts were within the dimensions ‘physical pain’ and ‘psychological discomfort’. It was found that 12.2% of LTC residents found it uncomfortable to eat any foods ‘fairly/very’ often compared to 7.7% in the community, and 11.6% of LTC residents reported being self-conscious ‘fairly/very often’ compared to 8.2% in the community. Of those residing in the community, pre-seniors (28.8%) reported significantly more impacts than seniors (22.0%); but there were no significant differences in OHRQoL between pre-seniors (21.2%) and seniors (25.3%) in LTC. Pre-seniors living in the community scored significantly higher than community dwelling seniors on prevalence, extent and severity of OHIP-14 scores. Logistic regression revealed that for the community dwelling sample, individuals living in rural areas in addition to those being born outside of Canada were approximately 2.0 times more likely to report an impact ‘fairly/very often’, whereas among the LTC sample, those having a high school education or less were 2.3 times more likely to report an impact. Conclusions: Findings indicate that the oral health and OHRQoL of both pre-seniors and seniors in LTC residents is poor. Community dwelling pre-seniors have the highest prevalence rate of oral impacts. Keywords: Oral health, Quality of life, Elderly, Aging, Seniors, Pre-seniors, Canada Keywords: Oral health, Quality of life, Elderly, Aging, Seniors, Pre-seniors, Canada population in 2033 is estimated to be 257,874, an in- crease of 86.3% from 2007 [2]. Due to the aging of the population and increased purchasing power of today’s elderly, more people are taking advantage of the advancements in dental healthcare, leading to a decrease in rates of edentulism [5-8]. Open Access Open Access Kotzer et al. Health and Quality of Life Outcomes 2012, 10:50 http://www.hqlo.com/content/10/1/50 Oral health-related quality of life in an aging Canadian population Robert D Kotzer1*, Herenia P Lawrence1†, Joanne B Clovis2† and Debora C Matthews3† Robert D Kotzer1*, Herenia P Lawrence1†, Joanne B Clovis2† and Debora C Matthews3† © 2012 Kotzer et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract As a result of living longer and retaining more of their natural teeth, more oral pro- blems arise and the treatment decisions of these patients becomes much more complex [5,9]. It is therefore im- perative that information regarding the current oral health status, treatment needs, as well as the oral health- related quality of life (OHRQoL) of aging Canadians is collected in order to guide oral health policy. In the field of dentistry, the term “oral health-related quality of life” is commonly used to describe the impact that one’s oral * Correspondence: r.kotzer@utoronto.ca †Equal contributors 1Discipline of Dental Public Health, Department of Biological and Diagnostic Sciences, Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, Ontario M5G 1G6, Canada Full list of author information is available at the end of the article Background Compared to previous decades, the elderly population today is much more predominant in Canada and con- tinues to rapidly increase due to longer life expectancy and the effects of the baby boom generation [1-3]. As these individuals (born between 1947 and 1966) begin to turn 65 years of age (in 2012), the number of seniors in Canada is estimated to jump from 4.2 million to 9.8 mil- lion from 2005 to 2036 [4]. In Nova Scotia, the seniors’ * Correspondence: r.kotzer@utoronto.ca †Equal contributors 1Discipline of Dental Public Health, Department of Biological and Diagnostic Sciences, Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, Ontario M5G 1G6, Canada Full list of author information is available at the end of the article Page 2 of 12 Kotzer et al. Health and Quality of Life Outcomes 2012, 10:50 http://www.hqlo.com/content/10/1/50 Kotzer et al. Health and Quality of Life Outcomes 2012, 10:50 http://www.hqlo.com/content/10/1/50 Kotzer et al. Health and Quality of Life Outcomes 2012, 10:50 http://www.hqlo.com/content/10/1/50 of Health. Nursing Homes (homes for the aged) meet the needs of people who require a high level of personal care and professional nursing care. These facilities are licensed and inspected by the Department of Health. Residential Care Facilities are homes for people in need of supervision and limited help with personal care. These facilities are also licensed and inspected by the Depart- ment of Health. Community Based Options provide a similar level of care that residential care facilities offer but only accommodate a maximum of three people in each home. These facilities are unlicensed, but are inspected and approved by the Department of Health. No Community Based Options were included in this study because they did not meet the requirement of ac- commodating at least 20 beds. This study did not distin- guish between Residential Care Facilities and Nursing Homes. LTC residents pay for accommodation charges (including salaries, benefits and operational costs of LTC employees) and personal expenses (including dental ser- vices and transportation) [17]. health can have on their everyday life experiences [10,11]. The shift towards the importance of measuring one’s oral health-related quality of life reflects the reality that modern dentistry is not just aiming to prolong life or eliminate oral disease, but ultimately is attempting to make life better [12,13]. Measurements l h l h Oral health-related quality of life was measured using the 14-item Oral Health Impact Profile questionnaire (OHIP-14) [18]. This questionnaire was administered through a random digit dialing telephone survey, com- pleted by a Toronto-based telephone marketing company (for those living independently in the community), or a face-to-face interview (for those in LTC). The OHIP-14 was also translated into Acadian French (the local dia- lect). For each of the 14 items contained in the OHIP-14, study members were asked how often they had experi- enced the problem in the past year. Responses were coded as ‘very often’ (scoring 4), ‘fairly often’ (scoring 3), ‘occasionally’ (scoring 2), ‘hardly ever’ (scoring 1) or ‘never’ (scoring 0). This self-report questionnaire con- tains seven domains including: functional limitation, physical pain, psychological discomfort, physical dis- ability, psychological disability, social disability and handicap. Study Design This cross-sectional study is part of a larger survey – The Oral Health of our Aging Population (TOHAP) – con- ducted in the province of Nova Scotia, Canada in 2008–09 [14]. The primary objective of TOHAP was to understand how the oral health and expectations of the baby boomer (45 yr-64 yr) generation differed from those preceding them (65 yr+) for the purpose of planning and creating policy. The participants were grouped by age (pre-seniors [aged 45 yr-64 yr] and seniors [65 yr+]), geographic loca- tion (urban or rural) and residential status (long-term care facility [LTC] or community dwelling). A pilot study was conducted prior to this survey to test the survey instru- ments and determine feasibility [15]. Ethics approval was received from the Health Sciences Research Ethics Board at Dalhousie University. Community sites were selected based on proximity to previously selected LTC facilities. In total, 22 Nova Sco- tian communities were chosen. Sample Size Determination A sample size calculation based on known population prevalence rates determined the minimal sample size required was 382 pre-seniors, 382 seniors and 359 LTC residents [16]. This was adjusted to allow for a 10% cancellation rate of appointments. Background This study examines the differ- ences in oral health-related quality of life between people aged 45 years and older living in the community and in long-term care (LTC) facilities in Nova Scotia. It also addresses the differences in oral health-related quality of life between pre-seniors and seniors within the commu- nity and LTC residences. Discussions regarding the dis- parities among these populations along with possible solutions to these problems are also explored. Sampling Frame and Sample Selection All private and government owned LTC facilities with at least 20 beds per facility were included in the sampling frame. A total of 102 LTC facilities were used to deter- mine the sampling frame. The LTC samples were pro- portionate to size (small, medium or large) as well as location (rural or urban). A small facility had 20–34 beds, a medium sized facility had 35–101 beds and a large facility had greater than 102 beds. The study was completed in 31 LTC facilities across 21 communities in Nova Scotia (NS), Canada. Four private facilities and 28 public facilities were sampled. In addition to the OHIP-14 questions, additional ques- tions were derived from the 2007–09 Canadian Health Measures Survey [19]. These questions included: demo- graphic information (age, sex, education, etc.), oral health questions (personal oral care habits and oral health care services utilization), general health questions, medication use, labour force activity (income and em- ployment status) and questions regarding smoking and alcohol exposure. There are three kinds of publicly funded LTC facilities that are all licensed and approved by the NS Department Comprehensive intra-oral examinations were per- formed after the interview by one of six dentists, Page 3 of 12 Kotzer et al. Health and Quality of Life Outcomes 2012, 10:50 http://www.hqlo.com/content/10/1/50 Kotzer et al. Health and Quality of Life Outcomes 2012, 10:50 http://www.hqlo.com/content/10/1/50 Kotzer et al. Health and Quality of Life Outcomes 2012, 10:50 http://www.hqlo.com/content/10/1/50 or ‘very often’; (iii) the extent, which is the number of items reported ‘fairly often’ or ‘very often’ ranging from 0 to 14 [21]. Descriptive and inferential statistics were computed using SPSS version 19. The non-parametric Mann-Whiney U test was used to compare the mean ex- tent and severity of oral health impacts between pre- seniors and seniors living in the community or in LTC residences. A chi-square analysis for categorical variables and logistic regression (using a stepwise and forward technique based on the Wald statistic) were used to identify factors related to prevalence of oral impacts for community dwelling and LTC residents. Statistical tests were two-tailed and interpreted at the 5% significance level. The variables that were inserted into the multivari- ate analysis and are thus being controlled for include: age, community type (rural vs. Sampling Frame and Sample Selection urban), sex, having a high school education, perceived general health, perceived quality of life, perceived mouth health, satisfaction with one’s teeth or dentures, frequency of dental visits, having dental insurance, smoking, household income, oral pain, dentate status and country of birth. The measures of se- verity and extent of OHRQoL impacts were not used as outcomes in multivariate analyses due to their skewed distributions. calibrated according to the standards of the World Health Organization (dentists were calibrated by a W.H. O. standardized dentist) [20] and the oral examination procedures used are reported elsewhere [14]. Results Since there is such a small portion of Nova Scotia residents who live in LTC facilities (approximately 5%) [2], LTC residents were over-sampled in this study in order to gain enough power to identify an effect dur- ing statistical analyses. They represented 22.6% of the study population (Table 1). LTC residents were signifi- cantly more likely to be aged 65 and older, be female, edentulous, have a high school education or less, a household income of less than $30,000/yr, visit a den- tal professional less than once per year, brush their teeth less than twice per day (dentate only), floss their teeth less than once per day (dentate only) and were less likely to have dental insurance or be daily smo- kers (Table 1). In addition, LTC residents were signifi- cantly more likely to perceive their general health, quality of life and mouth health as fair or poor but have less oral pain than their community-dwelling counterparts (Table 2). Procedures A call list was assembled for each community targeting pre-seniors and seniors living within a 20 km radius of the community. From each call list, numbers were chosen randomly and called until contact with the indi- vidual was made, or three calls were made without con- tact. Only those who were able to provide informed consent to complete the telephone survey and the clin- ical exam were included in the study. Informed consent was accepted in writing or verbally. Once informed consent was obtained for community dwellers, an interview was done over the phone in either English or French, using a script developed for the study. However, none of the community residents and only 5 of the LTC residents completed the interview in French. There were no problems specific to the OHIP interview by telephone. The same telephone interviewers were used throughout the data collection period. After the interview was conducted, participants sched- uled appointments for a clinical exam within two weeks of the interview. Appointments for the clinical exams were scheduled using an online appointment system and conducted at hospitals, local private dental offices, long- term care facilities and public health offices. Approxi- mately one-third of daily appointments were double booked to compensate for those who did not show up to their appointment. In LTC facilities interviews were con- ducted in person by a trained research assistant, followed immediately by an onsite clinical exam. As an incentive to complete the study, all participants were placed in contention to win one of two $250 prizes by means of a lottery (upon completion of the study). Data were col- lected in two different collection periods. The first col- lection period took place in the fall of 2008, and the second in the spring and summer of 2009. Further details related to the methodology of this study can be found in a separate publication [14]. Data Analysis S i d Health and Quality of Life Outcomes 2012, 10:50 http://www.hqlo.com/content/10/1/50 Table 1 Characteristics of study participants aged 45 years and older living in the community or long-term care in Nova Scotia, Canada Characteristic % (N) Total Community LTC 100 (1461) 77.4 (1131) 22.6 (330) Age (yrs) 45 – 64 45.3 (662) 55.6 (629) 10 (33) 65 and over 54.7 (799) 44.4 (502) 90 (297) Sex Male 34.5 (504) 37.1 (420) 25.5 (84) Female 65.5 (957) 62.9 (711) 74.5 (246) Education Level More than high school 49.3 (718) 57.6 (649) 21.0 (69) High school or less 50.7 (738) 42.4 (478) 79.0 (260) Community Type Urban 59.1 (864) 59.2 (669) 59.1 (195) Rural 40.9 (597) 40.8 (462) 40.9 (135) Annual Household Income More than $30,000 57.6 (643) 72.0 (618) 9.7 (25) Less than $30,000 42.4 (474) 28.0 (240) 90.3 (234) Dental Insurance Yes 43.5 (621) 50.7 (568) 17.3 (53) No 56.5 (806) 49.3 (553) 82.7 (253) Country of Birth Canada 90.3 (1316) 90.3 (1018) 90.3 (298) Other 9.7 (141) 9.7 (109) 9.7 (32) Frequency of Dental Visits 1+ times per year 59.9 (862) 70.1 (782) 24.7 (80) < 1 times per year 40.1 (578) 29.9 (334) 75.3 (244) Dentate status Dentate 81.8 (878) 91.9 (684) 59.0 (194) Edentulous 18.2 (195) 8.1 (60) 41.0 (135) Brushing Frequency (dentate only) 2+ times per day 74.2 (650) 79.3 (541) 56.2 (109) < 2 times per day 25.8 (226) 20.7 (141) 43.8 (85) Flossing Frequency (dentate only) 1+ times per day 35.2 (299) 40.3 (268) 16.8 (31) < 1 times per day 64.8 (551) 59.7 (397) 83.2 (154) Smoking Frequency Occasionally or not at all 88.4 (1288) 87.3 (985) 92.1 (303) Daily 11.6 (169) 12.7 (143) 7.9 (26) N.B. Bolded percentages are significant when p ≤0.05 using the Chi-square test. Data Analysis S i d Table 1 Characteristics of study participants aged 45 years and older living in the community or long-term care in Nova Scotia, Canada Table 2 Self-perceived oral health status of adults aged 45 and older in Nova Scotia, Canada Characteristic % (N) Total Community LTC 100 (1461) 77.4 (1131) 22.6 (330) Perceived General Health Excellent/ very good/ good 80.0 (1167) 84.1 (950) 66.0 (217) Fair or poor 20.0 (292) 15.9 (180) 34.0 (112) Perceived Quality of Life Excellent/ very good/ good 89.8 (1305) 92.8 (1049) 79.0 (256) Fair or poor 10.2 (149) 7.2 (81) 21.0 (68) Perceived Mouth Health Excellent/ very good/ good 79.9 (1161) 81.1 (916) 75.6 (245) Fair or poor 20.1 (292) 18.9 (213) 24.4 (79) Oral Pain No oral pain 69.0 (743) 66.0 (493) 75.8 (250) Mouth, dental, jaw or other pain 31.0 (334) 34.0 (254) 24.2 (80) Satisfaction with Teeth/Dentures Very satisfied/ satisfied/ neither 85.4 (1235) 85.4 (1235) 84.9 (269) satisfied or dissatisfied 14.6 (211) 14.6 (211) 15.1 (48) Dissatisfied or very dissatisfied N.B. Bolded percentages are significant when p ≤0.05 using the Chi-square test. Table 2 Self-perceived oral health status of adults aged 45 and older in Nova Scotia, Canada addition, 6.1% of LTC residents compared to 2% of com- munity dwellers reported impacts ‘fairly/very often’ with regards to difficulty pronouncing words. In terms of ‘prevalence’ of impact, 25.8% of the com- munity dwellers and 24.8% of LTC residents reported one or more OHIP problems ‘fairly/very often’. A larger percentage of LTC residents reported one or more impacts ‘fairly/very often’ in the functional limitation, physical pain, psychological disability and handicap dimensions. Regarding ‘extent’ of impact, (i.e., the mean number of OHIP items reported ‘fairly/very often’) the mean for community residents was 0.63 (SD = ±1.59) and 0.89 (SD = ±2.24) for LTC residents. Furthermore, in terms of ‘severity’ of impact, (i.e., the total OHIP score) the mean was 5.57 (SD = ±7.57) for community dwellers and 5.57 (SD = ±9.58) for LTC residents (Table 4). However, a statistically significant difference was reported in terms of the mean number of items reported ‘fairly/very often’ between community and LTC residents. Further analysis of prevalence, extent and severity were carried out by comparing pre-seniors with seniors in both LTC and community settings (Table 5). Data Analysis S i d The most commonly reported oral health quality of life impacts were within the dimensions ‘physical pain’ and ‘psychological discomfort’ (Table 3). It was found that 12.2% of LTC residents found it uncomfortable to eat any foods ‘fairly often’ or ‘very often’ compared to 7.7% in the community. Nearly 12% of LTC reported being self-conscious ‘fairly/very often’ compared to 8.2% in the community, while 9.7% of LTC residents reported being embarrassed by their teeth, mouth or dentures ‘fairly/ very often’ compared to 4% in the community. In Socio-demographic characteristics and self-perceived oral health status of community dwelling and LTC residents were summarized using descriptive statistics. Similarly, responses to individual OHIP-14 items were summarized according to place of residence. OHIP-14 overall scores were computed in three ways: (i) a total OHIP-14 score was calculated by summing responses over all 14 items, with possible scores ranging from 0 to 56 which indi- cates the severity of OHRQoL impacts; (ii) the preva- lence of people reporting one or more items ‘fairly often’ Page 4 of 12 Kotzer et al. Health and Quality of Life Outcomes 2012, 10:50 http://www.hqlo.com/content/10/1/50 Kotzer et al. n = no. unit 0.26 0.42) 0.45 0.33 0.34 0.27) 0.26 n = no. o unity 0.26) 0.42) 0.45) 0.33) 0.34) 0.27) 0.26) 1.59) Discussion The 2007–09 Canadian Health Measures Survey, in ac- cordance with Statistics Canada, released data regarding the oral health status and treatment needs of elderly Canadians, but did not do so at the provincial level [22]. The TOHAP study is the first to focus on the oral health of older adults living in the province of Nova Scotia. The findings of this study are not only important in assem- bling a complete picture of the oral health of Canadians, but they also provide important insight into the oral health-related quality of life of these individuals. Bivariate analyses were conducted for prevalence of impacts for both community and LTC residents. Com- munity residents who reported one or more impacts ‘fairly often’ or ‘very often’ were more likely to be pre-seniors, live in a rural area, be female, have a high school education or less, make less than $30,000 per year, visit the dentist less than once per year, smoke daily, have oral pain, perceive their general health, mouth health and quality of life to be fair or poor and be dissatisfied with their teeth or dentures (Table 6). LTC residents who reported one or more impacts ‘fairly often’ or ‘very often’ were more likely to have a high school education or less, have oral pain, perceive their general health, mouth health and quality of life to be fair or poor and be dissatisfied with their teeth or dentures (Table 7). The most interesting finding of this study was regard- ing the comparison of oral health impacts on pre-seniors and seniors. It was found that pre-seniors living in the community reported more oral health impacts than seniors even though the oral health of pre-seniors was better than that of seniors [23]. This reinforces the no- tion that individual expectations and experiences can greatly impact ones satisfaction or dissatisfaction with their oral health [12]. For example, one who experiences poor health but has low expectations may not perceive their health to have a significant impact on his/her life. Seniors living within the community may not feel as though oral health has a huge impact on their lives and may be more satisfied with the quality of their oral health compared to their general health, causing them to report less impacts ‘fairly often’ or ‘very often’. Data Analysis S i d It was found that in the community, pre-seniors scored significantly higher than seniors on prevalence (p = 0.009), extent (p = 0.007) and severity (p < 0.001). In the LTC residences, seniors scored higher than (SD) unity 35) 0. 46) 0. 54) 0. 61) 0. 61) 0. 59) 0. 53) 0. 41) 0. 51) 0. 49) 0. 46) 0. 35) 0. 44) 0. ) ty 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. nd mean item scores (n = 1460*) Distribution of responses% Mean (SD) ear, . . .” Never (0)/Hardly Ever (1) Occasionally (2) Fairly Often (3)/Very Often (4) Comm. LTC Comm. LTC Comm. LTC Community 92.9 86.7 5.2 7.3 2.0 6.1 0.09 (0.35) 88.5 88.8 7.5 5.9 4.0 5.3 0.16 (0.46) 79.7 83.6 15.2 11.8 5.2 4.5 0.25 (0.54) 74.8 73.3 17.5 14.6 7.7 12.2 0.33 (0.61) 78.7 74.5 13.1 14.0 8.2 11.6 0.29 (0.61) 80.2 84.4 12.4 8.0 7.4 7.6 0.27 (0.59) 86.3 84.5 7.8 7.9 5.9 7.6 0.20 (0.53) 91.0 88.8 6.0 6.7 2.9 4.6 0.12 (0.41) 85.4 88.4 9.6 6.4 5.1 5.2 0.20 (0.51) 83.8 82.4 12.2 7.9 4.0 9.7 0.20 (0.49) 86.1 89.1 10.6 7.3 3.3 3.6 0.17 (0.46) 93.8 93.9 3.9 3.1 2.3 3.1 0.09 (0.35) 89.7 87.2 6.6 6.7 3.7 6.1 0.14 (0.44) 96.1 95.1 2.6 2.4 1.3 2.4 0.05 (0.28) 4. .44) .28) an it Nev Com 92.9 88.5 79.7 mea . . .” (n = 1460) no. of items reported fairly/very often (SD) Seve munity LTC Co (0.26) 0.11 (0.37) 0. (0.42) 0.17 (0.44) 1 (0.45) 0.19 (0.51) 1 (0.33) 0.12 (0.44) 0. (0.34) 0.15 (0.44) 0 (0.27) 0.07 (0.33) 0. (0.26) 0.09 (0.34) 0. (1.59) 0.89 (2.24) 5 Sev Co 0 1 1 0 0 0 0 5 Sev C 0 0 0 0 0 5 Sev C 0 0 0 0 Sev C 0 0 0 0 Kotzer et al. Health and Quality of Life Outcomes 2012, 10:50 http://www.hqlo.com/content/10/1/50 Kotzer et al. Health and Quality of Life Outcomes 2012, 10:50 http://www.hqlo.com/content/10/1/50 Kotzer et al. Data Analysis S i d Health and Quality of Life Outcomes 2012, 10:50 http://www.hqlo.com/content/10/1/50 Page 7 of 12 Page 7 of 12 Table 5 Prevalence, extent and severity of impacts by OHIP-14 subscale grouped by pre-seniors and seniors Community LTC Pre-seniors (n=629) Seniors (n=501) P-value Pre-seniors (n=33) Seniors (n=297) P-value Prevalence: % reporting 1+ impacts fairly/very often (no.) 28.8 (181) 22.0 (110) 0.009* 21.2 (7) 25.3 (75) 0.610* Extent: mean no. of items reported fairly/very often (SD) 0.73 (1.73) 0.49 (1.40) 0.007** 0.45 (1.33) 0.94 (2.32) 0.456** Severity: mean OHIP-14 score (SD) 6.22 (8.0) 4.75 (6.92) <0.001** 4.30 (7.29) 5.71 (9.80) 0.867** *P-value obtained from the Chi-squared test. **P-value obtained from the Mann–Whitney U test. revalence, extent and severity of impacts by OHIP-14 subscale grouped by pre-seniors and seniors rity of impacts by OHIP-14 subscale grouped by pre-seniors and seniors report an impact ‘fairly often’ or ‘very often’ (Table 9). Those with fair or poor perceived mouth health were nearly 10 times more likely to report impacts ‘fairly often’ or ‘very often’. pre-seniors for prevalence, extent and severity but there was not a statistically significant difference in the OHIP-14 scores. Although not mentioned in the tables, pre-seniors from the community were com- pared with pre-seniors in LTC, and seniors from the community were compared with seniors from LTC. Pre-seniors in the community scored higher on preva- lence, extent and severity than pre-seniors in LTC residences, but severity was the only significant differ- ence (p = 0.033). Furthermore, seniors in LTC resi- dences scored higher than seniors in the community for prevalence, extent and severity, but the differences were not statistically significant. Discussion In con- trast, one who has excellent oral health but extremely high expectations might report being dissatisfied due to a minor oral health-related problem. Community dwell- ing pre-seniors who are generally in good health may be- come irritated by small oral health problems, and frustrated that dental visits can be expensive and cut into work hours [12]. Locker and Gibson’s (2005) study of community living individuals over the age of 50 reported that 16.5% of those who rated their oral health as either excellent, very good, or good were dissatisfied with their Logistic regression models controlling for all the factors (significant and non-significant) at the bivariate level of analysis were used to predict the prevalence of impacts ‘fairly often’ or ‘very often’ for community and LTC residents, separately. For the community dwelling sample, individuals living in rural areas and those born outside of Canada were approximately 2.0 times more likely to report an impact ‘fairly/very often’ (Table 8). Having oral pain, fair or poor per- ceived mouth health and dissatisfaction with teeth or dentures also caused community residents to report impacts. Among the LTC sample, those having a high school education or less were 2.3 times more likely to Kotzer et al. Health and Quality of Life Outcomes 2012, 10:50 http://www.hqlo.com/content/10/1/50 Kotzer et al. Discussion They found that those between the ages of 35–54 reported an 18.3% prevalence rate of oral impacts and those aged greater than or equal to 55 years Table 6 Bivariate analyses for prevalence of impacts (‘fairly often’ or ‘very often’) for community residents Characteristic % (N) No Impacts 1+ Impacts P-value Age (yrs) 45 – 64 71.2 (448) 28.8 (181) 0.009 65 and over 78.0 (391) 22.0 (110) Community Type Urban 79.2 (529) 20.8 (139) < 0.001 Rural 67.1 (310) 32.9 (152) Sex Male 78.3 (328) 21.7 (91) 0.017 Female 71.9 (511) 28.1 (200) Education Level More than high school 78.3 (508) 21.7 (141) < 0.001 High school or less 68.6 (328) 31.4 (150) Annual Household Income More than $30,000 77.0 (476) 23.0 (142) < 0.001 Less than $30,000 64.2 (154) 35.8 (86) Dental Insurance Yes 75.9 (431) 24.1 (137) 0.278 No 73.1 (404) 26.9 (149) Country of Birth Canada 74.8 (761) 25.2 (256) 0.341 Other 70.6 (77) 29.4 (32) Frequency of Dental Visits 1+ times per year 77.7 (608) 22.3 (174) < 0.001 < 1 time per year 65.6 (219) 34.4 (115) Dentate Status Dentate 76.3 (524) 23.7 (163) 0.169 Edentulous 68.3 (41) 31.7 (19) Smoking Frequency Occasionally or not at all 75.8 (747) 24.2 (238) 0.003 Daily 64.3 (92) 35.7 (51) Oral Pain No 82.8 (408) 17.2 (85) < 0.001 Yes 61.8 (157) 38.2 (97) Perceived General Health Excellent/ very good/ good 77.9 (740) 22.1 (210) < 0.001 Fair or poor 55.0 (99) 45.0 (81) Perceived Mouth Health Table 6 Bivariate analyses for prevalence of impacts (‘fairly often’ or ‘very often’) for community residents (Continued) Perceived Quality of Life Excellent/ very good/ good 76.5 (803) 23.5 (246) < 0.001 Fair or poor 44.4 (36) 55.6 (45) Table 6 Bivariate analyses for prevalence of impacts (‘fairly often’ or ‘very often’) for community residents Table 6 Bivariate analyses for prevalence of impacts (‘fairly often’ or ‘very often’) for community residents (Continued) Perceived Quality of Life Excellent/ very good/ good 76.5 (803) 23.5 (246) < 0.001 Fair or poor 44.4 (36) 55.6 (45) Table 6 Bivariate analyses for prevalence of impacts (‘fairly often’ or ‘very often’) for community residents (Continued) oral health [12]. Moreover, 50.8% of participants who rated their oral health as fair or poor reported that they were satisfied with mouth, teeth or gums. Discussion Health and Quality of Life Outcomes 2012, 10:50 http://www.hqlo.com/content/10/1/50 Page 8 of 12 Table 6 Bivariate analyses for prevalence of impacts (‘fairly often’ or ‘very often’) for community residents Characteristic % (N) No Impacts 1+ Impacts P-value Age (yrs) 45 – 64 71.2 (448) 28.8 (181) 0.009 65 and over 78.0 (391) 22.0 (110) Community Type Urban 79.2 (529) 20.8 (139) < 0.001 Rural 67.1 (310) 32.9 (152) Sex Male 78.3 (328) 21.7 (91) 0.017 Female 71.9 (511) 28.1 (200) Education Level More than high school 78.3 (508) 21.7 (141) < 0.001 High school or less 68.6 (328) 31.4 (150) Annual Household Income More than $30,000 77.0 (476) 23.0 (142) < 0.001 Less than $30,000 64.2 (154) 35.8 (86) Dental Insurance Yes 75.9 (431) 24.1 (137) 0.278 No 73.1 (404) 26.9 (149) Country of Birth Canada 74.8 (761) 25.2 (256) 0.341 Other 70.6 (77) 29.4 (32) Frequency of Dental Visits 1+ times per year 77.7 (608) 22.3 (174) < 0.001 < 1 time per year 65.6 (219) 34.4 (115) Dentate Status Dentate 76.3 (524) 23.7 (163) 0.169 Edentulous 68.3 (41) 31.7 (19) Smoking Frequency Occasionally or not at all 75.8 (747) 24.2 (238) 0.003 Daily 64.3 (92) 35.7 (51) Oral Pain No 82.8 (408) 17.2 (85) < 0.001 Yes 61.8 (157) 38.2 (97) Perceived General Health Excellent/ very good/ good 77.9 (740) 22.1 (210) < 0.001 Fair or poor 55.0 (99) 45.0 (81) Perceived Mouth Health Excellent/ very good/ good 79.7 (730) 20.3 (186) < 0.001 Fair or poor 50.7 (108) 49.3 (105) Satisfaction with Teeth or Dentures Satisfied 79.5 (768) 20.5 (198) < 0.001 Dissatisfied 42.9 (70) 57.1 (93) oral health [12]. Moreover, 50.8% of participants who rated their oral health as fair or poor reported that they were satisfied with mouth, teeth or gums. In addition, the frames of reference on which people base their oral health can naturally range depending on a host of variables. While some compare themselves to others who are close in age, others might use their phys- ical or emotional state to assess their oral health. Some people who have, or perceive themselves as having, poor oral health may actually be satisfied with the state of their oral health [12,24]. Sprangers and Schwartz (1999) explain this phenomenon through the process of re- sponse shift. Discussion This is when changes in internal standards, values and meanings of health contribute to the accept- ance of an individual’s illness or disability [24]. As indivi- duals age, they are more likely to consider minor or even severe oral health problems as insignificant at this point in their lives [12]. The theory of response shift may ex- plain why community dwelling seniors, and the elderly population in general, may report fewer impacts in cer- tain dimensions [24]. As these individuals age, they come to accept that their health is deteriorating and they may consider oral health problems as less significant [12]. Consequently, these oral health problems take a backseat to general health problems. A study completed in On- tario involving 61 residents in three long-term care facil- ities suggests that general health issues often overshadowed and minimized oral health issues in long- term care facilities. Chronic illnesses such as Alzheimer’s and Parkinson’s disease, which interfere with ones cogni- tive and communicative skills, cause barriers in identify- ing treatments needs for these residents [25]. In the study completed by Locker and Quinonez [26] telephone numbers for households (therefore those living in the community) were randomly sampled in a Canad- ian population. Discussion In addition, the frames of reference on which people base their oral health can naturally range depending on a host of variables. While some compare themselves to others who are close in age, others might use their phys- ical or emotional state to assess their oral health. Some people who have, or perceive themselves as having, poor oral health may actually be satisfied with the state of their oral health [12,24]. Sprangers and Schwartz (1999) explain this phenomenon through the process of re- sponse shift. This is when changes in internal standards, values and meanings of health contribute to the accept- ance of an individual’s illness or disability [24]. As indivi- duals age, they are more likely to consider minor or even severe oral health problems as insignificant at this point in their lives [12]. The theory of response shift may ex- plain why community dwelling seniors, and the elderly population in general, may report fewer impacts in cer- tain dimensions [24]. As these individuals age, they come to accept that their health is deteriorating and they may consider oral health problems as less significant [12]. Consequently, these oral health problems take a backseat to general health problems. A study completed in On- tario involving 61 residents in three long-term care facil- ities suggests that general health issues often overshadowed and minimized oral health issues in long- term care facilities. Chronic illnesses such as Alzheimer’s and Parkinson’s disease, which interfere with ones cogni- tive and communicative skills, cause barriers in identify- ing treatments needs for these residents [25]. g In the study completed by Locker and Quinonez [26] telephone numbers for households (therefore those living in the community) were randomly sampled in a Canad- ian population. They found that those between the ages of 35–54 reported an 18.3% prevalence rate of oral impacts, and those aged greater than or equal to 55 years reported a 19.5% prevalence rate [26]. In this national study an older population of community dwellers reported 1.2% more impacts than a younger population of community dwellers. Despite the slight difference in age groups, our results show that among those living within the community, pre-seniors reported a 28.8% Kotzer et al. Health and Quality of Life Outcomes 2012, 10:50 http://www.hqlo.com/content/10/1/50 Kotzer et al. Discussion Health and Quality of Life Outcomes 2012, 10:50 http://www.hqlo.com/content/10/1/50 Page 9 of 12 Table 7 Bivariate analyses for prevalence of impacts (‘fairly often’ or ‘very often’) for LTC residents % (N) Characteristic No Impacts 1+ Impacts P-value Age (yrs) 45 – 64 78.8 (26) 21.2 (7) 0.610 65 and over 74.7 (222) 25.3 (75) Community Type Urban 76.9 (150) 23.1 (45) 0.371 Rural 72.6 (98) 27.4 (37) Sex Male 73.8 (62) 26.2 (22) 0.742 Female 75.6 (186) 24.4 (60) Education Level More than high school 84.1 (58) 15.9 (11) 0.052 High school or less 72.7 (189) 27.3 (71) Annual Household Income More than $30,000 88.0 (22) 12.0 (3) 0.081 Less than $30,000 71.8 (168) 28.2 (66) Dental Insurance Yes 79.2 (42) 20.8 (11) 0.384 No 73.5 (186) 26.5 (67) Country of Birth Canada 74.8 (223) 25.2 (75) 0.682 Other 78.1 (25) 21.9 (7) Frequency of Dental Visits 1+ times per year 76.3 (61) 23.8 (19) 0.766 < 1 time per year 74.6 (182) 25.4 (62) Dentate Status Dentate 75.3 (146) 24.7 (48) 0.957 Edentulous 75.0 (102) 25.0 (34) Smoking Frequency Occasionally or not at all 76.2 (231) 23.8 (72) 0.096 Daily 61.5 (16) 38.5 (10) Oral Pain No 79.6 (199) 20.4 (51) 0.001 Yes 61.3 (49) 38.8 (31) Perceived General Health Excellent/ very good/ good 81.1 (176) 18.9 (41) 0.001 Fair or poor 64.3 (72) 35.7 (40) Perceived Mouth Health Excellent/ very good/ good 85.7 (210) 14.3 (35) < 0.001 Fair or poor 40.5 (32) 59.5 (47) Satisfaction with Teeth or Dentures Satisfied 81.0 (218) 19.0 (51) < 0.001 Dissatisfied 56.3 (27) 43.8 (21) Table 7 Bivariate analyses for prevalence of impacts (‘fairly often’ or ‘very often’) for LTC residents (Continued) Perceived Quality of Life Excellent/ very good/ good 80.5 (206) 19.5 (50) < 0.001 Fair or poor 54.4 (37) 45.6 (31) Table 7 Bivariate analyses for prevalence of impacts (‘fairly often’ or ‘very often’) for LTC residents (Continued) prevalence rate of oral impacts, whereas seniors reported a prevalence rate of 22.0%. Another important finding of our study indicates that approximately one in four pre-seniors and seniors report at least one or more impacts of their oral health on the quality of life ‘fairly’ or ‘very often’. This is slightly higher than a national study of adults aged 55 years and older where the finding was one in five (19.5%) [26]. Discussion The Yukon, Nunavut and Northwest Territories, which make up 0.3% of the Canadian population, were not included in that study. It is evident that no matter where you live in Canada, people in your community are going to report having oral impacts. But, it is important to note that older samples and edentulous samples will report having more OHRQoL impacts. Logistic regression models indicate that both socio- demographic factors and self-perceived oral health can have an effect on the prevalence of impacts. The findings that pre-seniors and seniors in rural areas have the poor- est OHRQoL suggest that a decreased access to dental care may be affecting their oral health and OHRQoL. Further findings show that elderly residents living in the community visit the dentist significantly less often if they live in rural areas as opposed to urban areas. Results in- dicate that 75.4% of Nova Scotia residents aged 45 and older who live in an urban area visit the dentist one or more times per year, whereas only 62.4% of rural resi- dents visit the dentist one or more times per year. According to the literature, “in dentistry, a functional definition of an elderly adult is based on his or her ability to travel to seek services” [5]. Many elderly patients who live in rural areas may have access to fewer dental clinics, or there may be barriers limiting their access to care. Barriers include lack of public transportation, cost of transportation and treatment, or mobility issues [27]. The reliance of many seniors on others for help may also limit their ability to receive dental care [27]. Furthermore, funding for retired employees must be developed by union negotiators, working Canadians must plan for retirement by saving money for dental care and family members and caregivers must be educated in the importance of dental care for the elderly [28]. Although many health economists believe that govern- ment funding may be insufficient to meet the increasing dental needs of the baby boom population [28], educa- tion is a relatively inexpensive, yet effective dental health Page 10 of 12 Page 10 of 12 Page 10 of 12 Kotzer et al. Health and Quality of Life Outcomes 2012, 10:50 http://www.hqlo.com/content/10/1/50 Kotzer et al. Discussion Health literacy has been defined as “the degree to which individuals have the capacity to obtain, process, and understand basic health information and services needed to make appropriate health decisions” [29]. Health literacy skills are essential in the maintenance of quality of life for the elderly population [30]. It has also been shown to be an important contributor to both gen- eral and oral health [28]. As individuals age, health liter- acy also becomes an important tool to help take or administer medications appropriately [31]. In addition, it was found that those who were born outside of Canada living within the community have greater oral health impacts, implying that oral health lit- eracy, understanding the Canadian health-care system and acculturation may be limiting their access to dental care. An increase in educational resources and training by dentists and dental hygienists can be essential in developing proper oral health care skills and routines for seniors, LTC nursing staff, and family members [27]. Education is also necessary so that they can provide care in a productive, cost-effective and timely manner [28]. Although our study, in addition to other similar stud- ies, have identified potential correlates of health literacy, few studies have attempted to recognize educational and learning pathways that increase health literacy skills throughout ones life [30]. The development and main- tenance of health literacy skills throughout ones life can be accomplished by the use of adult education, seminars, self-study, internet use, library use, daily reading and en- gagement in social networks [30]. A study found that practicing literacy at home by methods such as reading books, magazines and newspapers, had a stronger effect on ones health literacy than educational attainment [32]. These practices can be maintained throughout ones life The binary logistic regression model also indicated that for those living in the community, people with oral pain were 1.87 times more likely to report impacts, and those with fair or poor perceived mouth health were 2.19 times more likely to report impacts. These two variables are closely related to the outcome of oral health-related quality of life and it is therefore not surprising to find them in this model. Similarly, those with dissatisfaction with the appearance of their teeth and/or dentures were 5.16 times more likely to report impacts ‘fairly often’ or ‘very often’. Discussion Health and Quality of Life Outcomes 2012, 10:50 http://www.hqlo.com/content/10/1/50 Table 8 Logistic regression model for prevalence of impacts (‘fairly often’ or ‘very often’) for community residents Table 8 Logistic regression model for prevalence of impacts (‘fairly often’ or ‘very often’) for community residents Adjusted Odds Ratio 95% CI for Odds Ratio P- value Living in a rural area 2.07 1.35 – 3.17 0.001 Having oral pain 1.87 1.21 – 2.88 0.005 Born outside of Canada 1.97 1.01 – 3.85 0.048 Fair or poor perceived mouth health 2.19 1.30 – 3.71 0.003 Dissatisfaction with teeth or dentures 5.16 2.87 – 9.27 <0.001 CI = Confidence Interval. and will maintain and increase ones literacy in a rela- tively inexpensive manner. In addition, using the internet or computer to learn was found to be one of the stron- gest predictors of adequate health literacy [30]. Another issue that must be addressed is the current level of communication between dental care providers and their patients, which is important for the elderly population. Effective communication is critical for den- tists and hygienists in improving the oral health literacy of their patients [33]. Several findings suggest that the communication techniques used by dentists may not be effectively accommodating the literacy skills of certain patients [34]. One technique among others that has been proven to be effective in increasing the health literacy of patients is the teach-back method; therefore it is import- ant that a set of communications guidelines for prac- ticing dentists be developed [34]. care initiative because it is generally less expensive to prevent disease than cure it [28]. A large percentage of the Nova Scotian elderly population has a high school education or less. This is especially troubling because education is a social determinant of health and education is also highly related to health literacy. While some studies question whether literacy is really a problem in the context of health care, and suggest a need for more Canadian research in this area [35], edu- cation and health literacy can improve access to care for Canadian seniors and the general population by focusing on health, oral health, and quality of life issues [28]. Edu- cation that focuses and raises awareness on how oral health enhances self-image and social interactions can also positively affect attitudes towards care [28]. Acknowledgements Funding for this research was provided by the Canadian Institutes of Health Research – Institute of Musculoskeletal Health and Arthritis (ROP-86224), Health Canada (Office of the Chief Dental Officer) and the Nova Scotia Health Research Foundation. Authors' contributions RK carried out the data analysis and the interpretation of data as well as wrote the manuscript. HPL contributed to the study design, calculated the required sample size and random selection of the long-term care facilities. She also participated in the analysis and interpretation of the data and critically reviewed the manuscript. DCM and JBC were principal investigators of the TOHAP study. They contributed to the interpretation of the data and review of the manuscript. All authors read and approved the final manuscript. In addition to a high school education, LTC residents with fair or poor perceived mouth health were 9.49 times more likely to report impacts. LTC residents have poor oral hy- giene and limited access to routine dental care [40]. It has been shown that once a comprehensive dental program is implemented into LTC facilities, residents who receive den- tal care show improvements in caries rates, periodontal health, and other clinical oral disorders [40]. Living in an LTC facility is a barrier to treatment in and of itself. Abreviations Abreviations OHRQoL: Oral health-related quality of life; TOHAP: The Oral Health of our Aging Population; LTC: Long-term care; OHIP: Oral Health Impact Profile; NS: Nova Scotia. Competing interests The authors declare that they have no competing interests. It is clear that public health initiatives need to focus on Canadians with low levels of education. Even though ac- cess to education and literacy levels are for the most part managed outside of the health sector, they have a direct effect on health status. Therefore, multi-sectoral strat- egies must be implemented in order to improve the health of Canadians [38]. Conclusions This study has provided valuable information regarding the oral health-related quality of life of pre-seniors and seniors living in Nova Scotia, Canada. LTC residents are more likely to have poorer indicators of socio-demographic characteris- tics and self-perceived oral health status compared to com- munity dwellers. Having more oral health problems can have an effect on one’s OHRQoL and in turn, explain why LTC residents report impacts ‘fairly/very often’ on the OHIP-14. One in four pre-seniors and seniors living in the community and LTC facilities reported one or more impacts ‘fairly/very often’, however, pre-seniors in the com- munity experienced greater prevalence, extent and severity of oral impacts than seniors. This finding suggests that as people age, oral health problems may take a backseat to general health problems. The study findings also indicate that pre-seniors and seniors in rural areas have the poorest OHRQoL, suggesting that a decreased access to dental care may be affecting their oral health and OHRQoL. In addition, lower levels of education and health literacy over- all and especially among those who were born outside of Canada but are now living within the community have greater oral health impacts, suggesting that decreased oral health literacy and a lack of understanding of the Canadian health care system may be limiting their access to dental care. LTC residents with a low education level and low health literacy may be a group at higher risk in terms of greater impacts on their OHRQoL. Reported in the 2006 census, only 24% of adults aged 25– 64 had a high school diploma as their highest level of edu- cational attainment, while 15% did not graduate from high school. In addition, 32% of adults aged 55 to 64 years did not have a high school diploma [37]. Educational attain- ment is recognized as one of the key components of socioe- conomic status, and while income and education are highly correlated, education is an independent predictor of health status and visiting the dentist [38,39]. Regardless of age, people with low education levels have more disabilities and chronic illnesses [38]. People with a higher educational background tend to embrace positive health practices and have access to healthier physical environments [38]. Kotzer et al. Health and Quality of Life Outcomes 2012, 10:50 http://www.hqlo.com/content/10/1/50 conscious because of trouble with teeth, mouth or den- tures was one of the highest scoring items on the OHIP- 14. conscious because of trouble with teeth, mouth or den- tures was one of the highest scoring items on the OHIP- 14. Therefore it is imperative that dental programs be devel- oped in order to increase access to dental care for seniors in LTC, by providing transportation or by bringing oral care providers and dental equipment into the facilities. Moreover, LTC residents with a low education level may be a group at risk in terms of greater impacts on their OHRQoL. LTC residents have poorer indicators of socioeconomic status including household income and dental insurance. Since dental coverage is not covered by the Canadian healthcare system, out of pocket costs may deter people from seeking dental care or accepting recommended dental care when visiting the dentist [22]. The Canadian Health Measures Survey reported that as Canadians age they are less likely to have dental insur- ance. In addition, being born outside of Canada, annual income and level of education are also directly related to having dental insurance [22]. A 2006 study using Canadian health survey data from 2003, found that the probability of receiving any dental care throughout the course of a year increases dramatically with dental insur- ance, household income and level of education [36]. This study confirms these findings in the NS population as 79% of LTC residents have less than or equal to a high school education, 82.7% do not have dental insurance and 90.3% have an annual household income of less than $30,000. Discussion This readdresses the theme of how a compli- cation with ones teeth and/or dentures can have a sig- nificant impact on oral health-related quality of life. Dissatisfaction with the appearance of teeth and/or den- tures is directly related to variables on the OHIP-14 such as being self conscious and embarrassed. 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English
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Chondromalacia patella increases the risk of herpes zoster: a population-based study
BMC musculoskeletal disorders
2,022
cc-by
5,664
RESEARCH Open Access Abstract Background  The reactivation of herpes zoster (HZ) is associated with disease stress. However, the relationship between chondromalacia patella (CMP) and HZ remains poorly understood. This study investigated the relationship between CMP and the risk of developing HZ. Methods  Data were collected from the Taiwan’s National Health Insurance Research Database. Patients with CMP diagnosed between 2000 and 2017 were assigned to the case group; patients without CMP were randomly selected from the same database and paired with controls matched by age and sex. The primary outcome was a diagnosis of HZ. All patients were followed until their diagnosis of HZ, their withdrawal from the NHI program, their death, or the end of 2017, whichever was earliest. The risk of developing HZ was compared between the case and control groups. Results  In total, 22,710 patients with CMP and 90,840 matched controls were enrolled. The overall incidence rates of HZ in the CMP and control cohorts were 7.94 and 7.35 per 1,000 person-years, respectively. After potential confounders were controlled for, the case group exhibited a higher risk of HZ than did the control group [adjusted hazard ratio (aHR) = 1.06, p < 0.05]. In a stratification analysis by age, patients over 65 years old in the CMP group exhibited a higher risk of HZ than did those in the control group (aHR = 1.22, p < 0.01). In a stratification analysis by sex, women with CMP were at greater risk of developing HZ than women without CMP (aHR = 1.18, p < 0.01). Conclusion  Patients with CMP, especially elder adults and women, exhibited a higher risk of HZ. The HZ risk of patients with CMP should thus be assessed, and the necessity of HZ vaccination should be informed. Keywords  Chondromalacia patella, Herpes zoster, Women Keywords  Chondromalacia patella, Herpes zoster, Women q y corresponding authors. *Correspondence: Der-Shin Ke 07238@cych.org.tw Wen-Che Hsieh cych06085@gmail.com 1Department of Medical Education, Ditmanson Medical Foundation, Chia- Yi Christian Hospital, Chia-Yi, Taiwan 2Department of Medical Imaging, Ditmanson Medical Foundation, Chia-Yi Christian Hospital, Chia-Yi, Taiwan 3Department of Rehabilitation, Ditmanson Medical Foundation, Chia-Yi Christian Hospital, Chia-Yi, Taiwan 4Clinical Data Center, Department of Medical Research, Ditmanson Medical Foundation, Chia-Yi Christian Hospital, Chia-Yi, Taiwan Chondromalacia patella increases the risk of herpes zoster: a population-based study Chia-Hung Chen1,2, Yung-Chi Cheng1,3, Hsin-Yi Yang4,5, Ching-Fang Tsai5, Chao-Yu Hsu1,6,7,8,9, Der-Shin Ke10* and Wen-Che Hsieh11* 5Clinical Medicine Research Center, Department of Medical Research, Ditmanson Medical Foundation, Chia-Yi Christian Hospital, Chia-Yi, Taiwan 6Department of Optometry, Central Taiwan University of Science and Technology, Taichung, Taiwan 7Department of Medical Imaging and Radiological Sciences, Central Taiwan University of Science and Technology, Taichung, Taiwan 8Center for General Education, National Taichung University of Science and Technology, Taichung, Taiwan 9Department of General Education, National Chin-Yi University of Technology, Taichung, Taiwan 10Department of Neurology, Ditmanson Medical Foundation, Chia-Yi Christian Hospital, No 539 Zhongxia Road, Chia-Yi, Taiwan 11Department of Chinese Medicine, Ditmanson Medical Foundation, Chia-Yi Christian Hospital, No 539 Zhongxia Road, Chia-Yi, Taiwan Chen et al. BMC Musculoskeletal Disorders (2022) 23:961 https://doi.org/10.1186/s12891-022-05929-y Chen et al. BMC Musculoskeletal Disorders (2022) 23:961 https://doi.org/10.1186/s12891-022-05929-y BMC Musculoskeletal Disorders Main outcome and relevant variablesh The primary outcome of this study was a HZ diagnosis (ICD-9-CM: 053; ICD-10-CM: B02). Several diseases, namely diabetes (ICD-9-CM: 250; ICD-10-CM: E08- E13), chronic kidney disease (CKD; ICD-9-CM: 585, ICD-10-CM: N18), coronary artery disease (CAD; ICD- 9-CM: 410–414; ICD-10-CM: I20-I22, I24, I25), depres­ sion (ICD-9-CM: 296.2, 296.3, 300.4, 311; ICD-10-CM: F32, F33, F34.1), and cancer (ICD-9-CM: 140–208; ICD-10-CM: C00-C26, C30-C34, C37-C41, C43-C50, C53-C55, C4A, C7A, D03, Z51.12), were considered as comorbidities in this study. © The Author(s) 2022. 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The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Chen et al. BMC Musculoskeletal Disorders (2022) 23:961 Chen et al. BMC Musculoskeletal Disorders (2022) 23:961 Page 2 of 8 Page 2 of 8 Chen et al. BMC Musculoskeletal Disorders claims of enrolled residents are recorded in the NHI Research Database (NHIRD). In this study, we analyzed the 2000 Longitudinal Generation Tracking Database (LGTD2000), which is a subset of the NHIRD, and con­ tains the records of 2  million insured individuals ran­ domly selected from the NHIRD. The information of each patient enrolled in this study, including records of clinic visits, hospitalization and medication was well documented in the NHIRD. To protect the patient’s pri­ vacy, the patients’ personally identifiable information is encrypted in all NHI data. The diagnostic codes of CMP, HZ and all comorbidities were defined in accordance with the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) between 2000 and 2015, and the International Classification of Diseases, Tenth Revision, Clinical Modification (ICD- 10-CM) after 2015. This study was approved by the insti­ tutional review board of Chia-Yi Christian Hospital, in Chia-Yi, Taiwan (IRB2020113). All methods were car­ ried out in accordance with declaration of Helsinki in our manuscript. Background Chondromalacia patella (CMP) involves degenerative changes in cartilage due to a poor alignment of the knee­ cap, and is a highly common cause of chronic knee pain. In a postmortem study involving 59 persons, cartilagi­ nous change was discovered in 91 of the individuals’ 118 patellae [1]. In a study by Özdemir and Kavak involving military recruits with anterior knee pain, magnetic reso­ nance imaging examinations revealed that 58.7% of the recruits had CMP [2]. Perineural injection plus physical therapy is an effective conservative management strategy to relieve pain and stiffness [3]. f Herpes zoster (HZ) is a skin disease caused by the reactivation of the varicella zoster virus (VZV). The prevalence of HZ is 18.54 per 1,000 persons [4], and the incidence of HZ increased every year. In a population- based study, Kawai et al. reported that the incidence rates of HZ were 0.76 and 3.15 per 1000 person-years from 1945 to 1949 and from 2000 to 2007, respectively [5]. Thompson et al. reported that in the United States, the incidence of HZ increased from 286.0 to 579.6 per 100,000 person-years from 1994 to 2018, which is an average annual increase of 3.1% [6]. The incidence of post-herpetic neuralgia, a painful complication of HZ, was reported to be 57.5 per 100,000 person-years [6]. Several procedures, including steroid injection and radio­ frequency ablation, may be effective for treating post- herpetic neuralgia [7]. Study population h Patients with an initial diagnosis of CMP made between 2000 and 2017, (ICD-9-CM: 717.7; ICD-10-CM: M22.4) were enrolled in the case cohort. Patients with a previ­ ous history of HZ or who were younger than 20 years were excluded from the study. The patients in the control group were matched by age and sex with the patients in the case group at a 4:1 ratio. The index date was defined as the date of initial diagnosis of CMP in the case cohort. All the patients were followed until their diagnosis of HZ, their withdrawal from the NHI program, their death, or the end of 2017, whichever was earliest. CMP is characterized by chronic knee pain, and chronic pain is significantly associated with depression [8]. Rapti et al. reported that 22.5% of their participants with chronic pain had depression, as evaluated using the Patient Health Questionnaire-9 [9]. Chronic pain-related conditions, such as fatigue and frailty, also increase depression risk [10–13]. Thus, because depression increases HZ risk [14], the patients with chronic pain may have a higher risk to have HZ development. Further­ more, chronic pain-related diseases including chronic interstitial cystitis [15], varicocele [16] and endometriosis [17] in the urogenital system and also adhesive capsuli­ tis of the shoulder [18], sciatica [19], lateral epicondylitis [20], plantar fascial fibromatosis [21], and de Quervain syndrome [22] in the musculoskeletal system are strongly associated with HZ development. Therefore, patients with CMP are also likely to exhibit an increased risk of HZ. In this study, we investigated the risk of HZ reactiva­ tion in patients with CMP. Statistical analysish The distributions of the patients’ characteristics and comorbidities between the CMP and control cohorts were compared. A chi-square test and a t-test were used to analyze categorical and continuous variables. The hazard ratios and 95% confidence intervals (CIs) were The National Health Insurance (NHI) program, which employs a government-run single-payer model, has been in operation since 1995. More than 99% of Tai­ wanese people are enrolled in the program. All medical Page 3 of 8 Chen et al. BMC Musculoskeletal Disorders (2022) 23:961 (2022) 23:961 Fig. 1  Flowchart for data collection process Fig. 1  Flowchart for data collection process Fig. 1  Flowchart for data collection process Table 1  Demographic characteristics and comorbidities in the patients with and without chondromalacia of patella Table 1  Demographic characteristics and comorbidities in the patients with and without chondromalacia of patella Chondromalacia of patella p-value No Yes Variable N = 90,840  N = 22,710 Age ≤ 49 61,128 (67.29) 15,282 (67.29) 1.000 50–64 21,144 (23.28) 5286 (23.28) 65+ 8568 (9.43) 2142 (9.43) Mean ± SD& 41.69 ± 16.37 41.69 ± 16.37 1.000 Sex Female 55,092 (60.65) 13,773 (60.65) 1.000 Male 35,748 (39.35) 8937 (39.35) Comorbidity Diabetes 7429 (8.18) 1995 (8.78) 0.003 Chronic Kidney Disease 1053 (1.16) 265 (1.17) 0.923 Coronary Artery Disease 5938 (6.54) 1920 (8.45) < 0.001 Depression 4233 (4.66) 1598 (7.04) < 0.001 Cancer 2759 (3.04) 719 (3.17) 0.314 Chi-square test; &: t-test Results Women with CMP were Page 4 of 8 Page 4 of 8 (2022) 23:961 Chen et al. BMC Musculoskeletal Disorders Fig. 2  The cumulative incidence of HZ in the patients with CMP was significantly higher than the patients without CMP. Fig. 2  The cumulative incidence of HZ in the patients with CMP was significantly higher than the patients without CMP. 1.18 times (95% CI = 1.07, 1.31) more likely to develop HZ than were women without CMP. No significant dif­ ference in HZ risk was identified between the patients with CMP with or without comorbidities and the patients without CMP. However, the patients with CMP, whether with or without comorbidities, still exhibited a slightly higher incidence of HZ than did those without CMP (7.80 vs. 6.44 per 1,000 person-years among patients with no comorbidities; 13.09 vs. 12.81 per 1,000 person-years among patients with comorbidities) (Table 3).h 1.18 times (95% CI = 1.07, 1.31) more likely to develop HZ than were women without CMP. No significant dif­ ference in HZ risk was identified between the patients with CMP with or without comorbidities and the patients without CMP. However, the patients with CMP, whether with or without comorbidities, still exhibited a slightly higher incidence of HZ than did those without CMP (7.80 vs. 6.44 per 1,000 person-years among patients with no comorbidities; 13.09 vs. 12.81 per 1,000 person-years among patients with comorbidities) (Table 3).h significant predictor of the severity of chronic pain [24]. Al-Rawaf et al. used microRNAs as biomarkers of pain intensity to explore the correlation between patients’ expression of microRNAs and levels of inflammatory markers. MicroRNAs expression was significantly asso­ ciated with interleukin-6, tumor necrosis factor-α, and cyclooxygenase-2 levels in patients with fatigue [25]. Fatigue may commonly be due to increased levels of pro- inflammatory cytokines such as interleukin-1, interleu­ kin-6 and tumor necrosis factor-α. In addition, pain and behavioral symptoms might also be exacerbated by pro- inflammatory processes [26]. Thus, inflammation may serve as a mechanism underlying both pain and fatigue. The Schoenfeld residuals are calculated for each regres­ sion variable to see if each variable independently satis­ fies the assumptions of the Cox model (Table 4). We used Stratified Cox regression to check, the case group still has a higher risk (Table 5). Corfield et al. Results reported that the incidence of depres­ sion was significantly higher among patients with fatigue than among patients without fatigue, and that fatigue was a strong predictor of depression. Moreover, the authors determined that patients with fatigue or depres­ sion are twice more likely to have both conditions than to have only one. In addition, the symptoms of fatigue and depression may overlap [10]. Both conditions are associ­ ated with an increased activation of the immune system, which affects both the central and peripheral nervous system. Lee and Giuliani postulated that an immunopsy­ chiatric link must exist between fatigue and depression [11]. Results A total of 22,710 patients with CMP and 90,840 patients without CMP were enrolled in the final study (Fig. 1). The mean follow-up period was 7.88 ± 4.88 years for control group, and 8.01 ± 4.91 years for case group. The patients’ characteristics and comorbidities are listed in Table  1. After matching, the age and sex distributions of the cohorts were similar. The patients with CMP exhibited higher rates of CAD and depression than did the patients without CMP. The cumulative incidence of HZ among the patients with CMP was significantly higher than that among the patients without CMP (Fig. 2). The incidence of HZ was higher among the patients with CMP than among the patients without CMP (7.94 and 7.35 per 1000 person-years, respectively) (Table 2). The patients with CMP were 1.06 times more likely to develop HZ than were those without CMP. HZ risk increased with age: compared with patients aged ≤ 49 years, patients aged 50–64 and > 65 years were 2.41 (95% CI = 2.28, 2.55) and 2.69 (95% CI = 2.49, 2.91) times respectively, as likely to develop HZ. estimated using a Cox proportional hazards model. Mul­ tivariate analysis was conducted after age, sex and comor­ bidities were adjusted for. The Kaplan-Meier method was used to assess the cumulative incidence of HZ, and the differences between groups were evaluated using a log- rank test. A two-tailed p value of < 0.05 was considered statistically significant. All data were analyzed using SAS 9.4 for Windows (SAS Institute, Cary, NC, USA). estimated using a Cox proportional hazards model. Mul­ tivariate analysis was conducted after age, sex and comor­ bidities were adjusted for. The Kaplan-Meier method was used to assess the cumulative incidence of HZ, and the differences between groups were evaluated using a log- rank test. A two-tailed p value of < 0.05 was considered statistically significant. All data were analyzed using SAS 9.4 for Windows (SAS Institute, Cary, NC, USA). The effects of CMP on the development of HZ as related to age, sex and the presence of comorbidities are described in Table 3. The patients aged 50–64 and > 65 years, with CMP were 1.10 (95% CI = 1.00, 1.21) and 1.22 (95% CI = 1.06, 1.41) times respectively, more likely to develop HZ compared with patients without CMP in their respective age groups. Discussion In this study, we investigated the relationship between CMP and HZ, and discovered that patients with CMP, especially older adults and women, exhibited a signifi­ cantly higher risk of developing HZ. Chronic pain is a frequently complaint of patients with CMP and is associ­ ated with several conditions, including fatigue, frailty and depression that may exacerbate the stress of patients with CMP. Aili et al. reported that fatigue was a predictive factor for the onset of chronic pain by the 5th-year of follow-up [23]. Manning et al. reported that fatigue sensitivity was a Nakai et al. reported that a strong association between chronic pain and frailty (as defined by five items: Chen et al. BMC Musculoskeletal Disorders (2022) 23:961 Page 5 of 8 Page 5 of 8 Table 2  The incidence and risk factors of herpes zoster Variables Event PY Rate# (per 1000 PY) Crude HR Multivariate HR& (95% CI) (95% CI) Chondromalacia of patella No 5265 716251.9 7.35 1.00 1.00 Yes 1448 182321.0 7.94 1.08 (1.02–1.14)** 1.06 (1.00-1.13)* Age ≤ 49 3249 644208.3 5.04 1.00 1.00 50–64 2442 187042.5 13.06 2.63 (2.50–2.77)*** 2.41 (2.28–2.55)*** 65+ 1022 67322.1 15.18 3.10 (2.88–3.32)*** 2.69 (2.49–2.91)*** Sex Female 4663 548359.4 8.50 1.00 1.00 Male 2050 350213.5 5.85 0.69 (0.66–0.73)*** 0.81 (0.77–0.85)*** Diabetes No 5950 839308.0 7.09 1.00 1.00 Yes 763 59264.9 12.87 1.84 (1.71–1.99)*** 1.07 (0.99–1.16) CKD No 6615 891938.0 7.42 1.00 1.00 Yes 98 6634.9 14.77 2.03 (1.66–2.48)*** 1.19 (0.97–1.46) CAD No 5980 848569.0 7.05 1.00 1.00 Yes 733 50003.9 14.66 2.12 (1.96–2.29)*** 1.20 (1.10–1.30)*** Depression No 6316 862744.4 7.32 1.00 1.00 Yes 397 35828.5 11.08 1.54 (1.39–1.70)*** 1.18 (1.07–1.31)** Cancer No 6398 879227.5 7.28 1.00 1.00 Yes 315 19345.4 16.28 2.28 (2.03–2.55)*** 1.50 (1.34–1.68)*** Rate#, incidence rate, per 1,000 person-years; Crude HR, relative hazard ratio; Adjusted HR&: multivariable analysis including age, sex, and comorbidities of diabetes, CKD, CAD, depression and cancer. *p < 0.05, **p < 0.01, ***p < 0.001. Discussion compared VZV-specific responder cell frequency (RCF) in patients with and without depression to identify the groups’ respective levels of VZV-specific cellular immunity. VZV-specific RCF was significantly lower in the patients with depression than in patients without depression; therefore, the authors concluded that the VZV-specific cellular immunity of patients with depression was significantly lower than that of patients without depression [30]. Liao et al. conducted a population-based study to eval­ uate the incidence of HZ among patients with depres­ sion, and reported that the incidence of HZ was higher among patients with depression than among patients without depression (4.58 and 3.54 per 1,000 person- years, respectively; aHR = 1.1). The authors also discov­ ered that middle-aged patients exhibited the highest risk of HZ compared with any other age group [31]. Choi et al. conducted a study employing a sample more than twice the size of that employed by Liao et al. to assess the incidence of HZ among patients with depression and similarly reported that the incidence of HZ was higher among patients with depression than among patients without depression (6.8% and 6.3%, respectively). In addi­ tion, they reported that patients with depression were 1.09 times more likely to develop HZ than were patients without depression, and that middle-aged women exhib­ ited the highest risk of developing HZ compared with other age and sex group [32]. Table 5  Checking by stratified Cox regression, the case group still has a higher risk Adjusted HR (95% CI) p- value Group No 1.00 Yes 1.06 (1–1.12) 0.047 Age ≦ 49 1.00 50–64 2.41 (2.28–2.54) < 0.001 65+ 2.69 (2.49–2.91) < 0.001 DM No 1.00 Yes 1.07 (0.99–1.16) 0.097 CKD No 1.00 Yes 1.19 (0.97–1.45) 0.096 CHD No 1.00 Yes 1.2 (1.1–1.3) < 0.001 Depression No 1.00 Yes 1.19 (1.07–1.32) 0.001 Cancer No 1.00 Yes 1.5 (1.34–1.69) < 0.001 Table 5  Checking by stratified Cox regression, the case group still has a higher risk In our study, the incidence of depression was higher among the case group than among the control group (Table  1), and according to the univariate analysis, HZ risk was higher among patients with any comorbidities than among those without comorbidities (Table 2). How­ ever, the incidence of HZ among the patients with CMP with or without comorbidities was higher than that of the patients without CMP (Table 3). CMP must be stressful for involving persons. Discussion CKD: chronic kidney disease; CAD: coronary artery disease Table 3  Incidence of herpes zoster by age, sex and comorbidities, and Cox model measured hazards ratio for patients with chondromalacia of patella Variables Chondromalacia of patella No Yes Crude HR* Multivariate HR& Event PY Rate# Event PY Rate# (95% CI) (95% CI) Age ≤ 49 2595 513934.4 5.05 654 130273.8 5.02 0.99 (0.91–1.08) 0.98 (0.90–1.06) 50–64 1901 149089.8 12.75 541 37952.7 14.25 1.12 (1.02–1.23)* 1.10 (1.00-1.21)* 65+ 769 53227.6 14.45 253 14094.5 17.95 1.24 (1.08–1.43)** 1.22 (1.06–1.41)** Sex Female 3693 436871.9 8.45 970 111487.5 8.70 1.20 (1.08–1.33)*** 1.18 (1.07–1.31)** Male 1572 279380.0 5.63 478 70833.6 6.75 1.03 (0.96–1.10) 1.01 (0.94–1.08) Comorbidity§ No 3950 613587.1 6.44 1016 149318.1 6.80 1.06 (0.99–1.13) 1.06 (0.99–1.14) Yes 1315 102664.7 12.81 432 33002.9 13.09 1.02 (0.91–1.14) 1.05 (0.94–1.17) Rate#, incidence rate, per 1,000 person-years; Crude HR*, relative hazard ratio; Adjusted HR&: multivariable analysis including age, sex, and comorbidities§. Patients with any comorbidities of diabetes, CKD, CAD, depression, and cancer were classified into the comorbidity group. *p < 0.05, **p < 0.01, ***p < 0.001 f herpes zoster by age, sex and comorbidities, and Cox model measured hazards ratio for patients with pain were 50% and 37% among patients with frailty and pre-frailty, respectively. Furthermore, the authors found that patients with chronic pain were a 1.85 times more likely to have frailty than those without chronic pain [28]. Otones Reyes et al. investigated the relationship between exhaustion, slowness, weakness, low physical activity, and weight loss) or pre-frailty [27]. A systematic review and meta-analysis reported that prevalence rates of frailty and pre-frailty in patients with chronic pain were 18% and 43%, respectively. The prevalence rates of chronic Chen et al. BMC Musculoskeletal Disorders (2022) 23:961 Page 6 of 8 Page 6 of 8 Chen et al. BMC Musculoskeletal Disorders Table 4  Cox proportional-hazards model assumption test chisq df p Group 1.86 1 0.172 Sex 9.67 1 0.002 Age 1.97 1 0.160 Diabetes 0.28 1 0.596 CAD 0.23 1 0.628 CHD 1.55 1 0.213 Depression 0.26 1 0.608 cancer 0.22 1 0.642 Globle 14.33 8 0.074 CKD: chronic kidney disease; CAD: coronary artery disease psychosocial factors [13]. On the basis of the aforemen­ tioned reports, frailty and depression can be concluded to be frequently concomitant conditions.i Irwinet et al. Data availability Data are available from the National Health Insurance Research Database which provided by the Ministry of Health and Welfare (MOHW), Taiwan. However, data cannot be disclosed because of data protection laws. Data requests should be applied following a formal procedure (http://dep.mohw. gov.tw). Please contact the staff of MOHW (Email: stdlwu@mohw.gov.tw) for assistance. References 1. Stougård J. Chondromalacia of the patella. Incidence, macroscopical and radiographical findings at autopsy. Acta Orthop Scand. 1975;46(5):809–22. Ö 1. Stougård J. Chondromalacia of the patella. Incidence, macroscopical and radiographical findings at autopsy. Acta Orthop Scand. 1975;46(5):809–22. Conflict of interest l The authors declare that they have no conflict of interest. The authors declare that they have no conflict of interest. Received: 2 May 2022 / Accepted: 29 October 2022 Received: 2 May 2022 / Accepted: 29 October 2022 Funding This study was supported by Ditmanson Medical Foundation, Chia-Yi Christian Hospital (Research program R110-10). The fund was not involved in research design, data collection, analysis, and interpretation of data, writing reports, or deciding to submit article for publication. Acknowledgements Not Applicable. impropriety in diagnosis, drug prescription or examina­ tion is punishable by law. Therefore, although diagnosis codes were issued by different specialists, diagnoses ana­ lyzed in this study were still generally accurate. Second, the severity of a disease affects its subsequent treatment and results in different prognoses. The ICD-10-CM has been officially adopted since 2016; however, because the diagnostic codes used in this study are mainly ICD-9-CM codes, the severity of each patient’s disease cannot be distinguished. Third, NHIRD data do not fully cover the life-style of the patients. Each patient’s dietary (vegetar­ ian or non-vegetarian), smoking (number of cigarettes), drinking (beer or spirits), and exercise (number of exer­ cises and length of time) habits may affect the appear­ ance, severity and prognosis of their disease. Fourth, self-financed treatments are not consistently recorded in the NHIRD. Few patients with CMP who experience chronic pain may seek alternative treatments, such as herbal medicine, acupuncture or therapeutic massage. These self-financed treatments may not be recorded in the NHIRD, but may not affect the overall incidence of CMP. Author contributions Conceptualization, C.-H.C., Y.-C.C., C.-Y.H., D.-S.K., W.-C.H.; Methodology, C.-H.C., Y.-C.C., H.-Y.Y., C.-F.T., C.-Y.H.; Formal analysis, H.-Y.Y., C.-F.T.; Project administration, C.-H.C., Y.-C.C., H.-Y.Y., C.-F.T., C.-Y.H., D.-S.K., W.-C.H.; Writing- original draft, C.-H.C., Y.-C.C., C.-Y.H.; Writing- review and editing, D.-S.K., W.-C.H.; Supervision, D.-S.K., W.-C.H. All authors have read and agreed to the published version of the manuscript. Conceptualization, C.-H.C., Y.-C.C., C.-Y.H., D.-S.K., W.-C.H.; Methodology, C.-H.C., Y.-C.C., H.-Y.Y., C.-F.T., C.-Y.H.; Formal analysis, H.-Y.Y., C.-F.T.; Project administration, C.-H.C., Y.-C.C., H.-Y.Y., C.-F.T., C.-Y.H., D.-S.K., W.-C.H.; Writing- original draft, C.-H.C., Y.-C.C., C.-Y.H.; Writing- review and editing, D.-S.K., W.-C.H.; Supervision, D.-S.K., W.-C.H. All authors have read and agreed to the published version of the manuscript. Discussion CMP can seriously affect patient’ wellbeing, and the risk of HZ should be assessed in patients with CMP, especially women. chronic pain and frailty, and discovered that 45% of patients with frailty also had chronic pain. The authors concluded that chronic pain was a predictor of frailty [29]. This study does have some limitations. First, the diag­ nosis codes were obtained from Taiwan’s NHIRD. CMP could be diagnosed by patient’s medical history and phys­ ical examination or imaging findings. Most patients with HZ were diagnosed by the appearance of painful herpeti­ form vesicles in a restricted dermatomal distribution. The patients’ diagnosis of CMP or HZ were made by differ­ ent specialists; therefore, diagnosis bias may be existed. However, Taiwan’s NHI Administration (NHIA) has a strict review system. When a prescription or results of an examination are returned to the NHIA with a diagno­ sis, the diagnosis is reviewed under strict protocols. Any Soysal et al. reported a reciprocal interaction exists between depression and frailty. The prevalence of depres­ sion among the patients with frailty enrolled in their study was 38.6%, and the patients with frailty were 2.64 times as likely to develop depression. Moreover, the prevalence of frailty was 40.4% among the patients with depression, and the patients with depression were 3.72 times as likely to have frailty [12]. Oyon et al. observed a dose-response relationship between severity of depres­ sion and risk of frailty; the severity of depression exhib­ ited a higher association with frailty than with other Page 7 of 8 Page 7 of 8 Chen et al. BMC Musculoskeletal Disorders (2022) 23:961 Chen et al. BMC Musculoskeletal Disorders (2022) 23:961 Chen et al. BMC Musculoskeletal Disorders (2022) 23:961 Ethics approval and consent to participate This study was approved by the institutional review board of Ditmanson Medical Foundation, Chia-Yi Christian Hospital, in Chia-Yi, Taiwan (approval number: IRB2020113, Octorber 21, 2020). The need for informed consent was waived by the Institutional Review Board of Ditmanson Medical Foundation, Chia-Yi Christian Hospital, because of the retrospective nature of the study. Due to the unique insurance system with high acces­ sibility, most Taiwanese will seek medical care whenever having any discomfort. Therefore, the diagnosis of HZ in this study is highly trustable and will not be affected by presence or not of CMP. This is a retrospective cohort study, the temporality between diagnosis of CMP and occurrence of HZ is preserved, therefore possibility of a causal association is present. Despite the limitations of this study, our finding of a strong positive correlation between CMP and HZ from analysis of NHIRD data, contributes to the literature and clinical practice. More­ over, the results can provide a reference for population medicine research in the future. Conclusion Co-occurrence and symptomatology of fatigue and depression. Compr Psychiatry. 2016;71:1–10. 25. Al-Rawaf HA, Alghadir AH, Gabr SA. MicroRNAs as biomarkers of pain intensity intensity in patients with chronic fatigue syndrome. Pain Pract. 2019;19(8):848–60. fatigue and depression. Compr Psychiatry. 2016;71:1–10. 11. Lee CH, Giuliani F. The role of inflammation in depression and fatigue. Front Immunol. 2019;10:1696. 12. Soysal P, Veronese N, Thompson T, Kahl KG, Fernandes BS, Prina AM, et al. Relationship between depression and frailty in older adults: a systematic review and meta-analysis. Ageing Res Rev. 2017;36:78–87. 26. Louati K, Berenbaum F. Fatigue in chronic inflammation - a link to pain path­ ways. Arthritis Res Ther. 2015;17:254. 27. Nakai Y, Makizako H, Kiyama R, Tomioka K, Taniguchi Y, Kubozono T, et al. Association between chronic pain and physicial fraility in community-dwell­ ing older adults. Int J Environ Res Public Health. 2019;16(8):1330. 13. Oyon J, Serra-Prat M, Ferrer M, Llinares A, Pastor N, Limón E, et al. Psychosocial factors associated with frailty in the community-dwelling aged population with depression. A cross-sectional study. Aten Primaria. 2021;53(5):102048. 28. Lin T, Zhao Y, Xia X, Ge N, Yue J. Association between frailty and chronic pain among older adults: a systematic review and meta-analysis. Eur Geriatr Med. 2020;11(6):945–59. 14. Marin M, Harpaz R, Zhang J, Wollan PC, Bialek SR, Yawn BP. Risk factors for herpes zoster among adults. Open Forum Infect Dis. 2016;3(3):ofw119. 15. Hsu CY, Lin CL, Kao CH. Association between chronic interstitial cystitis and herpes zoster. Int J Environ Res Public Health. 2020;17(7):2228. 29. Otones Reyes P, García Perea E, Pedraz Marcos A. Chronic pain and frailty in community-dwelling older adults: a systematic review. Pain Manag Nurs. 2019;20(4):309–15. 29. Otones Reyes P, García Perea E, Pedraz Marcos A. Chronic pain and frailty in community-dwelling older adults: a systematic review. Pain Manag Nurs. 2019;20(4):309–15. 16. Hsu CY, Ke DS, Lin CL, Kao CH. Risk of herpes zoster infection in men with varicocele. Postgrad Med. 2021;133(6):599–603. 30. Irwin M, Costlow C, Williams H, Artin KH, Chan CY, Stinson DL, et al. Cellular immunity to varicella-zoster virus in patients with major depression. J Infect Dis. 1998;178(Suppl 1):104-8. 30. Irwin M, Costlow C, Williams H, Artin KH, Chan CY, Stinson DL, et al. Cellular immunity to varicella-zoster virus in patients with major depression. J Infect Dis. 1998;178(Suppl 1):104-8. 17. Hsu CY, Ke DS, Lin CL, Kao CH. Conclusion To investigate the risk of herpes zoster in women with endometriosis: a Taiwan national population-based cohort study. Front Med (Lausanne). 2021;8:584322. 31. Liao CH, Chang CS, Muo CH, Kao CH. High prevalence of herpes zoster in patients with depression. J Clin Psychiatry. 2015;76(9):e1099-104. 31. Liao CH, Chang CS, Muo CH, Kao CH. High prevalence of herpes zoster in patients with depression. J Clin Psychiatry. 2015;76(9):e1099-104. 18. Hsu CY, Ke DS, Lin CL, Kao CH. Risk of herpes zoster in patients with adhesive capsulitis of the shoulder. Int J Environ Res Public Health. 2020;17(10):3592. 32. Choi HG, Kim EJ, Lee YK, Kim M. The risk of herpes zoster virus infection in patients with depression: a longitudinal follow-up study using a national sample cohort. Med (Baltim). 2019;98(40):e17430. 32. Choi HG, Kim EJ, Lee YK, Kim M. The risk of herpes zoster virus infection in patients with depression: a longitudinal follow-up study using a national sample cohort. Med (Baltim). 2019;98(40):e17430. 19. Ke DS, Hsu CY, Lin CL, Hsu CY, Kao CH. Herpes zoster in patients with sciatica. BMC Musculoskelet Disord. 2020;21(1):813. 20. Hsu CY, Ke DS, Lin CL, Kao CH. Association between lateral epicondylitis and the risk of herpes zoster development. Postgrad Med. 2021;133(1):96–101. 21. Hsu CY, Ke DS, Lin CL, Kao CH. Plantar fascial fibromatosis and herpes zoster. PLoS ONE. 2021;16(11):e0259942. Conclusion 2. Özdemir M, Kavak RP. Chondromalacia patella among military recruits with anterior knee pain: prevalence and association with patellofemoral malalign­ ment. Indian J Orthop. 2019;53(6):682–8. In this study, the patients with CMP, especially older adults and women, exhibited a significantly higher risk of HZ reactivation. The risk of HZ should be carefully assessed in patients with CMP, and the necessity of HZ vaccination should be informed. 3. García-Triana SA, Toro-Sashida MF, Larios-González XV, Fuentes-Orozco C, Mares-País R, Barbosa-Camacho FJ, et al. The benefit of perineural injection treatment with Dextrose for treatment of chondromalacia patella in partici­ pants receiving home physical therapy: a pilot randomized clinical trial. J Altern Complement Med. 2021;27(1):38–44. 4. Cheong C, Lee TJ. Prevalence and healthcare utilization of herpes zoster and postherpetic neuralgia in South Korea: disparity among patients with differ­ ent immune statuses. Epidemiol Health. 2014;36:e2014012. Abbreviations CAD Coronary artery disease CKD Chronic kidney disease CMP Chondromalacia patella RCF Responder cell frequency HZ Herpes zoster ICD-9-CM International classification of diseases, ninth revision, clinical modification ICD-10-CM International classification of diseases, tenth revision, clinical modification LGTD Longitudinal generation tracking database NHI National health insurance NHIA National health insurance administration NHIRD National health insurance research database VZV Varicella zoster virus 5. Kawai K, Yawn BP, Wollan P, Harpaz R. Increasing incidence of herpes zoster over a 60-year period from a population-based study. Clin Infect Dis. 2016;63(2):221–6. 6. Thompson RR, Kong CL, Porco TC, Kim E, Ebert CD, Acharya NR. Herpes zoster and post-herpetic neuralgia: changing incidence rates from 1994 to 2018 in the United States. Clin Infect Dis. 2021;73(9):e3210-7. 7. Aggarwal A, Suresh V, Gupta B, Sonthalia S. Post-herpetic neuralgia: a system­ atic review of current interventional pain management strategies. J Cutan Aesthet Surg. 2020;13(4):265–74. 8. Kostov S, Schug SA. Depression and chronic pain. Saudi J Anaesth. 2018;12(3):377–8. Page 8 of 8 Page 8 of 8 Chen et al. BMC Musculoskeletal Disorders (2022) 23:961 (2022) 23:961 9. Rapti E, Damigos D, Apostolara P, Roka V, Tzavara C, Lionis C. Patients with chronic pain: evaluating depression and their quality of life in a single center study in Greece. BMC Psychol. 2019;7(1):86. 24. Manning K, Kauffman BY, Rogers AH, Garey L, Zvolensky MJ. Fatigue severity severity and fatigue sensitivity: relations to anxiety, depression, pain catastro­ phizing, and pain severity among adults with severe fatigue and chronic low back pain. Behav Med. 2020:1–9. 10. Corfield EC, Martin NG, Nyholt DR. 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. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 22. Hsu CY, Ke DS, Lin CL, Kao CH. Association between de Quervain syn­ drome and herpes zoster: a population-based cohort study. BMJ Open. 2021;11(12):e046891. 23. Aili K, Andersson M, Bremander A, Haglund E, Larsson I, Bergman S. Sleep problems and fatigue as predictors for the onset of chronic widespread pain over a 5- and 18-year perspective. BMC Musculoskelet Disord. 2018;19(1):390.
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Categorization of 77 dystrophinexons into 5 groups by a decision tree using indexes of splicing regulatory factors as decision markers
BMC genomic data
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RESEARCH ARTICLE Open Access Abstract Background: Duchenne muscular dystrophy, a fatal muscle-wasting disease, is characterized by dystrophin deficiency caused by mutations in the dystrophin gene. Skipping of a target dystrophin exon during splicing with antisense oligonucleotides is attracting much attention as the most plausible way to express dystrophin in DMD. Antisense oligonucleotides have been designed against splicing regulatory sequences such as splicing enhancer sequences of target exons. Recently, we reported that a chemical kinase inhibitor specifically enhances the skipping of mutated dystrophin exon 31, indicating the existence of exon-specific splicing regulatory systems. However, the basis for such individual regulatory systems is largely unknown. Here, we categorized the dystrophin exons in terms of their splicing regulatory factors. Results: Using a computer-based machine learning system, we first constructed a decision tree separating 77 authentic from 14 known cryptic exons using 25 indexes of splicing regulatory factors as decision markers. We evaluated the classification accuracy of a novel cryptic exon (exon 11a) identified in this study. However, the tree mislabeled exon 11a as a true exon. Therefore, we re-constructed the decision tree to separate all 15 cryptic exons. The revised decision tree categorized the 77 authentic exons into five groups. Furthermore, all nine disease- associated novel exons were successfully categorized as exons, validating the decision tree. One group, consisting of 30 exons, was characterized by a high density of exonic splicing enhancer sequences. This suggests that AOs targeting splicing enhancer sequences would efficiently induce skipping of exons belonging to this group. Conclusions: The decision tree categorized the 77 authentic exons into five groups. Our classification may help to establish the strategy for exon skipping therapy for Duchenne muscular dystrophy. Keywords: Splicing, Dystrophin, Exon, Splicing enhancer, Decision tree © 2012 Malueka 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. Categorization of 77 dystrophin exons into 5 groups by a decision tree using indexes of splicing regulatory factors as decision markers Rusdy Ghazali Malueka1, Yutaka Takaoka2, Mariko Yagi1, Hiroyuki Awano1, Tomoko Lee1, Ery Kus Dwianingsih1, Atsushi Nishida1,3, Yasuhiro Takeshima1 and Masafumi Matsuo1,4* Malueka et al. BMC Genetics 2012, 13:23 http://www.biomedcentral.com/1471-2156/13/23 Malueka et al. BMC Genetics 2012, 13:23 http://www.biomedcentral.com/1471-2156/13/23 * Correspondence: mmatsuo@reha.kobegakuin.ac.jp 1Department of Pediatrics, Graduate School of Medicine, Kobe University, Chuo, Kobe 6500017, Japan Full list of author information is available at the end of the article Background At the next node, SF2/ASF-D was used as the splitting variable, with a cut-off point of less than 10.44, generating a group of 32 authentic exons. In this way, seven nodes were used to separate clearly the 77 authentic exons from the 14 cryptic exons. The authentic exons were categorized into four groups, comprising 43, 32, 1, and 1 exon, respec- tively; similarly, the cryptic exons were also categorized into four groups. to one of the downstream authentic dystrophin exons, pro- ducing a tissue-specific dystrophin isoform [4,5]. Further- more, alternative splicings of some exons lead to the production of additional isoforms of the tissue-specific transcripts [6-8]. Remarkably, 14 cryptic exons that resem- ble authentic exons in terms of length and splice site strength, but are very rarely if ever spliced, have been reported within the huge introns [9,10]. Splicing is the process that removes introns from pre- mRNA, and is performed in the spliceosome, a ribonucleo- protein assembly. The spliceosome is one of the most com- plex cellular machineries, comprising approximately 150 proteins and five small nuclear RNAs (snRNAs U1, U2, U4, U5, and U6) [11]. Three sites, the splice donor site (5’ss), the splice acceptor site (3’ss), and the branch point sequences are the core splice site signals that are present in every intron. Despite the high potential for errors, the spli- cing process appears to occur with high fidelity, implying the widespread involvement of additional transcript fea- tures. These exonic elements are conventionally classified as exonic splicing enhancers (ESEs) or silencers (ESSs) and they function to promote or inhibit inclusion of the exon in which they reside, respectively. These splicing regulatory elements function by recruiting trans-acting splicing fac- tors that activate or suppress splice site recognition or spli- ceosome assembly by various mechanisms [12]. AOs that induce skipping of dystrophin exons have been mainly designed against ESEs of target exons to hamper recruit- ment of splicing factors [2,3]. We evaluated the decision tree by analyzing a novel cryptic exon 11a that was identified in this study. Exon 11a was found inserted into dystrophin mRNA in one DMD case who had a two-nucleotide (CA) deletion at the 5th and 6th nucleotides of exon 12 (c.1336_1337del). RT- nested PCR amplification of a fragment spanning exons 10 to 14 from this individual revealed two products: one cor- responding to the normal size and the other larger than expected (Figure 2). Background produce in-frame dystrophin mRNA from the out-of- frame mRNA by inducing exon skipping during splicing [1]. The newly generated in-frame dystrophin mRNA is expected to produce semi-functional, internally deleted dystrophin protein. Currently, induction of exon skipping with AOs is considered one of the most promising treat- ments for DMD [2,3]. Duchenne muscular dystrophy (DMD), a fatal muscle- wasting disease, is the most common inherited muscle dis- ease, affecting one in every 3500 male births. DMD is characterized by dystrophin deficiency caused by muta- tions in the dystrophin gene, the largest human gene that spans over 2500 kb on the X-chromosome. For the treat- ment of DMD, antisense oligonucleotides (AOs) against splicing regulatory sequences have been proposed to The dystrophin gene encodes a 14-kb mRNA consisting of 79 exons and is characterized by its huge intron size; the largest, intron 44, is 248 kb long. In addition, eight alternative promoters that are activated in a tissue-specific manner have been identified. Each tissue-specific exon 1 under the control of a cryptic promoter is spliced correctly * Correspondence: mmatsuo@reha.kobegakuin.ac.jp 1Department of Pediatrics, Graduate School of Medicine, Kobe University, Chuo, Kobe 6500017, Japan Full list of author information is available at the end of the article Malueka et al. BMC Genetics 2012, 13:23 http://www.biomedcentral.com/1471-2156/13/23 Page 2 of 10 Malueka et al. BMC Genetics 2012, 13:23 http://www.biomedcentral.com/1471-2156/13/23 splicing regulatory factors as decision markers. Cryptic exons within the dystrophin gene resemble authentic exons in terms of length and splice site strength, but are very rarely if ever spliced [10]. Therefore, analyzing the exon recognition parameters of these exons compared to the authentic dystrophin exons can give insight into which splicing regulatory elements actually play a critical role in the splicing of dystrophin exons. The goal of the decision tree was to determine the critical parameters that provided the most accurate categorization of authen- tic exons and cryptic exons. A preliminary decision tree was constructed to discriminate 77 authentic exons from 14 known cryptic exons. To classify these exons, we used 26 indexes that have been reported as important in proper splicing (see Methods). The decision tree system output a simple data structure (Figure 1). The decision tree revealed that the strength of the 3’ss calculated by maximum entropy (ME3’ss) was the first splitting vari- able, with a cut-off point of 1.39. At this node, four cryp- tic exons were classified into one group. Background Sequencing of the two products revealed that the normal-size band had the predicted exon content with the two-nucleotide deletion, and the larger product contained a 157-bp unknown insertion between exons 11 and 12. The inserted 157-bp sequence was iden- tical to part of the 29.7-kb-long intron 11. The corre- sponding intronic sequence was present 5244 nt downstream of the 3’ end of exon 11 and 24,278 nt upstream of exon 12 (Figure 2c). The inserted sequence maintained the AG and GT dinucleotide consensus sequences for splicing acceptor and donor sites at either end. Although the sequences flanking the inserted sequence were examined in this individual, no nucleotide change was found. The inserted sequence was named exon 11a. Recently, we reported that a small chemical enhances skipping of mutated dystrophin exon 31 in a sequence- specific manner, not altering the splicing of other dystro- phin exons [13]. It was strongly suggested that dystrophin exons have their own splicing regulatory systems. How- ever, the characteristics of the splicing regulatory systems of individual dystrophin exons are not well understood. Decision trees are classifiers that predict class labels for data items and can make very accurate predictions [14,15]. They have been used to establish an integrated method that is one of the best available ways to find genes in the human genome [15]. Here, we identified a novel cryptic exon in intron 11 of the dystrophin gene in a DMD patient, and constructed decision trees to discriminate authentic exons from cryp- tic exons. Finally, we categorized 77 authentic exons into five groups based on indexes of their splicing regulatory factors. From the decision tree, we suppose that one group of exons showing high density for ESE sequences are a good target for exon skipping therapy. Results When we tested exon 11a on our decision tree, it was classified not as a cryptic exon but as a real exon. There- fore, we decided that this tree was not suitable to classify To examine the splicing regulatory factors that character- ize particular exons, we constructed decision trees classi- fying authentic from cryptic exons using indexes of Malueka et al. BMC Genetics 2012, 13:23 http://www.biomedcentral.com/1471-2156/13/23 Malueka et al. BMC Genetics 2012, 13:23 Page 3 of 10 Figure 1 Preliminary decision tree to classify 77 authentic and 14 cryptic dystrophin exons. Exons are passed down the tree beginning at the top, where a “yes” result on any test means that it should be passed down to the left. The features tested in this tree are the maximum entropy at the 3’ splice site (ss) (ME3’ss), the SF2/ASF density (SF2/ASF-D), the GC content at the 5’ss (GC5’ss), the maximum entropy at the 5’ss (ME5’ss), the free energy at the 5’ss U1 snRNP binding site (FE), the number of exonic splicing silencer (FESS), and the Shapiro score at the 3’ss (SH3’ss). The internal nodes of the tree represent index values that are tested for each exon as it is passed through the tree. Each successive node in the tree represents a decision that is based on those values, until a final classification is reached (the leaves). Authentic and cryptic exons were classified into four groups each. Figure 1 Preliminary decision tree to classify 77 authentic and 14 cryptic dystrophin exons. Exons are passed down the tree beginning at the top, where a “yes” result on any test means that it should be passed down to the left. The features tested in this tree are the maximum entropy at the 3’ splice site (ss) (ME3’ss), the SF2/ASF density (SF2/ASF-D), the GC content at the 5’ss (GC5’ss), the maximum entropy at the 5’ss (ME5’ss), the free energy at the 5’ss U1 snRNP binding site (FE), the number of exonic splicing silencer (FESS), and the Shapiro score at the 3’ss (SH3’ss). The internal nodes of the tree represent index values that are tested for each exon as it is passed through the tree. Each successive node in the tree represents a decision that is based on those values, until a final classification is reached (the leaves). Authentic and cryptic exons were classified into four groups each. Results Both the AG and GT splicing consensus dinucleotides are present adjacent to exon 11a. Figure 2 Cloning of cryptic exon 11a. a. RT-nested PCR products. A fragment spanning exons 10 to 14 was amplified by RT-nested PCR. Two amplified products were obtained from peripheral lymphocytes of a DMD patient (P) but not a control (C). A schematic representation of the exon structure of the amplified fragments is shown on the right. b. Sequences at the exon junctions. Subcloning and sequencing of the amplified products revealed that the larger product contained a 157-bp insertion (exon 11a) between exons 11 and 12. c. Schematic description of the location of exon 11a. The 5’ and 3’ ends of exon 11a (hatched box) are 5.2 kb downstream of exon 11, and 24.3 kb upstream of exon 12, respectively. Both the AG and GT splicing consensus dinucleotides are present adjacent to exon 11a. Therefore, we consider our decision tree a suitable clas- sifier of pathological dystrophin exons. and 78; group E). On the “no” branch at the fourth node, SIZE with a cut-off point of 144 was next used, categoriz- ing the final data into one exon group consisting of exons 2 and 3 (group D) and one group containing four cryptic exons (group d). Seven indexes were used in common by both the preli- minary and re-constructed trees. However, their values in each tree were different, except for one (≤1.39 ME3’ss score at the first node). When we compared the two trees, we found that three exons (2, 70, and 78) and two exons (40 and 56) were re-categorized into small groups (groups E and D) and the largest group (group C), respectively. This indicated slight changes in the categorizing pathways between the two trees. The cryptic exons were ultimately categorized into four groups, and exon 11a belonged to group d, which comprises exons 1a, 2a, and 3a-l. It was interesting that group D was categorized by a SIZE score of ≤114 (Figure 3). This indicates that a small exon can be real if the splicing silencer density is high. We evaluated our re-constructed tree using nine dis- ease-causing exons that have been identified in the dys- trophin mRNAs of dystrophinopathy patients. These pathological exons are created by the effect of deep- intron single nucleotide mutations. Results ME5’ss with a cut-off point of 5.58 was used to divide the data into two subnodes. On the “yes” branch, FE with a cut-off point of -2.6 split the group: one exon group con- sisting of five cryptic exons (group b) and one exon group containing only exon 27 (group B). On the other, “no” branch, FESS-D with a cut-off point of 4.45 divided the group into two subnodes. On the “yes” branch, GC5’ss with a cut-off point of 46.8 was used at the fifth node. One big exon group (group C), comprising 42 exons, formed the “yes” branch. On the other, “no” branch, SH3’ss with a cut-off point of 0.79 was used at the sixth node, generating a cryptic exon group (group c) and a group containing two authentic exons (exons 70 exon 11a. We reconstructed the decision tree, including exon 11a as an additional cryptic exon using the same 26 indexes that were used to construct the first decision tree. The final tree, which had eight nodes, was able to separate all 15 cryptic exons from all 77 authentic exons with 100% accuracy (Figure 3). ME3’ss was the first split- ting variable, with a cut-off point of 1.39. At this node, four cryptic exons were classified into one group (group a; Figure 3). SF2/ASF-D was the splitting variable at the second node, with a cut-off point of 10.53. A group of 32 exons (group A) was categorized on its “no” branch. Group A, therefore, was characterized by a high-density of ESEs recognized by SF2/ASF. At the third node, Malueka et al. BMC Genetics 2012, 13:23 http://www.biomedcentral.com/1471-2156/13/23 Malueka et al. BMC Genetics 2012, 13:23 Page 4 of 10 http://www.biomedcentral.com/1471-2156/13/23 Figure 2 Cloning of cryptic exon 11a. a. RT-nested PCR products. A fragment spanning exons 10 to 14 was amplified by RT-nested PCR. Two amplified products were obtained from peripheral lymphocytes of a DMD patient (P) but not a control (C). A schematic representation of the exon structure of the amplified fragments is shown on the right. b. Sequences at the exon junctions. Subcloning and sequencing of the amplified products revealed that the larger product contained a 157-bp insertion (exon 11a) between exons 11 and 12. c. Schematic description of the location of exon 11a. The 5’ and 3’ ends of exon 11a (hatched box) are 5.2 kb downstream of exon 11, and 24.3 kb upstream of exon 12, respectively. Results These mutations confer the characteristics of an authentic exon on a por- tion of intronic sequence, and thus the intron segment becomes recognized as an exon during the splicing pro- cess. Therefore, the decision tree should categorize these pathological exons as authentic exons. Remark- ably, all nine pathological exons were correctly categor- ized into one of the four exon groups: four in group A, three in group C, and one in each of groups D and E. Among the 26 indexes used as candidates for the decision markers, only eight were used in the tree. Furthermore, five Malueka et al. BMC Genetics 2012, 13:23 Malueka et al. BMC Genetics 2012, 13:23 http://www.biomedcentral.com/1471-2156/13/23 Page 5 of 10 Figure 3 Final decision tree to classify 77 authentic and 15 cryptic dystrophin exons. The structure of the tree is as described for Figure 1. The features tested in this tree include seven indexes used in the preliminary tree (Figure 1) and one additional index, exon size (SIZE). This tree classified the authentic dystrophin exons into five groups (groups A to E), containing 30, 1, 42, 2, and 2 exons, respectively. The cryptic exons were classified into four groups (groups a to d). Figure 3 Final decision tree to classify 77 authentic and 15 cryptic dystrophin exons. The structure of the tree is as described for Figure 1. The features tested in this tree include seven indexes used in the preliminary tree (Figure 1) and one additional index, exon size (SIZE). This tree classified the authentic dystrophin exons into five groups (groups A to E), containing 30, 1, 42, 2, and 2 exons, respectively. The cryptic exons were classified into four groups (groups a to d). splicing errors caused by intra-exon mutation. We have previously reported nonsense mutation-induced exon skipping in seven exons (exons 14, 15, 17, 27, 41, 42, and 70) [16]. Among these, only four (exons 17, 27, 41, and 42) cause disruption of an ESE. Remarkably, exons 17, 41, and 42 were categorized in group A, while all those that do not disrupt an ESE were categorized in other groups. This indicates that, for exons in group A, nonsense muta- tion-induced exon skipping is caused by disruption of an ESE, while for exons in other groups, nonsense mutation- induced exon skipping is caused by other factors. Results Third, we examined exons that splice to tissue-specific exon 1 (exons 2, 30, 45, 56, and 63). We did not identify any par- ticular categorization characteristics for these exons. of these eight were those determining the strength of splice sites (Table 1). This indicates that recognition of the spli- cing sites is very important for proper splicing. Group E (exons 70 and 78) was categorized through six nodes; the final one discriminating it from group c (cryptic exons 2b and 77a). For this categorization, the strength of the 3’ss was tested twice using different indexes (ME3’ss and SH3’ss). This indicates the importance of the 3’ss strength relative to the 5’ss strength in dystrophin splicing. Indexes of ESE density (SF2/ASF-D) and ESS density (FESS-D) were also used in the tree. This indicates that both the ESE and ESS densities are important in the splicing process. Next, we used the tree to characterize exons that are subjected to specific splicing patterns. First, we marked exons known to be alternatively spliced (data not shown). However, they were found in all five groups. This indicates that no particular cis-elements predispose to alternative splicing. Second, we examined exons that were prone to Discussion It is important to understand more about the splicing regulatory mechanisms for each exon of the dystrophin gene because treatment with exon skipping is a promis- ing therapeutic approach. tissue-specific promoters, 15 cryptic exons, and gener- ates many alternatively spliced products. It has been reported that there is a gradient in exon and intron defi- nition at the level of pre-mRNA splicing [17]. For exam- ple, efficient use of intrinsically weak cryptic splice sites in exons is facilitated by a higher-than-average density of ESSs and a high density of SF2/ASF ESE motifs [17]. This suggests that each exon has a specific splicing reg- ulatory mechanism in which particular splicing factors could compensate for the lack of other splicing factors. It is important to understand more about the splicing regulatory mechanisms for each exon of the dystrophin gene because treatment with exon skipping is a promis- ing therapeutic approach. Our exon categorization suggested that there are at least five different patterns of splicing regulatory mechanisms for dystrophin pre-mRNAs (Table 1). At the extreme, one group contained only one exon (exon 27; group B), imply- ing that this exon has a unique splicing regulatory mechanism. In fact, alteration of exon 27 splicing has been reported for two nonsense mutations, resulting in a mild phenotype [19]. Exon 27 may be particularly vulnerable to splicing alterations caused by intra-exonic mutations. Decision trees have been used in many kinds of classifi- cations, such as for the management of Parkinson’s dis- ease, disease severity profiling, large-scale proteomic studies, and microarray data classification [14,18]. Here, we classified 77 authentic and 15 cryptic dystrophin exons into five groups based on their splicing regulatory factor characteristics. In this study, we needed to modify our pre- liminary decision tree (Figure 1) based on data from the novel exon 11a (Figure 2). This suggested a possibility that the tree needs further modification when another cryptic exon is identified in the future construction. However, we believe any modifications, if necessary, will be minimal as exon 11a was additionally analyzed (Figure 3). Moreover, our trees were constructed using indexes obtained only from cis-elements; further modification incorporating information from trans-elements may further improve the reliability of the tree. On the other hand, two groups (A and C) contained 72 out of 77 exons, suggesting that most dystrophin exons are under similar splicing control. Discussion The dystrophin gene produces many mRNAs because it is so complex: it contains 79 exons, huge introns, eight Malueka et al. BMC Genetics 2012, 13:23 http://www.biomedcentral.com/1471-2156/13/23 Page 6 of 10 Table 1 Summary of categorization of exons by the decision tree Number of Exon Indexes ME3’ss SF2/ASF-D ME5’SS FE FESS-D GC5’ss SH3’ss SIZE Group A 30 H H - - - - - - B 1 H L L H - - - - C 42 H L H - L L - - D 2 H L H - H - - L E 2 H L H - L H H - Results of categorization of 77 dystrophin exons are tabulated. Indexes determining the strength of splice sites are marked bold. H and L represent high and low at each node, respectively. Bars indicate not applicable. Table 1 Summary of categorization of exons by the decision tree Results of categorization of 77 dystrophin exons are tabulated. Indexes determining the strength of splice sites are marked bold. H and L represent high and low at each node, respectively. Bars indicate not applicable. Results of categorization of 77 dystrophin exons are tabulated. Indexes determining the strength of splice sites are marked bold. H and L represent high and low at each node, respectively. Bars indicate not applicable. sequence as indicated by maximum entropy score is the most critical, as all authentic dystrophin exons must have a maximum entropy score of more than 1.39 at the 3’ss. The next most important deciding factor is ESE density, particularly for SF2/ASF; almost half (30) of dystrophin exons can be classified based on this parameter and 3’ss strength only. These findings are in line with those of a previous report, which showed that exons that are skipped because of splice site mutations have a weaker 3’ss and a lower-than-average density of ESEs [17]. tissue-specific promoters, 15 cryptic exons, and gener- ates many alternatively spliced products. It has been reported that there is a gradient in exon and intron defi- nition at the level of pre-mRNA splicing [17]. For exam- ple, efficient use of intrinsically weak cryptic splice sites in exons is facilitated by a higher-than-average density of ESSs and a high density of SF2/ASF ESE motifs [17]. This suggests that each exon has a specific splicing reg- ulatory mechanism in which particular splicing factors could compensate for the lack of other splicing factors. Numbers and densities of ESEs and ESSs Numbers and densities of ESEs and ESSs The number of ESEs in each exon was calculated using the prediction algorithm at http://genes.mit.edu/burge- lab/rescue-ese/ (RESE). The number of ESSs was calcu- lated using two algorithms: at http://genes.mit.edu/ fas-ess/ (FESS) and http://cubweb.biology.columbia. edu/pesx/ (PESS) [17,36-38]. To calculate the densities of ESSs/ESEs, the RESE, FESS, and PESS numbers were divided by the sequence length (in nucleotides) and this figure was multiplied by 100 [17] to give the RESE-D, FESS-D, and PESS-D scores. Interest in the splicing regulation of dystrophin pre- mRNA was first aroused when exon skipping caused by a small intra-exonic deletion was identified in a DMD patient [25]. Subsequently it was revealed that the deleted region was an ESE sequence within exon 19 [1] and that AOs against this ESE successfully induced the skipping of exon 19 [26]. This has led to the establish- ment of exon-skipping therapy [27]. In this study, we demonstrated that exon 19 was categorized into group A, characterized by a high ESE density; hence it is rea- sonable that AOs against the exon 19 ESE would induce exon skipping efficiently. Currently exon skipping is recognized as the most promising way to express dystro- phin in DMD. The main targets for exon skipping ther- apy are exons 44, 45, 51, and 53 [28] and AOs against exon 51 are now in phase II or III clinical trials [2,3]. Because exons 45, 51, and 53 belong to group A, we would expect AOs against the ESEs in these exons to work well. The numbers of binding sites for the four SR proteins (SF2/ASF, SRp40, SC35, and SRp55) were obtained using ESEfinder (v. 3.0) available at http://rulai.cshl.edu/cgi- bin/tools/ESE3/esefinder.cgi?process=home (SF2/ASF-N, SRp40-N, SC35-N, and SRp55-N) [39]. The density of SR protein-binding sites (SF2/ASF-D, SC35-D, SRp40-D, SRp55-D) was obtained by dividing each number by its nucleotide length then multiplying it by 100 [17]. The numbers of binding sites for the four SR proteins (SF2/ASF, SRp40, SC35, and SRp55) were obtained using ESEfinder (v. 3.0) available at http://rulai.cshl.edu/cgi- bin/tools/ESE3/esefinder.cgi?process=home (SF2/ASF-N, SRp40-N, SC35-N, and SRp55-N) [39]. The density of SR protein-binding sites (SF2/ASF-D, SC35-D, SRp40-D, SRp55-D) was obtained by dividing each number by its nucleotide length then multiplying it by 100 [17]. Discussion Group A, consisting of 32 exons, was characterized by a high density of ESEs, particu- larly SF2/ASF (more than 10.53) (Figure 3) (Table 1), indi- cating that ESE density plays a critical role in the splicing of these exons. Disruption of ESEs for exons in this group is more likely to cause exon skipping compared with dis- ruption of ESEs in the other exon groups. Nonsense muta- tions within exons 17, 41, and 42, all belonging to group A, have been shown to induce exon skipping [16]. Accord- ingly, the nonsense mutations in these three exons disrupt an ESE sequence and thus cause exon skipping, while non- sense mutations that induce skipping of exons in other groups do not disrupt an ESE [16]. However, not all ESE- disrupting nonsense mutations identified in these exons induce exon skipping [16]. It may be necessary to consider trans-elements to explain these differences, along with the strength of splicing factor binding to the ESEs and their positions in the pre-mRNA secondary structure. We used our decision tree to determine the parameters that are most useful for the discrimination of authentic and cryptic dystrophin exons. We separated the two cate- gories completely using eight variables: (1) strength of the 3’ss measured by maximum entropy score; (2) density of ESEs particularly SF2/ASF; (3) strength of the 5’ss mea- sured by maximum entropy score; (4) free energy in U1- snRNP binding to the 5’ss; (5) density of ESSs as identified by FESS-D; (6) GC content at the 5’ss; (7) exon size; and (8) strength of the 3’ss as measured by the Shapiro score. In other words, the strength of the 3’ss consensus Intronic pseudo-exons that look like perfect exons, maintaining splicing consensus sequences, are now under intensive investigation [10,20-22]. There is Malueka et al. BMC Genetics 2012, 13:23 http://www.biomedcentral.com/1471-2156/13/23 Page 7 of 10 Malueka et al. BMC Genetics 2012, 13:23 http://www.biomedcentral.com/1471-2156/13/23 Conclusions evidence that inclusion of many of these sequences in mRNAs is actively inhibited because of the presence of intrinsic defects, the presence of silencer elements, or the formation of an inhibitory RNA secondary structure [23]. We categorized the cryptic exons into four groups (Figure 3), indicating a heterogeneous contribution of splicing factors required for their activation. evidence that inclusion of many of these sequences in mRNAs is actively inhibited because of the presence of intrinsic defects, the presence of silencer elements, or the formation of an inhibitory RNA secondary structure [23]. We categorized the cryptic exons into four groups (Figure 3), indicating a heterogeneous contribution of splicing factors required for their activation. The decision tree categorized the 77 authentic dystrophin exons into five groups. Our classification may help to establish the strategy for exon skipping therapy for DMD. Indexes of splicing regulatory factors Group C contained 42 exons and was separated at the fifth node by a score of less than 46.8% GC content at the 5’ss. More than half of the dystrophin exons belonged to this group and are therefore presumed to be subject to similar splicing regulation. From the decision tree we can see that this group is characterized also by an ESS density ≤4.45 (FESS-D), indicating that splicing silencer signals play a critical role in the splicing of these exons. Indeed, we showed previously that nonsense mutation-induced exon skipping of exon 31 was caused by the creation of a splicing silencer-binding site for hnRNP A1 [13]. It has been reported that negative elements play important roles in distinguishing real splicing signals from the vast num- ber of false-positive splicing signals [9]. The sequences of 14 known cryptic exons, and nine dis- ease-causing mutations were obtained from our previous report [10] and the literature [29,30]. Twenty six indexes of splicing regulatory factors of each exon were obtained as described below (Table 2). Splice site strength Splice site strength was determined in three ways: Sha- piro’s splicing probability matrix scores at the 5’ss and the 3’ss were calculated using published formulae [17,31] (SH5’ss and SH3’ss, respectively). Maximum entropy scores at the 5’ss and the 3’ss were obtained using online tools available at http://genes.mit.edu/burgelab/maxent/ Xmaxentscan_scoreseq.html (ME5’ss and ME3’ss, respec- tively) [32]. Information content values at the 5’ss and the 3’ss were obtained using the Delila server at https://splice. uwo.ca/ (Ri5’ss and Ri3’ss, respectively) [33]. Because the dystrophin gene is so complex, it has the potential to produce many alternatively spliced transcripts that are translated into protein variants [24]. However, studies on alternative splicing are limited [6-8]. In one study, Sironi et al. identified 16 alternative transcripts and examined splicing regulatory factors such as the 3’ and 5’ consensus values and exonic splicing enhancer scoring matrices; however, no reasonable explanation of the alter- native splicing was identified [7]. This is consistent with our findings that the cryptic exons fell into five different groups. It is possible that alternative splicing does not rely completely on specific sequence elements and is regulated by trans-acting factors. y Free energy of U1 snRNA binding to the 5’ss U1 snRNA binding to constitutive splice sites has lower free energy than that of its binding to alternatively spliced exon splice sites [34]. Free energy was analyzed using the SROOGLE server at http://sroogle.tau.ac.il/ (FE) [35]. Dystrophin mRNA analysis A 1-year-old Japanese boy (KUCG 952) without any family history of neuromuscular disorders was referred to Kobe University Hospital (Kobe, Japan) because of a high serum creatine kinase level (37,110 IU/l). DMD was tentatively diagnosed, and mutation in the dystrophin gene was ana- lyzed after obtaining informed consent from his parents. Dystrophin mRNA expressed in his peripheral lympho- cytes was analyzed as described previously [10,25]. A frag- ment spanning exons 10 to 14 was amplified by reverse transcription (RT)-nested PCR using two sets of primers. The outer primer set comprised a forward primer, 1A 5’- TTTTTATCGCTGCCTTGATATACA-3’ and a reverse primer, 1B 5’-ACTCTGCAACACAGCTTCTGAG-3’; the inner primer set comprised a forward primer, 1E 5’- TTGCAAGCACAAGGAGAGATT-3’ and a reverse pri- mer, c14r 5’-ACGTTGCCATTTGAGAAGGAT-3’. The Exon size The number of nucleotides in each exon was also taken into account (SIZE). Numbers and densities of ESEs and ESSs Minimum free energy value of pre-mRNA secondary structure and GC content around splice sites Minimum free energy values of the pre-mRNA second- ary structure at the 5’ss and 3’ss were obtained using the free energy minimization program RNAfold http:// rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi using the 70 Minimum free energy value of pre-mRNA secondary structure and GC content around splice sites Minimum free energy values of the pre-mRNA second- ary structure at the 5’ss and 3’ss were obtained using the free energy minimization program RNAfold http:// rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi using the 70 Malueka et al. BMC Genetics 2012, 13:23 http://www.biomedcentral.com/1471-2156/13/23 Page 8 of 10 Table 2 Indexes of splicing regulatory factors No Features Symbol Reference 1 5’ splice site strength (Shapiro score) SH5’ss [31] 2 3’ splice site strength (Shapiro score) SH3’ss [31] 3 5’ spice site strength (maximum entropy) ME5’ss [32] 4 3’ spice site strength (maximum entropy) ME3’ss [32] 5 5’ splice site strength (information content/Ri) Ri5’ss [33] 6 3’ splice site strength (information content/Ri) Ri3’ss [33] 7 U1 SnRNA binding free energy FE [35] 8 ESE density (RESCUE ESE/RESE) RESE-D [36] 9 ESE density (PESE) PESE-D [38] 10 ESS density (FAS-ESS/FESS) FESS-D [37] 11 ESS density (PESS) PESS-D [38] 12 SF2/ASF number SF2/ASF-N [39] 13 SF2/ASF (IgM/BRCA1) number SF2/ASF (IgM, BRCA1)-N [39] 14 SRp40 number SRp40-N [39] 15 SC35 number SC35-N [39] 16 SRp55 number SRp55-N [39] 17 SF2/ASF score density SF2/ASF-D [39] 18 SF2/ASF (IgM/BRCA1) score density SF2/ASF (IgM-BRCA1)-D [39] 19 SC35 score density SC35-D [39] 20 SRp40 score density SRp40-D [39] 21 SRp55 score density SRp55-D [39] 22 5’ splice site pre-mRNA secondary structure free energy RSS5’ss [40] 23 3’ splice site pre-mRNA secondary structure free energy RSS3’ss [40] 24 5’ splice site GC content GC5’ss [40] 25 3’ splice site GC content GC3’ss [40] 26 The number of nucleotides in exon SIZE Table 2 Indexes of splicing regulatory factors We used 92 out of 94 data points (79 exons and 15 cryptic exons) for decision tree construction. We excluded two exons, exons 1 and 79, because they lacked some feature values. nucleotides both up- and down-stream of each splice site (RSS5’ss and RSS3’ss, respectively) [40,41]. GC content around splice sites has been reported to affect splicing [40]. Thus, the percentage of GC content for each 70 nucleotides both up- and down-stream of the 5’ss and the 3’ss was calculated (GC5’ss and GC3’ss, respectively) [40]. Abbreviations DMD D h Abbreviations DMD: Duchenne muscular dystrophy; AO: Antisense oligonucleotide; ESE: Exonic splicing enhancer; ESS: Exonic splicing silencer; ME3’ss: Maximum entropy of 3’ splice site; SF2/ASF-D: SF2/ASF score density; ME5’SS: Maximum entropy of 5’ splice site; FE: Free energy of U1 SnRNA binding; FESS-D: Fluorescence-activated screen-for exonic splicing silencers density; GC5’ss: GC content of 5’ splice site; SH3’ss: Shapiro score of 3’ splice site; SIZE: Size of exon. DMD: Duchenne muscular dystrophy; AO: Antisense oligonucleotide; ESE: Exonic splicing enhancer; ESS: Exonic splicing silencer; ME3’ss: Maximum entropy of 3’ splice site; SF2/ASF-D: SF2/ASF score density; ME5’SS: Maximum entropy of 5’ splice site; FE: Free energy of U1 SnRNA binding; FESS-D: Fluorescence-activated screen-for exonic splicing silencers density; GC5’ss: GC content of 5’ splice site; SH3’ss: Shapiro score of 3’ splice site; SIZE: Size of exon. 11. Valadkhan S: Role of the snRNAs in spliceosomal active site. RNA Biol 2010, 7:345-353. 11. Valadkhan S: Role of the snRNAs in spliceosomal active site. RNA Biol 2010, 7:345-353. 12. Matlin AJ, Clark F, Smith CW: Understanding alternative splicing: towards a cellular code. Nat Rev Mol Cell Biol 2005, 6:386-398. 12. Matlin AJ, Clark F, Smith CW: Understanding alternative splicing: towards a cellular code. Nat Rev Mol Cell Biol 2005, 6:386-398. 13. Nishida A, Kataoka N, Takeshima Y, Yagi M, Awano H, Ota M, Itoh K, Hagiwara M, Matsuo M: Chemical treatment enhances skipping of a mutated exon in the dystrophin gene. Nat Commun 2011, 2:308. 13. Nishida A, Kataoka N, Takeshima Y, Yagi M, Awano H, Ota M, Itoh K, Hagiwara M, Matsuo M: Chemical treatment enhances skipping of a mutated exon in the dystrophin gene. Nat Commun 2011, 2:308. Author details 1 1Department of Pediatrics, Graduate School of Medicine, Kobe University, Chuo, Kobe 6500017, Japan. 2Division of Medical Informatics and Bioinformatics, Kobe University Hospital, Chuo, Kobe 6500017, Japan. 3Department of Clinical Pharmacy, Kobe Pharmaceutical University, Higashinada, Kobe 6588558, Japan. 4Department of Medical Rehabilitation, Faculty of Rehabilitation, Kobegakuin University, 518 Arise, Ikawadani, Nishi, Kobe 651-2180, Japan. 1Department of Pediatrics, Graduate School of Medicine, Kobe University, Chuo, Kobe 6500017, Japan. 2Division of Medical Informatics and Bioinformatics, Kobe University Hospital, Chuo, Kobe 6500017, Japan. 3Department of Clinical Pharmacy, Kobe Pharmaceutical University, 17. Královicová J, Vorechovsky I: Global control of aberrant splice-site activation by auxiliary splicing sequences: evidence for a gradient in exon and intron definition. Nucleic Acids Res 2007, 35:6399-6413. 18. David MP, Concepcion GP, Padlan EA: Using simple artificial intelligence methods for predicting amyloidogenesis in antibodies. BMC Bioinforma 2010, 11:79. Higashinada, Kobe 6588558, Japan. Department of Medical Rehabilitation, Faculty of Rehabilitation, Kobegakuin University, 518 Arise, Ikawadani, Nishi, Kobe 651-2180, Japan. 19. Shiga N, Takeshima Y, Sakamoto H, Inoue K, Yokota Y, Yokoyama M, Matsuo M: Disruption of the splicing enhancer sequence within exon 27 of the dystrophin gene by a nonsense mutation induces partial skipping of the exon and is responsible for Becker muscular dystrophy. J Clin Invest 1997, 100:2204-2210. Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (B), and a Grant-in-Aid for Exploratory Research from the Japan Society for the Promotion of Science, a Health and Labour Sciences Research Grant for Research on Psychiatric and Neurological Diseases and Mental Health, and a research grant for Nervous and Mental Disorders from the Ministry of Health, Labour and Welfare, Japan. 14. Kingsford C, Salzberg SL: What are decision trees? Nat Biotechnol 2008, 26:1011-1013. 14. Kingsford C, Salzberg SL: What are decision trees? Nat Biotechnol 2008, 26:1011-1013. 15. Allen JE, Majoros WH, Pertea M, Salzberg SL: JIGSAW, GeneZilla, and GlimmerHMM: puzzling out the features of human genes in the ENCODE regions. Genome Biol 2006, 7(Suppl 1:S9):1-13. 15. Allen JE, Majoros WH, Pertea M, Salzberg SL: JIGSAW, GeneZilla, and GlimmerHMM: puzzling out the features of human genes in the ENCODE regions. Genome Biol 2006, 7(Suppl 1:S9):1-13. 16. Nishiyama A, Takeshima Y, Zhang Z, Habara Y, Tran TH, Yagi M, Matsuo M: Dystrophin nonsense mutations can generate alternative rescue transcripts in lymphocytes. Ann Hum Genet 2008, 72:717-724. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. 22. Vorechovsky I: Transposable elements in disease-associated cryptic exons. Hum Genet 2010, 127:135-154. Received: 18 January 2012 Accepted: 31 March 2012 Received: 18 January 2012 Accepted: 31 March 2012 23. Dhir A, Buratti E, van Santen MA, Luhrmann R, Baralle FE: The intronic splicing code: multiple factors involved in ATM pseudoexon definition. EMBO J 2010, 29:749-760. Published: 31 March 2012 24. Wei B, Jin JP: Troponin T isoforms and posttranscriptional modifications: Evolution, regulation and function. Arch Biochem Biophys 2011, 505:144-154. Authors’ contributions RGM carried out the molecular genetic studies, participated in the bioinformatics analysis and drafted the manuscript. YT carried out the bioinformatics analysis. MY, HA, TL and EKD participated in the clinical and genetic analysis. YT participated in the design of the study. MM conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. 20. Buratti E, Chivers M, Královicová J, Romano M, Baralle M, Krainer A, Vorechovsky I: Aberrant 5’ splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization. Nucleic Acids Res 2007, 35:4250-4263. 21. Dhir A, Buratti E: Alternative splicing: role of pseudoexons in human disease and potential therapeutic strategies. FEBS J 2010, 277:841-855. Construction of decision trees The C4.5 algorithm was used to construct decision trees. The C4.5 decision tree algorithm is an approach for pattern recognition and data mining [42,43] and was developed by Quinlan [44,45]. The algorithm uses infor- mation gain, which is an entropy-based criterion in information theory for decision tree construction. In this study, the conditions were as follows: (1) at least one object was to be contained in each branch, (2) the pruning confidence level was set to 100%, and (3) the iterative mode was used to avoid a local optimum. Malueka et al. BMC Genetics 2012, 13:23 http://www.biomedcentral.com/1471-2156/13/23 Malueka et al. BMC Genetics 2012, 13:23 http://www.biomedcentral.com/1471-2156/13/23 Page 9 of 10 6. Feener CA, Koenig M, Kunkel LM: Alternative splicing of human dystrophin mRNA generates isoforms at the carboxy terminus. Nature 1989, 338:509-511. amplified fragments were resolved by agarose gel electro- phoresis. Sequencing of the amplified products was per- formed by subcloning sequencing as described previously [46]. 7. Sironi M, Cagliani R, Pozzoli U, Bardoni A, Comi GP, Giorda R, Bresolin N: The dystrophin gene is alternatively spliced throughout its coding sequence. FEBS Lett 2002, 517:163-166. The mutation study was approved by our ethical com- mittee and mutation analysis of the dystrophin gene was done after obtaining the informed consent from the par- ents of the patient. 8. Kubokawa I, Takeshima Y, Ota M, Enomoto M, Okizuka Y, Mori T, Nishimura N, Awano H, Yagi M, Matsuo M: Molecular characterization of the 5’-UTR of retinal dystrophin reveals a cryptic intron that regulates translational activity. Mol Vis 2010, 16:2590-2597. 8. Kubokawa I, Takeshima Y, Ota M, Enomoto M, Okizuka Y, Mori T, Nishimura N, Awano H, Yagi M, Matsuo M: Molecular characterization of the 5’-UTR of retinal dystrophin reveals a cryptic intron that regulates translational activity. Mol Vis 2010, 16:2590-2597. y 9. Sun H, Chasin LA: Multiple splicing defects in an intronic false exon. Mol Cell Biol 2000, 20:6414-6425. 10. Zhang Z, Habara Y, Nishiyama A, Oyazato Y, Yagi M, Takeshima Y, Matsuo M: Identification of seven novel cryptic exons embedded in the dystrophin gene and characterization of 14 cryptic dystrophin exons. J Hum Genet 2007, 52:607-617. 10. Zhang Z, Habara Y, Nishiyama A, Oyazato Y, Yagi M, Takeshima Y, Matsuo M: Identification of seven novel cryptic exons embedded in the dystrophin gene and characterization of 14 cryptic dystrophin exons. J Hum Genet 2007, 52:607-617. References 1. Takeshima Y, Nishio H, Sakamoto H, Nakamura H, Matsuo M: Modulation of in vitro splicing of the upstream intron by modifying an intra-exon sequence which is deleted from the dystrophin gene in dystrophin Kobe. J Clin Invest 1995, 95:515-520. 1. Takeshima Y, Nishio H, Sakamoto H, Nakamura H, Matsuo M: Modulation of in vitro splicing of the upstream intron by modifying an intra-exon sequence which is deleted from the dystrophin gene in dystrophin Kobe. J Clin Invest 1995, 95:515-520. 25. Matsuo M, Masumura T, Nishio H, Nakajima T, Kitoh Y, Takumi T, Koga J, Nakamura H: Exon skipping during splicing of dystrophin mRNA precursor due to an intraexon deletion in the dystrophin gene of Duchenne muscular dystrophy Kobe. J Clin Invest 1991, 87:2127-2131. 2. Aartsma-Rus A: Antisense-mediated modulation of splicing: Therapeutic implications for duchenne muscular dystrophy. RNA Biol 2010, 7(4):453-461. uchenne muscular dystrophy Kobe. J Clin Invest 1991, 87:2127-2131. 26. Pramono ZA, Takeshima Y, Alimsardjono H, Ishii A, Takeda S, Matsuo M: Induction of exon skipping of the dystrophin transcript in lymphoblastoid cells by transfecting an antisense oligodeoxynucleotide complementary to an exon recognition sequence. Biochem Biophys Res Commun 1996, 226:445-449. 3. Lu QL, Yokota T, Takeda S, Garcia L, Muntoni F, Partridge T: The Status of Exon Skipping as a Therapeutic Approach to Duchenne Muscular Dystrophy. Mol Ther 2011, 19:9-15. 4. Ahn AH, Kunkel LM: The structural and functional diversity of dystrophin. Nat Genet 1993, 3:283-291. 27. Takeshima Y, Yagi M, Wada H, Ishibashi K, Nishiyama A, Kakumoto M, Sakaeda T, Saura R, Okumura K, Matsuo M: Intravenous infusion of an antisense oligonucleotide results in exon skipping in muscle dystrophin mRNA of Duchenne muscular dystrophy. Pediatr Res 2006, 59:690-694. 27. Takeshima Y, Yagi M, Wada H, Ishibashi K, Nishiyama A, Kakumoto M, Sakaeda T, Saura R, Okumura K, Matsuo M: Intravenous infusion of an antisense oligonucleotide results in exon skipping in muscle dystrophin mRNA of Duchenne muscular dystrophy. Pediatr Res 2006, 59:690-694. 5. Nishio H, Takeshima Y, Narita N, Yanagawa H, Suzuki Y, Ishikawa Y, Minami R, Nakamura H, Matsuo M: Identification of a novel first exon in the human dystrophin gene and of a new promoter located more than 500 kb upstream of the nearest known promoter. J Clin Invest 1994, 94:1037-1042. 28. Malueka et al. 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Cartegni L, Wang J, Zhu Z, Zhang MQ, Krainer AR: ESEfinder: A w 39. Cartegni L, Wang J, Zhu Z, Zhang MQ, Krainer AR: ESEfinder: A web resource to identify exonic splicing enhancers. Nucleic Acids Res 2003, 31:3568-3571. 40. Zhang J, Kuo CC, Chen L: GC content around splice sites affects splicing through pre-mRNA secondary structures. BMC Genomics 2011, 12:90. g p y 41. Gruber AR, Lorenz R, Bernhart SH, Neubock R, Hofacker IL: The Vienna RNA websuite. Nucleic Acids Res 2008, 36(Web Server):W70-74. 42. Han J, Kamber M, Pei J: Data Mining: Concepts and Techniques Waltham, Massachusetts: Morgan Kaufmann Publishers; 2011. 42. Han J, Kamber M, Pei J: Data Mining: Concepts and Techniques Waltham, Massachusetts: Morgan Kaufmann Publishers; 2011. Massachusetts: Morgan Kaufmann Publishers; 2011. 43. References Takeshima Y, Yagi M, Okizuka Y, Awano H, Zhang Z, Yamauchi Y, Nishio H, Matsuo M: Mutation spectrum of the dystrophin gene in 442 Duchenne/ 28. Takeshima Y, Yagi M, Okizuka Y, Awano H, Zhang Z, Yamauchi Y, Nishio H, Matsuo M: Mutation spectrum of the dystrophin gene in 442 Duchenne/ Page 10 of 10 Malueka et al. BMC Genetics 2012, 13:23 http://www.biomedcentral.com/1471-2156/13/23 Witten IH, Frank E: Practical Machine Learning Tools and Techniques Waltham, Massachusetts: Morgan Kaufmann Publishers; 2005. 43. Witten IH, Frank E: Practical Machine Learning Tools and Techniques Waltham, Massachusetts: Morgan Kaufmann Publishers; 2005. 43. Witten IH, Frank E: Practical Machine Learning Tools and Techn Waltham, Massachusetts: Morgan Kaufmann Publishers; 2005. 44. Quinlan JR: C4.5: Programs for Machine Learning Waltham, Massachusetts: Morgan Kaufmann Publishers; 1993. 44. Quinlan JR: C4.5: Programs for Machine Learning Waltham, Massachusetts: Morgan Kaufmann Publishers; 1993. 45. Quinlan JR: Improved Use of Continuous Attributes in C. J Artif Intell Res 1996, 4:77-90. 45. Quinlan JR: Improved Use of Continuous Attributes in C. J Artif Intell Res 1996, 4:77-90. 46. Surono A, Takeshima Y, Wibawa T, Ikezawa M, Nonaka I, Matsuo M: Circular dystrophin RNAs consisting of exons that were skipped by alternative splicing. Hum Mol Genet 1999, 8:493-500. 46. Surono A, Takeshima Y, Wibawa T, Ikezawa M, Nonaka I, Matsuo M: Circular dystrophin RNAs consisting of exons that were skipped by alternative splicing. Hum Mol Genet 1999, 8:493-500. doi:10.1186/1471-2156-13-23 Cite this article as: Malueka et al.: Categorization of 77 dystrophin exons into 5 groups by a decision tree using indexes of splicing regulatory factors as decision markers. BMC Genetics 2012 13:23. 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Bulletin of Electrical Engineering and Informatics Vol. 12, No. 2, April 2023, pp. 1103~1114 ISSN: 2302-9285, DOI: 10.11591/eei.v12i2.4524 Bulletin of Electrical Engineering and Informatics Vol. 12, No. 2, April 2023, pp. 1103~1114 ISSN: 2302-9285, DOI: 10.11591/eei.v12i2.4524  1103  1103 Keywords: An ameliorated Round Robin algorithm Average turnaround time Average waiting time Cloud computing Round Robin Task scheduling This is an open access article under the CC BY-SA license. This is an open access article under the CC BY-SA license. This is an open access article under the CC BY-SA license. Corresponding Author: Nermeen Ghazy Department of Mathematics, Faculty of Science (Girls), Al-Azhar University Cairo, Egypt Email: Nermeena207@gmail.com Nermeen Ghazy Department of Mathematics, Faculty of Science (Girls), Al-Azhar University Cairo, Egypt Email: Nermeena207@gmail.com Nermeen Ghazy Department of Mathematics, Faculty of Science (Girls), Al-Azhar University Cairo, Egypt Email: Nermeena207@gmail.com Email: Nermeena207@gmail.com An ameliorated Round Robin algorithm in the cloud computing for task scheduling Nermeen Ghazy1, Afaf Abdelkader1, Mervat S. Zaki1, Kamal A. Eldahshan2 1Department of Mathematics, Faculty of Science (Girls), Al-Azhar University, Cairo, Egypt 2Department of Mathematics, Faculty of Science, Al-Azhar University, Cairo, Egypt ABSTRACT Cloud computing is an advanced technology that offers types of assistance on requests. Because of the huge measure of requests got from cloud clients, all requests should be managed efficiently. Therefore, the task scheduling is critical in cloud computing. The provision of computational resources in cloud is controlled by a cloud provider. It is necessary to design high- efficiency scheduling algorithms that are compatible with the corresponding computing paradigms. This paper introduces a new task scheduling method for cloud computing called an ameliorated Round Robin algorithm (ARRA). The proposed algorithm develops an optimal time quantum based on the average of task burst time using fixed and dynamic manners. The experimental results showed that the ARRA significantly outperformed other algorithms including improved RR, enhanced RR, dynamic time quantum approach (ARR) and enhanced RR (RAST ERR) in terms of the average waiting time, average turnaround time and response time. Received Aug 6, 2022 Revised Oct 6, 2022 Accepted Oct 18, 2022 Corresponding Author: Nermeen Ghazy Department of Mathematics, Faculty of Science (Girls), Al-Azhar University Cairo, Egypt Email: Nermeena207@gmail.com 2. RELATED WORK According to Sangwan et al. [23], an improved RR algorithm is proposed. At the arrival of the CPU's arbitrary requests, the method calculates the mean of the burst time. The TQ is the mean of the burst times. The first process in the queue is selected, and the CPU is allocated for the chosen TQ. Then the remaining burst times are moved to the tail of the ready queue [21]. Sometimes the first process is too large and the rest processes are too small, so the response time decreases which causes an increase in waiting time and turnaround time. Research by Mittal et al. [24], an enhanced RR algorithm is offered. It consists of two other existing algorithms, namely (resource allocation scheduling algorithm) RASA and (shortest job first) SJF. SJF coordinates all procedures in the prepared line to minimize burst time, and RASA aids in preventing system starvation and reducing response time for larger tasks. The TQ is updated after each cycle using the same hybrid algorithm. y g According to Pradhan et al. [4], a modified RR algorithm is suggested. This algorithm starts with a time equal to the burst time of the first request, which changes once it is completed. The algorithm calculates the average of the requests waiting in the ready queue, including the newly arrived request. The disadvantage of this algorithm is that if the first processes are too small, it will cause many context switches and therefore will increase the AWT and ATT. According to Fataniya and Patel [25], a dynamic time quantum approach (ARR) to improve the RR is presented here. It is based on the MRRA [4] and SRBRR [27] algorithms. It has been offered to improve cloud computing resource allocation. It is a dynamic RR in which the TQ is always changing. TQ is calculated as the sum of the mean and median divided by two for each round. The disadvantage of this algorithm is that if the first processes are too small, it will cause many context switches and therefore will increase the average waiting time and ATT. Mora et al. [28] offered another RR algorithm called modified median RR algorithm (MMRRA). The TQ is dynamically allocated by determining a modified median of tasks. Mayuree et al. [28] introduced a RR based on remaining time and a median algorithm (RR_RT&M). Based on remaining time and task median, the TQ is modified. 1. INTRODUCTION And on the other hand, if the TQ is set too long, the algorithm trends toward the FCFS algorithm and may result in a slow response time. So, it is important to enhance the RR algorithm to reduce AWT, ATT and response time. p p g p The main contribution of this paper is to improve the RR algorithm by enhancing the TQ in cloud computing for solving task scheduling problem. A novel algorithm for changing TQ in a progressive manner at various states of the ready queue is proposed. A mathematical model has been created to prove that the proposed algorithm outperforms the traditional RR algorithm in terms of several performance metrics such as AWT, ATT and response time (ART). According to the experimental results, the proposed improved version of the RR algorithm outperforms the traditional RR algorithm. This proposed algorithm solves the problem by using a progressive TQ, which is based on the average task burst time using fixed and dynamic manners. Furthermore, the processes are sorted in ascending order of their burst time, and then the proposed algorithm is applied to each process to improve turnaround time, waiting time and response time. In comparison to the other RR techniques discussed in this work, the drawbacks of the discussed algorithms like Improved RR [23], enhanced RR [24], ARR [25] and enhanced RR (RAST ERR) [26] are that they give a large AWT, ATT and ART. The contribution of this work is to: i) minimize AWT, ii) minimize ATT, iii) minimize response time. This paper is divided into six sections. The related work is in section 2, the proposed algorithm in section 3, the method in section 4 and the results in section 5. The conclusion in section 6. 1. INTRODUCTION Cloud computing is a new computing technology that represents a significant step forward in the development and deployment of a growing variety of applications. Cloud computing is a model built on the use of clouds. The cloud is a collection of software and hardware that work together to provide various aspects of computing to the end-user as online services [1]. Cloud computing applications may be accessed anytime and from anywhere [2]. Many popular services and websites are hosted in the cloud [3], [4]. Task scheduling is one of the main issues with cloud computing [5]. It means using an efficient algorithm to map the tasks of the clients to the available and appropriate resources [6], [7]. Scheduling n tasks on m resources is characterized as an NP-hard problem with O (mn) run time complexity [8]. So it is important to use an effective task scheduling algorithm to enhance the performance of the system [8]. In a cloud computing system, three different task scheduling techniques are available: i) traditional algorithms [9], [10], like Round Robin (RR) [11], ii) heuristic algorithms, like (MCT), (MET) [12] and max-min [13], and iii) meta-heuristic algorithms, like ACO [14], PSO [15] and GA [16]. The RR algorithm is a popular scheduling algorithm in the cloud computing [17], [18]. The RR algorithm [19]: it is a preemptive algorithm [10]. It works well in time sharing [20]. For interactive users, these environments must ensure reasonable response times. In the RR scheduling algorithm, every process in the ready queue receives a time slice (time quantum (TQ)) [21]. The current process is placed at the end of this ready queue when the TQ expires. RR decreases the average turnaround time (ATT) Journal homepage: http://beei.org 1104  ISSN: 2302-9285 and average waiting time (AWT) [6]. The most significant issue with the RR method is the TQ length [19], [22]. Setting a TQ too short causes too many context switches, which lowers CPU efficiency. And on the other hand, if the TQ is set too long, the algorithm trends toward the FCFS algorithm and may result in a slow response time. So, it is important to enhance the RR algorithm to reduce AWT, ATT and response time. and average waiting time (AWT) [6]. The most significant issue with the RR method is the TQ length [19], [22]. Setting a TQ too short causes too many context switches, which lowers CPU efficiency. Bulletin of Electr Eng & Inf, Vol. 12, No. 2, April 2023: 1103-1114 2. RELATED WORK If the number of tasks is less than or equal to 3, the TQ is the maximum remaining time of task. Otherwise, TQ is the median of task time [29]. According to Tani and Amrani [30], a variant on RR (VORR), which is one of the improvements of the RR algorithm. It effectively exploits the CPU by setting up an effective TQ based on the median of burst times. Hicham et al. [30] introduced a smarter SRR algorithm which uses the concept of RR but the TQ is changed dynamically depending on the number of tasks in a queue [31]. The SJF algorithm is used to distribute the time if there are fewer than or equal to three tasks. However, it utilizes the average of the burst time of tasks if the number of tasks is larger than 3 and even. Otherwise, it uses the median of task burst time instead. According to Stephen et al. [26], an enhanced RR algorithm (RAST ERR technique) that has been suggested uses mean for dynamic time quantum. Burst time (the amount of time it takes to complete a task) is used to compute the mean for the provided tasks and is then set as the time quantum. For the first iteration, initial burst time and mean are used. The finished tasks will be deleted from the queue after the initial iteration; otherwise, the next iteration will begin. The remaining burst time of the second iteration is used to calculate the mean value and the remaining burst time for each task will determine how long it takes to complete. Then, in the following iteration, the mean is once more determined using the remaining burst time Bulletin of Electr Eng & Inf, Vol. 12, No. 2, April 2023: 1103-1114  1105 1105  ISSN: 2302-9285 Bulletin of Electr Eng & Inf for task and set as the time quantum. Until there are no tasks waiting in the queue to be completed, the procedure of obtaining the mean and establishing the time quantum is repeated. The waiting time and task turnaround time of the suggested method are distinguishing features. for task and set as the time quantum. Until there are no tasks waiting in the queue to be completed, the procedure of obtaining the mean and establishing the time quantum is repeated. The waiting time and task turnaround time of the suggested method are distinguishing features. 2. RELATED WORK gg g g All of the previous RR CPU scheduling enhancements have some drawbacks. The system's incoming processes might have different burst times, which mean that their CPU execution times might also change. The turnaround time and waiting time can be reduced if all of the processes are sent to the CPU for execution in ascending order. The RR algorithm uses a fixed time quantum to operate (TQ). There are two results for the RR algorithm: either time quantum is high or low. The RR method will operate on a first-come, first-served (FCFS) basis if the time quantum is large. If the time quantum is small, the algorithm will fail and result in a significant number of context switches. So, this paper proposes an optimal time quantum that solves this problem, which uses fixed and dynamic manners and enhances the performance of the system by: maximizing CPU utilization, minimizing waiting time, turnaround time and response time. Many algorithms have been developed for enhancing RR algorithm such as improved RR [23], enhanced RR [24], ARR [25] and enhanced RR (RAST ERR) [26]. However, these algorithms still have higher AWT, ATT and response time. The main focus of this work is the suggestion of a novel algorithm called an ameliorated RR algorithm (ARRA) in cloud computing that minimizes the drawbacks of the RR algorithm by raising the performance metrics by decreasing the AWT, ATT and response time for some algorithms. This is done by choosing an optimal TQ that achieves low waiting time, turnaround time and response time. Thus, it ends starvation. If tasks arrive at the same time, it uses a fixed TQ and a dynamic TQ if tasks arrive at different times. 3. THE PROPOSED ALGORITHM This study aims to improve system efficiency by introducing an ameliorated RR scheduling algorithm by minimizing metrics like waiting time, turnaround time and response time in the cloud. To optimize the task scheduling process, the intended algorithm has put the main focus on computing the time quantum effectively. The ARRA uses both fixed and dynamic manners according to the task arrivals. When the tasks arrive at the same time, a fixed TQ is applied. On the other hand, when the tasks arrive at different times, a dynamic TQ is applied. All the processes that are present in the ready queue are arranged in an increasing order. When two or more processes occur in the ready queue with the same burst time, they are rearranged in the ready queue according to when they occurred. The average is then computed for all of the tasks in the ready queue. After that, the TQ is calculated according to (1) as determined by [32]: 𝑇𝑄𝑖= 3 4 × ∑ 𝐵𝑇𝑖 𝑛 𝑖=1 𝑛 (1) Where TQi is the time quantum for tasks, BTi is the burst time for task i and n is the number of tasks. At the beginning, the algorithm allocates system resources to the first task in the ready queue. When the task process or TQ length is finished, the associated mechanism verifies the statutes of the task. If the remaining burst time for the currently executed task is less than half of the current set time quantum, the algorithm allocates the CPU to the same task; otherwise, the task is put to the back of the queue. With minimal average waiting and turnaround times, the newly implemented algorithm effectively enhances task scheduling. The pseudo code for the ARRA is given below. The flowchart of the ARRA algorithm is shown in Figure 1. 1 Tasks arrive in the cloud p g g 1- Tasks arrive in the cloud. 2- Load distribution: load the processes in the datacenter using circular load distribution algorithm. 2- Load distribution: load the processes in the datacenter using circular load distribution algorithm. For all datacenters, 3- If (ready queue! =0), then arrange all the tasks in an increasing order by their burst time in the ready queue. 4- Calculate the average of all the burst times. 4- Calculate the average of all the burst times. 3. THE PROPOSED ALGORITHM nsider the (TQ) to be equal to ¾ average of burst time 6- Assign this TQ to all tasks inserted in the ready queue. 6- Assign this TQ to all tasks inserted in the ready queue. 7- If the current task has a burst time less than half of the time quantum, then allocate it again. Else 9- Repeat steps 3-7 for all the tasks until the TQ expires for each one. 9- Repeat steps 3-7 for all the tasks until the TQ expires for each one. 10- If a new (task) arrives, then update the counter and go to step 4. 11- Calculate the (AWT), (ATT) and average response time. 12- Stop and exit. An ameliorated Round Robin algorithm in the cloud computing for task scheduling (Nermeen Ghaz  1106 ISSN: 2302-9285 Figure 1. Flowchart of the ARRA algorithm Figure 1. Flowchart of the ARRA algorithm 4. METHOD In cloud computing, users send multiple requests at the same time. The RR task scheduling algorithm works on both different and same arriving times. To evaluate the effectiveness of the proposed algorithm, various tasks are applied to RR [19], improved RR [23], enhanced RR [24], ARR [25], enhanced RR (RAST ERR) [26] and the ARRA algorithms. A comparative study among these algorithms is presented here. The comparison criteria is restricted to AWT, ATT and response time. The AWT is calculated as (2): AWT=Ʃ (TAT of Pi – BT of Pi) / N (2) The ATT is calculated as (3): Bulletin of Electr Eng & Inf, Vol. 12, No. 2, April 2023: 1103-1114 4.1. Case 1 (zero arrival time) ( ) In case 1, seven tasks which are T1, T2, T3, T4, T5, T6 and T7 are integrated with CPU burst time and zero arrival time. These values of burst time are shown in Table 1. Figures 2-7 shows of the RR, improved RR, enhanced RR, ARR, enhanced RR (RAST ERR) and ARRA. p , , , ( ) Table 1. The burst time for tasks Tasks Burst time(ms) T1 105 T2 60 T3 120 T4 48 T5 75 T6 160 T7 145 T1 T2 T3 T4 T5 T6 T7 T1 T3 T6 T7 0 103 163 266 314 389 492 595 597 614 671 713 Figure 2. Gantt chart for RR algorithm [19] T1 T2 T3 T4 T5 T6 T7 T1 T3 T6 T7 0 101 161 262 310 385 486 587 591 610 669 713 Figure 3. Gantt chart for improved RR algorithm [23] T4 T6 T2 T7 T5 T3 T1 T2 T6 T5 T7 T1 T3 T5 T6 T1 T7 T3 0 48 96 144 192 240 288 336 348 360 372 384 396 408 423 438 453 468 483 TQ=48 TQ=12 TQ=15 T1 T6 T3 T7 T3 T6 T7 T7 T6 T6 513 543 543 573 603 618 633 648 673 698 713 TQ=30 TQ=15 TQ=25 TQ=15 Figure 4. Gantt chart for the enhanced RR algorithm [24] T4 T2 T5 T1 T3 T7 T6 T1 T3 T7 T6 T7 T6 T6 0 48 108 183 286 389 492 595 597 614 643 672 685 705 713 TQ=103 TQ=29 TQ=20 TQ=8 Figure 5. Gantt chart for ARR algorithm [25] T4 T2 T5 T1 T3 T7 T6 T1 T3 T7 T6 T7 T6 T6 0 48 108 183 284 385 486 587 591 610 641 672 685 705 713 TQ=101 TQ=31 TQ=20 TQ=8 Figure 6. Gantt chart for enhanced RR (RAST ERR) algorithm [26] T4 T2 T5 T1 T1 T3 T7 T6 T3 T7 T6 T6 0 48 108 183 258 288 363 438 513 558 628 703 713 Figure 7. Gantt chart for ARRA algorithm The turn-around time, waiting time and response time of the ARRA algorithm for the above example are calculated as shown in Table 2. A comparative study among the RR, Improved RR, Enhanced RR, ARR, Enhanced RR (RAST ERR) and ARRA algorithms with respect to TQ, AWT, ATT and average response time (ART) as shown in Table 3. ATT=Ʃ (Ti – AT of Pi) / N ATT=Ʃ (Ti – AT of Pi) / N (3) Where ATT of Pi is the turnaround time of task Pi, BT of Pi is the burst time of task Pi, Ti is the exit time for task, AT of Pi is the arrival time of task and N is the number of all tasks. To test the effectiveness of the proposed algorithm, two scenarios are used. Bulletin of Electr Eng & Inf, Vol. 12, No. 2, April 2023: 1103-1114  1107 Bulletin of Electr Eng & Inf ISSN: 2302-9285 4.1. Case 1 (zero arrival time) Comparative study of RR, improved RR, enhanced RR, ARR, enhanced RR (RAST ERR) and ARRA algorithms (case 1) Algorithm TQ AWT ATT ART RR 103 392.57 494.42 246.7 Improved RR 101 389.42 491.28 243.5 Enhanced RR 48,12,15,30, 15,25,15 374.71 476.57 144 ARR 103,29,20,8 319.28 421.14 215.1 Enhanced RR (RAST ERR) 101,31,20,8 317.85 419.71 213.4 ARRA 75 259 360.85 204 Figure 8. Comparison graph for AWT, ATT and ART for (case 1) 387,85 494,42 246,7 389,42 491,28 243,5 374,71 476,57 144 317,857143 421.14 215,1 319,28 360.85 213,4 259 360,85 204 0 100 200 300 400 500 600 AWT ATT average response time Time (ms) Tasks vs Time RR Improved RR Enhanced RR Enhanced RR (RAST ERR) ARR ARRA Table 3. Comparative study of RR, improved RR, enhanced RR, ARR, enhanced RR (RAST ERR) and ARRA algorithms (case 1) Figure 8. Comparison graph for AWT, ATT and ART for (case 1) 387,85 494,42 246,7 389,42 491,28 243,5 374,71 476,57 144 317,857143 421.14 215,1 319,28 360.85 213,4 259 360,85 204 0 100 200 300 400 500 600 AWT ATT average response time Time (ms) Tasks vs Time RR Improved RR Enhanced RR Enhanced RR (RAST ERR) ARR ARRA Figure 8. Comparison graph for AWT, ATT and ART for (case 1) 4.1. Case 1 (zero arrival time) Figure 8 compares the performance of the preceding algorithms using AWT, ATT and ART. Table 1. The burst time for tasks Tasks Burst time(ms) T1 105 T2 60 T3 120 T4 48 T5 75 T6 160 T7 145 T1 T2 T3 T4 T5 T6 T7 T1 T3 T6 T7 0 103 163 266 314 389 492 595 597 614 671 713 Figure 2. Gantt chart for RR algorithm [19] T1 T2 T3 T4 T5 T6 T7 T1 T3 T6 T7 0 101 161 262 310 385 486 587 591 610 669 713 Figure 3. Gantt chart for improved RR algorithm [23] Figure 4. Gantt chart for the enhanced RR algorithm [24] T4 T2 T5 T1 T3 T7 T6 T1 T3 T7 T6 T7 T6 T6 0 48 108 183 286 389 492 595 597 614 643 672 685 705 713 TQ=103 TQ=29 TQ=20 TQ=8 Figure 5. Gantt chart for ARR algorithm [25] T4 T2 T5 T1 T3 T7 T6 T1 T3 T7 T6 T7 T6 T6 0 48 108 183 284 385 486 587 591 610 641 672 685 705 713 TQ=101 TQ=31 TQ=20 TQ=8 Figure 6. Gantt chart for enhanced RR (RAST ERR) algorithm [26] T4 T2 T5 T1 T1 T3 T7 T6 T3 T7 T6 T6 0 48 108 183 258 288 363 438 513 558 628 703 713 Figure 7. Gantt chart for ARRA algorithm Figure 7. Gantt chart for ARRA algorithm The turn-around time, waiting time and response time of the ARRA algorithm for the above example are calculated as shown in Table 2. A comparative study among the RR, Improved RR, Enhanced RR, ARR, Enhanced RR (RAST ERR) and ARRA algorithms with respect to TQ, AWT, ATT and average response time (ART) as shown in Table 3. Figure 8 compares the performance of the preceding algorithms using AWT, ATT and ART. An ameliorated Round Robin algorithm in the cloud computing for task scheduling (Nermeen Ghazy)  1108 ISSN: 2302-9285 ISSN: 2302-9285 Table 2. T.A.T and W.T for ARRA algorithm Tasks WT TAT Response time T1 183 288 183 T2 48 108 48 T3 438 558 288 T4 0 48 0 T5 108 183 108 T6 553 713 438 T7 483 628 363 Table 2. T.A.T and W.T for ARRA algorithm T k WT TAT R i Table 3. 4.2. Case 2(non-zero arrival time) In case 2, the tasks have non-zero arrival times in the ready queue, so a dynamic TQ is applied for the proposed algorithm in this case. TQ is updated each round. Five tasks, which are T1, T2, T3, T4 and T5 are integrated with CPU burst time with non-zero arrival times as shown in Table 4. Figures 9-14 shows of the RR, improved RR, enhanced RR, ARR, enhanced RR (RAST ERR) and ARRA algorithms. Table 4. The arrival time and burst time for tasks Tasks Arrival time(ms) Burst time(ms) T1 0 40 T2 5 25 T3 10 60 T4 15 100 T5 20 75 T1 T2 T3 T4 T5 T3 T4 T5 T5 0 40 65 105 145 185 205 240 280 300 Figure 9. Gantt chart for RR algorithm [19] T1 T2 T3 T4 T5 T4 T5 0 40 65 125 190 255 290 300 TQ=40 TQ=65 Figure 10. Gantt chart for improved RR algorithm [23] Figure 9. Gantt chart for RR algorithm [19] Figure 10. Gantt chart for improved RR algorithm [23] Bulletin of Electr Eng & Inf, Vol. 12, No. 2, April 2023: 1103-1114  1109 ISSN: 2302-9285 Bulletin of Electr Eng & Inf T1 T2 T3 T5 T4 T3 T5 T4 T4 T5 T5 0 40 65 90 115 140 175 210 245 260 275 300 TQ=40 TQ=25 TQ=35 TQ=15 Figure 11. Gantt chart for enhanced RR algorithm [24] T1 T2 T3 T5 T4 T5 T4 T4 0 40 65 125 191 257 266 287 300 TQ=40 TQ=66 TQ=21 TQ= Figure 12. Gantt chart for ARR algorithm [25] T1 T2 T3 T5 T4 T5 T4 T4 0 40 65 125 190 255 265 287 300 TQ=40 TQ=65 TQ=22 TQ= Figure 13. Gantt chart for enhanced RR (RAST ERR) algorithm [26] T1 T2 T3 T3 T5 T4 T5 T4 T4 0 40 65 113 125 173 221 248 296 300 TQ1=40 TQ2=48 Figure 14. Gantt chart for ARRA algorithm T1 T2 T3 T5 T4 T3 T5 T4 T4 T5 T5 0 40 65 90 115 140 175 210 245 260 275 300 TQ=40 TQ=25 TQ=35 TQ=15 TQ=40 TQ=25 TQ=35 TQ=15 Figure 11. Gantt chart for enhanced RR algorithm [24] T1 T2 T3 T5 T4 T5 T4 T4 0 40 65 125 191 257 266 287 300 TQ=40 TQ=66 TQ=21 TQ= Figure 12. 4.4. Simulation To evaluate the effectiveness of the proposed model, the benchmark was simulated using the same parameters while using various algorithms, considering the burst time and task arrival time. A simulation is implemented in C++ and made for the proposed algorithm(ARRA), improved RR [23], enhanced RR [24], ARR [25], enhanced RR (RAST ERR) [26] and all of these algorithms are compared with the RR algorithm in order to evaluate their performance. The model is a single resource in the form of virtual machines with the same random data set. The comparison of these algorithms is based on AWT, ATT and ART. Because the number of tasks in the ready queue determines average waiting and turnaround time, an increase in time results in a rise in cost. 4.3. Percentage of improvements Percentage of improvement for ART comparing with ARRA algorithm RR (%) Improved RR (%) Enhanced RR (%) ARR (%) Enhanced RR (RAST ERR) (%) Case 1 17.3 16.2 0 5.1 4.4 Case 2 0 2.28 0 4.27 4.04 4.2. Case 2(non-zero arrival time) Gantt chart for ARR algorithm [25] T1 T2 T3 T5 T4 T5 T4 T4 0 40 65 125 190 255 265 287 300 TQ=40 TQ=65 TQ=22 TQ= Figure 13. Gantt chart for enhanced RR (RAST ERR) algorithm [26] Figure 11. Gantt chart for enhanced RR algorithm [24] Figure 13. Gantt chart for enhanced RR (RAST ERR) algorithm [26] Figure 13. Gantt chart for enhanced RR (RAST ERR) algorithm [26] Figure 14. Gantt chart for ARRA algorithm Turnaround time, waiting time and response time for above example of the ARRA algorithm is calculated as shown in Table 5. Table 6 presents a comparative study among the RR, improved RR, enhanced RR, ARR, enhanced RR (RAST ERR) and ARRA algorithms with respect to TQ, AWT, ATT and ART. Figure 15 shows the comparison of AWT, ATT and ART for the previous algorithms in (case 2). Table 5. T.A.T, W.T and response time for ARRA algorithm Table 5. T.A.T, W.T and response time for ARRA algorithm Tasks WT TAT Response time T1 0 40 0 T2 35 60 40 T3 135 195 65 T4 125 225 105 T5 205 280 145 An ameliorated Round Robin algorithm in the cloud computing for task scheduling (Nermeen Ghazy) Table 6. Comparative study of RR, enhanced RR, ARR, enhanced RR (RAST ERR) and ARRA algorithms (case 2) Algorithm TQ AWT ATT ART RR 40 100 160 71 Improved RR 40,65 94 154 84 Enhanced RR 40,25,35,15 98 158 62 ARR 40,66,21,13 89.2 149.2 84.2 Enhanced RR (RAST ERR) 40,65,22,13 89 149 84 ARRA 40, 48 85.6 145.6 80.6 Figure 15. Comparison graph for AWT, ATT and ART (case 2) 100 160 71 94 154 84 98 158 62 89 149 84,2 89,2 149,2 84 85,6 145,6 80,6 0 100 200 AWT ATT average response time Time (ms) Tasks vs Time RR Improved RR Enhanced RR Enhanced RR (RAST ERR) ARR ARRA Table 6. Comparative study of RR, enhanced RR, ARR, enhanced RR (RAST ERR) and ARRA algorithms (case 2) Algorithm TQ AWT ATT ART RR 40 100 160 71 Improved RR 40,65 94 154 84 Enhanced RR 40,25,35,15 98 158 62 ARR 40,66,21,13 89.2 149.2 84.2 Enhanced RR (RAST ERR) 40,65,22,13 89 149 84 ARRA 40, 48 85.6 145.6 80.6 An ameliorated Round Robin algorithm in the cloud computing for task scheduling (Nermeen Ghazy)  1110  1110 ISSN: 2302-9285 1110 4.3. 4.2. Case 2(non-zero arrival time) Percentage of improvements Th f h f 4.3. Percentage of improvements 4.3. Percentage of improvements The percentage of each performance age o p ove e s ge of each performance metric improvement [29] when compared to ARRA is in (4): g p The percentage of each performance metric improvement [29] when compared to ARRA is in (4): The percentage of each performance metric improvement [29] when compared to ARRA is in (4): Improvement percentage= (𝑋𝑜𝑡ℎ𝑒𝑟 )−(𝑋𝑝𝑟𝑜𝑝𝑜𝑠𝑒𝑑) 𝑋𝑜𝑡ℎ𝑒𝑟 × 100 (4) (4) Where x is a performance metric that is AWT or ATT or ART and other refers to other algorithms that are used to compare with ARRA. The scheduling algorithm will be improved if the AWT, ATT and ART are lower. Table 7 and Table 8 show the improvement percentage for each metric for all algorithms compared with the ARRA algorithm in case 1 and case 2. The percentage of improvement for AWT ranges between 3.8-34.02%. Similarly, the improvement percentage of ATT varies between 7.84-27.01% and the ART varies between 0-17.3% as shown in Table 9. Therefore, compared to other algorithms, the ARRA performs the best in case 1 and case 2. Table 7. Percentage of improvement for AWT comparing with ARRA algorithm RR (%) Improved RR (%) Enhanced RR (%) ARR (%) Enhanced RR (RAST ERR) (%) Case 1 34.02 33.49 30.87 18.87 22.72 Case 2 14.4 4.04 12.6 4.03 3.82 Table 8. Percentage of improvement for ATT comparing with ARRA algorithm RR (%) Improved RR (%) Enhanced RR (%) ARR (%) Enhanced RR (RAST ERR) (%) Case 1 27.01 26.54 24.28 14.31 14.02 Case 2 9 2.28 7.84 2.41 2.82 Table 9. Percentage of improvement for ART comparing with ARRA algorithm RR (%) Improved RR (%) Enhanced RR (%) ARR (%) Enhanced RR (RAST ERR) (%) Case 1 17.3 16.2 0 5.1 4.4 Case 2 0 2.28 0 4.27 4.04 Table 7. Percentage of improvement for AWT comparing with ARRA algorithm RR (%) Improved RR (%) Enhanced RR (%) ARR (%) Enhanced RR (RAST ERR) (%) Case 1 34.02 33.49 30.87 18.87 22.72 Case 2 14.4 4.04 12.6 4.03 3.82 Table 8. Percentage of improvement for ATT comparing with ARRA algorithm RR (%) Improved RR (%) Enhanced RR (%) ARR (%) Enhanced RR (RAST ERR) (%) Case 1 27.01 26.54 24.28 14.31 14.02 Case 2 9 2.28 7.84 2.41 2.82 Table 9. 5. RESULTS AND DISCUSSION When compared to RR, the enhanced RR (RAST ERR), ARR, and enhanced RR produce significant results, while the improved RR produces reasonable improvement results. The proposed algorithms act similarly, however while increasing in number of tasks, the performance of ARRA showed an upward trend in ATT compared to other algorithms. In comparison to suggested algorithms, the ATT for RR is consistently increasing, as seen in the line chart. Figure 16. Comparative graph of AWT 0 100000 200000 300000 2000 4000 6000 8000 Time(ms) Number of tasks Improved RR Enhanced RR Enhanced RR (RAST ERR) ARR ARRA RR Figure 16. Comparative graph of AWT 0 100000 200000 300000 2000 4000 6000 8000 Time(ms) Number of tasks Improved RR Enhanced RR Enhanced RR (RAST ERR) ARR ARRA RR Figure 16. Comparative graph of AWT Figure 17. Comparative graph for ATT 0 100000 200000 300000 2000 4000 6000 8000 Time(ms) Number of tasks Improved RR Enhanced RR Enhanced RR (RAST ERR) ARR ARRA RR 0 100000 200000 300000 2000 4000 6000 8000 Time(ms) Number of tasks RR Improved RR Enhanced RR Enhanced RR (RAST ERR) ARR ARRA Figure 16. Comparative graph of AWT Figure 16. Comparative graph of AWT Figure 17. Comparative graph for ATT 0 100000 200000 300000 2000 4000 6000 8000 Time(ms) Number of tasks RR Improved RR Enhanced RR Enhanced RR (RAST ERR) ARR ARRA Improved RR Enhanced RR (RAST ERR) ARRA Figure 17. Comparative graph for ATT The comparison of algorithms in terms of ART is shown in Figure 18. For tasks (2000 to 8000), the stacked line chart is plotted. The ART of the tasks is provided in milliseconds and plotted by the y-axis, while the number of tasks in the ready queue is plotted by the x-axis. The proposed algorithm (ARRA) gives better results than Enhanced RR (RAST ERR), ARR, and Improved RR. A substantial improvement is given by these algorithms when compared to the RR algorithm. As the number of tasks increases in the ready queue, the performance of the algorithms is enhanced. When compared to RR, the enhanced RR (RAST ERR), ARR and improved RR produce significant results, while the enhanced RR gives better results than the proposed algorithm. Whereas the proposed algorithm shows more significant improvement results than other algorithms. 5. RESULTS AND DISCUSSION The experimental data used varied data sets from (2000-8000) tasks. The values of burst time tasks are generated randomly in the range of 1–100, and all tasks arrive at the same time. Several experiments were carried out to validate the proposed model, which is repeated numerous times while the number of tasks is increasing. The comparison of algorithms in terms of AWT is shown in Figure 16. For tasks (2000 to 8000), the stacked line chart is plotted. The AWT of the tasks is provided in milliseconds and plotted by the y-axis, while the number of tasks in the ready queue is plotted by the x-axis. The proposed algorithm (ARRA) gives better results, followed by enhanced RR (RAST ERR) [26], ARR [25], enhanced RR [24] and improved RR [23]. A substantial improvement is given by these algorithms when compared to the RR algorithm. As the number of tasks increases in the ready queue, the performance of the algorithms is enhanced. When compared to RR, the enhanced RR (RAST ERR) [26], ARR [25], and enhanced RR [24] produce significant results, while the improved RR [23] produces reasonable improvement results. Whereas the proposed algorithm shows more significant improvement results than other algorithms. While inceasing in number of tasks, the performance of ARRA showed an upward trend in AWT compared to other algorithms. In comparison to suggested algorithms, the AWT for RR is consistently increasing, as seen in the line chart. p gg g y g The behaviour of algorithms in terms of ATT exhibits a similar pattern to that shown in Figure 17. For tasks (2000 to 8000), the stacked line chart is plotted. The ATT of the tasks is provided in milliseconds and plotted by the y-axis, while the number of tasks in the ready queue is plotted by the x-axis. The proposed Bulletin of Electr Eng & Inf, Vol. 12, No. 2, April 2023: 1103-1114  1111  1111 ISSN: 2302-9285 Bulletin of Electr Eng & Inf algorithm (ARRA) gives better results, followed by enhanced RR (RAST ERR) [26], ARR [25], enhanced RR [24] and improved RR [23]. A substantial improvement is given by these algorithms when compared to the RR algorithm. As the number of tasks in the ready queue increases, the performance of the algorithms is enhanced. 5. RESULTS AND DISCUSSION While inceasing in number of tasks, the performance of ARRA showed an upward trend in ART compared to other algorithms.Table 10 shows the high percentage of improvement for the ARRA compared with the benchmarked algorithms, where the proposed algorithm enhanced the AWT by 13.61-38.20% and the ATT by 13.61-38.19%. The ART is enhanced by 0-28%. An ameliorated Round Robin algorithm in the cloud computing for task scheduling (Nermeen Ghazy)  1112 ISSN: 2302-9285  112 Figure 18. Comparative graph for ART 0 50000 100000 150000 200000 2000 4000 6000 8000 RR Improved RR Enhanced RR ARR Enhanced RR (RAST RR) ARRA Improved RR ARR ARRA Figure 18. Comparative graph for ART Table 10. Percentage of improvement for AWT, ATT and ART comparing with ARRA Number of tasks RR (%) Improved RR (%) Enhanced RR (%) ARR (%) Enhanced RR (RAST ERR) (%) AW AT AR AW AT AR AW AT AR AW AT AR AW AT AR N=200 37.8 37.8 18.8 33.0 33.0 27.3 37.6 37.6 0 13.8 13.8 8.3 13.88 13.8 8.3 N=400 38.2 38.1 19.2 34.1 34.0 28 37.8 37.8 0 13.6 13.6 8.1 13.62 13.6 8.1 N=600 38.1 38.1 18.3 33.8 33.8 27.3 37.8 37.8 0 13.7 13.6 8.1 13.70 13.6 8.1 N=800 35.9 35.8 20 33.9 33.9 27.7 36.3 36.2 0 13.6 13.6 8.4 13.61 13.6 8.4 Table 10. Percentage of improvement for AWT, ATT and ART comparing with ARRA REFERENCES Usman, “Performance comparison of heuristic algorithms for task scheduling in IaaS cloud computing environment,” PLoS ONE, vol. 12, no. 5, pp. 1–26, 2017, doi: 10.1371/journal.pone.0176321. [10] M. Kumar Mishra and F. Rashid, “An Improved Round Robin CPU Scheduling Algorithm with Varying Time Quantum,” International Journal of Computer Science, Engineering and Applications, vol. 4, no. 4, pp. 1–8, 2014, doi: 10.5121/ijcsea.2014.4401. j 1] K. Eldahshan, A. Abdelkader, and N. Ghazy, “Round Robin based Scheduling Algorithms, A Comparative Study,” Automa Control and System Engineering Journal, vol. 17, no. 2, pp. 29–42, 2017. y g g pp [12] I. A. Thiyeb and Dr. S. A. Alhomdy, “HAMM A Hybrid Algorithm of Min-Min and Max-Min Task Scheduling Algorithms in Cloud Computing,” International Journal of Recent Technology and Engineering (IJRTE), vol. 9, no. 4, pp. 209–218, 2020, doi: 10.35940/ijrte.d4874.119420. j [13] S. Devipriya and C. Ramesh, “Improved max-min heuristic model for task scheduling in cloud,” Proceedings of the 2013 International Conference on Green Computing, Communication and Conservation of Energy, ICGCE 2013, pp. 883–888, 2013. [14] M. A. Tawfeek, A. El-Sisi, A. E. Keshk, and F. A. Torkey, “Cloud task scheduling based on ant colony optimization,” Proceedings - 2013 8th International Conference on Computer Engineering and Systems, ICCES 2013, vol. 12, no. 2, pp. 64–69, 2013, doi: 10.1109/ICCES.2013.6707172. [15] M. Masdari, F. Salehi, M. Jalali, and M. Bidaki, “A Survey of PSO-Based Scheduling Algorithms in Cloud Computing,” Journal of Network and Systems Management, vol. 25, no. 1, pp. 122–158, 2017, doi: 10.1007/s10922-016-9385-9. 6] F. A. Emara, A. A. A. Gad-Elrab, A. Sobhi, and K. R. Raslan, “Genetic-Based Multi-objective Task Scheduling Algorithm Cloud Computing Environment,” International Journal of Intelligent Engineering and Systems, vol. 14, no. 5, pp. 571–582, 202 p g , f g g g y , , , pp , [17] T. Balharith and F. Alhaidari, “Round Robin Scheduling Algorithm in CPU and Cloud Computing: A review,” 2nd International Conference on Computer Applications and Information Security, ICCAIS 2019, pp. 1–7, 2019, doi: 10.1109/CAIS.2019.8769534. [18] S T l “T k h d li i i i f h l d i ” I i l j l f d d i i [17] T. Balharith and F. Alhaidari, “Round Robin Scheduling Algorithm in CPU and Cloud Computing: A review,” 2nd International Conference on Computer Applications and Information Security, ICCAIS 2019, pp. 1–7, 2019, doi: 10.1109/CAIS.2019.8769534. [18] S. REFERENCES A. Sharma and A. Sahu, A Modified Round Robin Method to Enhance the Performance in Cloud Computing. Springer Singapo 2021, doi: 10.1007/978-981-15-5243-4_49. [2] A. Razaque, N. R. Vennapusa, N. Soni, G. S. Janapati, and K. R. Vangala, “Task scheduling in Cloud computing,” 2016 IEEE Long Island Systems, Applications and Technology Conference, LISAT 2016, pp. 1–5, 2016, doi: 10.1109/LISAT.2016.7494149. g y pp gy f , pp , , S. Banerjee, M. Adhikari, and U. Biswas, “Design and analysis of an efficient QoS improvement policy in cloud computin Service Oriented Computing and Applications, vol. 11, no. 1, pp. 65–73, 2017, doi: 10.1007/s11761-016-0196-3. [3] S. Banerjee, M. Adhikari, and U. Biswas, Design and analysis of an efficient QoS improvement policy in cloud computing, Service Oriented Computing and Applications, vol. 11, no. 1, pp. 65–73, 2017, doi: 10.1007/s11761-016-0196-3. j g y Q p p y p Service Oriented Computing and Applications, vol. 11, no. 1, pp. 65–73, 2017, doi: 10.1007/s11761-016-0196-3. P. Pradhan, P. K. Behera, and B. N. B. Ray, “Modified Round Robin Algorithm for Resource Allocation in Cloud Computing Procedia Computer Science, vol. 85, no. Cms, pp. 878–890, 2016, doi: 10.1016/j.procs.2016.05.278. [5] B. Taneja, “An empirical study of most fit, max-min and priority task scheduling algorithms in cloud computing,” International Conference on Computing, Communication and Automation, ICCCA 2015, pp. 664–667, 2015, doi: 10.1109/CCAA.2015.7148457. [6] F. Alhaidari and T. Z. Balharith, “Enhanced round-robin algorithm in the cloud computing environment for optimal task scheduling,” Computers, vol. 10, no. 5, 2021, doi: 10.3390/computers10050063. g p p F. Alhaidari, T. Balharith, and E. Al-Yahyan, “Comparative analysis for task scheduling algorithms on cloud computing,” 20 I i l C f C d I f i S i ICCIS 2019 1 6 2019 d [7] F. Alhaidari, T. Balharith, and E. Al-Yahyan, “Comparative analysis for task scheduling algorithms on cloud computing,” 2019 International Conference on Computer and Information Sciences, ICCIS 2019, pp. 1–6, 2019, doi: 10.1109/ICCISci.2019.8716470. y p y g g p g International Conference on Computer and Information Sciences, ICCIS 2019, pp. 1–6, 2019, d 10.1109/ICCISci.2019.8716470. [8] R. Abu Khurma, H. Al Harahsheh, and A. Sharieh, “Task scheduling algorithm in cloud computing based on modified round robin algorithm,” Journal of Theoretical and Applied Information Technology, vol. 96, no. 17, pp. 5869–5888, 2018. [9] S. H. H. Madni, M. S. Abd Latiff, M. Abdullahi, S. M. Abdulhamid, and M. J. Bulletin of Electr Eng & Inf Bulletin of Electr Eng & Inf Given from TQ=(1.5*average) to TQ=(1.65*average) are decreased, and the results from TQ=(1.85*average) to TQ=(1.95 *average) are non-trusted values or have the same value. So whether the burst times are large or small, TQ=(0.75* average) is the ideal time quantum as shown in Figure 20. 6. CONCLUSION Task scheduling is so important in cloud computing. In this work, an ameliorated scheduling algorithm (ARRA) has been proposed to enhance the performance metrics; AWT, ATT and ART in cloud computing. For tasks that come at the same time and at different times, the TQ is determined. The proposed ARRA algorithm showed that (0.75*average) is the ideal time quantum that should be allocated to the tasks in increasing order. It is simulated and compared with the RR, Improved RR, Enhanced RR, ARR and Enhanced RR (RAST ERR) algorithms. From the experiments, the results showed that the ARRA algorithm has considerably reduced the AWT by 3.8-38.20% and the ATT by 2.28-38.19% compared to other algorithms. In future work, the proposed algorithm can be modified to conclude other criteria. 5.1. Why choosing TQ=(3/4)* average? 5.1. Why choosing TQ=(3/4)* average? Different ratios of average have been studied for the TQ to determine which ratio is the best. Different random datasets have been taken; dataset 1, dataset 2 and dataset 3 where the number of tasks n=4000, 10 and 5 tasks, with random burst times in the range (1 to 100). When taking ratios from TQ=(0.85*average) to TQ=(1.25*average) this gives non trusted values, which might either produce high or low AWT and ATT, or they could produce stable results with the same value as shown in Figures 19-20. Figures 19-20 show that the resulting AWT is stable and has the same value from TQ=(1.35*average) to TQ=1.95*average. It is obvious above that the results. Bulletin of Electr Eng & Inf, Vol. 12, No. 2, April 2023: 1103-1114 Figure 19. Comparison of several average ratios in dataset 1 Figure 20. Comparison of several average ratios in dataset 1 and dataset 2 0 100000 200000 0,15 0,35 0,55 0,75 0,95 1,15 1,35 1,55 1,75 1,95 AWT(ms) Ratios of average dataset 1 0 200 400 0,15 0,25 0,35 0,55 0,75 0,85 0,95 1,05 1,15 1,25 1,35 1,45 1,55 1,65 1,75 1,85 1,95 AWT(ms) Ratios of average dataset 2 dataset 3 Fi 19 C i f l i i d 1 0 100000 200000 0,15 0,35 0,55 0,75 0,95 1,15 1,35 1,55 1,75 1,95 AWT(ms) Ratios of average dataset 1 Figure 19. Comparison of several average ratios in dataset 1 Bulletin of Electr Eng & Inf, Vol. 12, No. 2, April 2023: 1103-1114  1113  1113 ISSN: 2302-9285 REFERENCES Matarneh, “Self-Adjustment Time Quantum in Round Robin Algorithm Depending on Burst Time of the Now Runni Processes,” American Journal of Applied Sciences, vol. 6, no. 10, pp. 1831–1837, 2009, doi: 10.3844/ajassp.2009.1831.1837. Processes, American Journal of Applied Sciences, vol. 6, no. 10, pp. 1831–1837, 2009, doi: 10.3844/ajassp.2009.1831.1837. [28] H. Mora, S. E. Abdullahi, and S. B. Junaidu, “Modified Median Round Robin Algorithm (MMRRA),” 2017 13th International Conference on Electronics, Computer and Computation, ICECCO 2017, vol. 2018-Janua, pp. 1–7, 2018, doi: , f pp , , , pp , , j p [28] H. Mora, S. E. Abdullahi, and S. B. Junaidu, “Modified Median Round Robin Algorithm (MMRRA),” 2017 13th International Conference on Electronics, Computer and Computation, ICECCO 2017, vol. 2018-Janua, pp. 1–7, 2018, doi: 10 1109/ICECCO 2017 8333325 , f pp , , , pp , , j p H. Mora, S. E. Abdullahi, and S. B. Junaidu, “Modified Median Round Robin Algorithm (MMRRA),” 2017 13th International , f pp pp j p 8] H. Mora, S. E. Abdullahi, and S. B. Junaidu, “Modified Median Round Robin Algorithm (MMRRA),” 2017 13th Internation Conference on Electronics Computer and Computation ICECCO 2017 vol 2018-Janua pp 1–7 2018 d Conference on Electronics, Computer and Computation, ICECCO 2017, vol. 2018-Janua, pp. 1–7, 2018, d 10.1109/ICECCO.2017.8333325. [29] M. Runsungnoen and T. Anusas-amornkul, Round Robin Scheduling Based on Remaining Time and Median (RR_RT&M) for Cloud Computing, vol. 165. Springer Singapore, 2020, doi: 10.1007/978-981-15-0077-0_3. p g p g g p [30] A. A. Abdelhafiz, “Al-Azhar Bulletin of Science: Section B,” vol. 32, no. 1-B, pp. 27–44, 2021. [31] H. G. Tani and C. EL Amrani, “Smarter Round Robin Scheduling Algorithm for Cloud Computing and Big Data,” Journal of Data Mining & Digital Humanities, vol. Special Is, 2018, doi: 10.46298/jdmdh.3104. g g , p , , j [32] K. ElDahshan, A. Abd, and N. Ghazy, “Achieving Stability in the Round Robin Algorithm,” International Journal of Computer Applications, vol. 172, no. 6, pp. 15–20, 2017, doi: 10.5120/ijca2017915161. REFERENCES Tayal, “Tasks scheduling optimization for the cloud computing systems,” International journal of advanced engineering sciences and technologies, vol. 5, no. 2, pp. 111-115., 2011. f p pp f y, , pp , , [18] S. Tayal, “Tasks scheduling optimization for the cloud computing systems,” International journal of advanced engineering sciences and technologies, vol. 5, no. 2, pp. 111-115., 2011. g pp 9] O. H. M. Dorgham and M. O. Nassar, “Improved Round Robin Algorithm: Proposed Method to Apply {SJF} using Geomet Mean,” International Journal of Advanced Studies in Computers, Science and Engineering, vol. 5, no. 11, pp. 112–119, 2016. 0] P. Banerjee, B. Kumar, and P. Banerjee, “Mixed Round Robin Scheduling for Real Time Systems,” International Journal Computer Trends and Technology, vol. 49, no. 3, pp. 189–195, 2017, doi: 10.14445/22312803/ijctt-v49p130. 1] S. Z. Iqbal et al., “Relative time quantum-based enhancements in Round robin scheduling,” Computer Systems Science a Engineering, vol. 41, no. 2, pp. 461–477, 2022, doi: 10.32604/csse.2022.017003. [21] S. Z. Iqbal et al., “Relative time quantum-based enhancements in Round robin scheduling,” Computer Systems Science and Engineering, vol. 41, no. 2, pp. 461–477, 2022, doi: 10.32604/csse.2022.017003. [22] S. Banerjee, A. Chowdhury, S. Mukherjee, and U. Biswas, “An approach towards development of a new cloudlet allocation g g pp S. Banerjee, A. Chowdhury, S. Mukherjee, and U. Biswas, “An approach towards development of a new cloudlet allocation policy with dynamic time quantum,” Automatic Control and Computer Sciences, vol. 52, no. 3, pp. 208–219, 2018, doi: 10.3103/S0146411618030033. [23] P. Sangwan, M. Sharma, and A. Kumar, “Improved round robin scheduling in cloud computing,” Advances in Computational Sciences and Technology, vol. 10, no. 4, pp. 639–644, 2017. An ameliorated Round Robin algorithm in the cloud computing for task scheduling (Nermeen Ghaz  4  1114 ISSN: 2302-9285 [24] S. Mittal, S. Singh, and R. Kaur, “Enhanced Round Robin Technique for Task Scheduling in Cloud Computing Environment,” International Journal of Engineering Research and, vol. V5, no. 10, pp. 525–529, 2016. 5] B. Fataniya and M. Patel, “Dynamic Time Quantum Approach to Improve Round Robin Scheduling Algorithm in Clo Environment,” Ijsrset, vol. 4, no. 4, pp. 963–969, 2018. j pp [26] A. Stephen, B. J. H. Shanthan, and D. Ravindran, “Enhanced Round Robin Algorithm for Cloud Computing,” Int J Sci Res Comput Sci Appl Manag Stud, vol. 7, no. 4, pp. 1–5, 2018. 7] R. J. BIOGRAPHIES OF AUTHORS Nermeen Ghazy received B.S. Degree in pure math and Computer Science, from the Department of Mathematics, Faculty of Science, Al-Azhar University, Cairo, Egypt in 2011. She received the master degree in enhancing scheduling algorithms in operating systems from the Department of Mathematics, Faculty of Science, Al-Azhar University, Cairo, Egypt, in 2017. She can be contacted at email: nermeena207@gmail.com. Afaf Abdelkader received the M.Sc degree in Computer science from Math. Dept., Faculty of Science, Al-Azhar University, Egypt, in 2006 and Ph.D in Computer Science from Math. Dept. Faculty of Science, Al-Azhar University, Egypt in 2011. She is currently lecturer at Al-Azhar University. She can be contacted at email: afaf2azhar@azhar.edu.eg. Mervat S. Zaki She is a professor in pure and operations research (OR) at department of mathematics, Faculty of science (girls), Al-Azhar University in Cairo, Egypt. She is a reviewer and supervisor in a lot of masters and phd theses at (Cairo, Helwan, Banha, Suez Canal) University. She can be contacted at email: Mervatzaki.1959@azhar.edu.eg. Kamal A. Eldahshan He is a Professor of Computer Science andInformation Systems at Al-Azhar University in Cairo, Egypt. An Egyptiannational and graduate of Cairo University, he obtained his doctoral degreefrom the Universitè de Technologie de Compiègne in France, where he alsotaught for several years. During his extended stay in France, he also workedat the prestigious Institute National de Tèlècommunications in Paris.Professor ElDahshanâAZs extensive international research, teaching, andconsulting experiences have spanned four continents and include academicinstitutions as well as government and private organizations. He taught atVirginia Tech as a Visiting Professor; he was a Consultant to the EgyptianCabinet Information and decision support centre (IDSC), and he was aSenior Advisor to the Ministry of Education and Deputy Director of theNational Technology Development Centre. Professor ElDahshan is aprofessional Fellow on Open Educational Resources as recognized by the United States Department of State and an Expert at ALECSO as recognizedby the League of Arab States. He can be contacted at email: dahshan@gmail.com. Bulletin of Electr Eng & Inf, Vol. 12, No. 2, April 2023: 1103-1114
https://openalex.org/W2946537597
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Evaluating and Diagnosing Road Intersection Operation Performance Using Floating Car Data
Sensors
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cc-by
13,578
Received: 9 April 2019; Accepted: 13 May 2019; Published: 15 May 2019 Abstract: Urban road intersections play an important role in deciding the total travel time and the overall travel efficiency. In this paper, an innovative traffic grid model has been proposed, which evaluates and diagnoses the traffic status and the time delay at intersections across whole urban road networks. This method is grounded on a massive amount of floating car data sampled at a rate of 3 s, and it is composed of three major parts. (1) A grid model is built to transform intersections into discrete cells, and the floating car data are matched to the grids through a simple assignment process. (2) Based on the grid model, a set of key traffic parameters (e.g., the total time delay of all the directions of the intersection and the average speed of each direction) is derived. (3) Using these parameters, intersections are evaluated and the ones with the longest traffic delays are identified. The obtained intersections are further examined in terms of the traffic flow ratio and the green time ratio as well as the difference between these two variables. Using the central area of Beijing as the case study, the potential and feasibility of the proposed method are demonstrated and the unreasonable signal timing phases are detected. The developed method can be easily transferred to other cities, making it a useful and practical tool for traffic managers to evaluate and diagnose urban signal intersections as well as to design optimal measures for reducing traffic delay and increase operation efficiency at the intersections. Keywords: intersections; operational state evaluation; grid model; floating car data sensors sensors Evaluating and Diagnosing Road Intersection Operation Performance Using Floating Car Data Deqi Chen 1, Xuedong Yan 1,* , Feng Liu 2, Xiaobing Liu 1, Liwei Wang 1 and Jiechao Zhang 3 1 MOT Key Laboratory of Transport Industry of Big Data Application Technologies for Comprehensive Transport, School of Traffic and Transportation, Beijing Jiaotong University, Beijing 100044, China; 17114230@bjtu.edu.cn (D.C.); 16114222@bjtu.edu.cn (X.L.); 16114207@bjtu.edu.cn (L.W.) 2 Transportation Research Institute (IMOB), Hasselt University, Wetenschapspark 5, bus 6, B-3590 Diepenbee Belgium; feng.liu@uhasselt.be 3 Deqi Chen 1, Xuedong Yan 1,* , Feng Liu 2, Xiaobing Liu 1, Liwei Wang 1 and Jiechao Zhang 3 1 MOT Key Laboratory of Transport Industry of Big Data Application Technologies for Comprehensive Transport, School of Traffic and Transportation, Beijing Jiaotong University, Beijing 100044, China; 17114230@bjtu.edu.cn (D.C.); 16114222@bjtu.edu.cn (X.L.); 16114207@bjtu.edu.cn (L.W.) 2 Transportation Research Institute (IMOB), Hasselt University, Wetenschapspark 5, bus 6, B-3590 Diepenbeek Belgium; feng.liu@uhasselt.be 3 Civil Engineering Department, University of Central Florida, Orlando, FL 32816-2450, USA; zlyjsl123@knights.ucf.edu * Correspondence: xdyan@bjtu.edu.cn 1. Introduction Intersections are the meeting points of pedestrians, bicycles and vehicle flow, and junctions where road users change their directions. They are also the bottlenecks of urban roads, contributing a great deal to the loss of travel time due to traffic interference, management and control [1]. Traditionally, intersection delay has been defined as the vehicle running time loss when turning or crossing intersections from upstream to downstream traffic flow [2]. Based on floating car data, Wang further characterized travel time delay as a floating car’s travel time in excess of that of user-specified free flow travel time [3]. The delay of intersections does not only affect the travel efficiency of road users but also the logic of signal control [4]. It is estimated that delay at traffic signals contributes 5–10% of all traffic delay worldwide, exemplified by the statistics that 295 million vehicle-hours of delay occurred on major roadways in the USA each year [5]. Thus, it is of striking importance for both real-time route planning and traffic control and management to accurately estimate time-dependent delay at intersections on urban road networks [6]. www.mdpi.com/journal/sensors www.mdpi.com/journal/sensors Sensors 2019, 19, 2256; doi:10.3390/s19102256 Sensors 2019, 19, 2256 2 of 20 In the existing traffic delay research, a wide range of traffic parameters has been explored, including the total travel time [7], average waiting time [8,9], queue length [10–12], throughput [13], time delay [14,15], and number of stops [16,17]. The data sources mainly originated from simulation platforms or fixed road sensors (e.g., cameras and loops) [18–21]. Nevertheless, the traditional data have a number of intrinsic limitations [22,23], making it a challenge to the comprehensive evaluation of operation status at intersections based on the data. The development of GPS (Global Position System) technologies enables us to utilize floating cars (i.e., vehicles equipped with GPS devices) to track vehicle trajectories and collect real-time traffic data across the entire road networks. The vehicles can be regarded as ubiquitous mobile sensors, probing a city’s rhythm and pulse with high data collection efficiency [24–26]. Compared with the conventional data sources, the floating car data (FCD) provide three important advantages. First, real-time traffic data can be collected, and automatically sent to a processing center where information about traffic conditions is extracted [27]. 1. Introduction Secondly, the intersections of a large part of the road network can be monitored, having a much larger coverage than sensor data by which only a limited number of areas can be observed [28]. Thirdly, via GPS-equipped vehicles, high-quality data are collected with low costs [29]. All these characteristics have led to FCD gradually becoming the mainstream data collection method in transportation research [30]. Studies have been conducted to extract traffic parameters from the FCD data, based on map-matching and Geographic Information System (GIS) techniques [31,32]. However, there are two major obstacles in applying these techniques to the analyses of intersections. First, it has been generally acknowledged that map-matching is a complicated and time consuming tool [33], making it challenging to match massive amounts of FCD across whole urban intersections [34]. Secondly, it is difficult to acquire a high quality and timely updated map which ensures the accuracy of the matches. Thus, despite the fact that FCD provide important information about traffic patterns of urban road networks [35], there has been a lack of methodologies which focus on the comprehensive evaluation of operation status at intersections using the data. To fill in this gap, in this study, a novel traffic grid model has been proposed, which is based on the FCD data but assesses the operational status at intersections more efficiently. The aim is to provide accurate estimation of traffic operation states at intersections across the whole urban road networks, and evaluate and diagnose serious problems (e.g., time delay) at the intersections. The final goal is to assist traffic managers in designing optimal measures to increase traffic operation efficiency and reduce traffic delay at urban intersections. Compared to the existing literature, the proposed method has the following major advantages. (1) It is completely based on the data, without the aid of any additional tools such as complex map-matching and digital maps. This makes this approach particularly practical for researchers and engineers who are not familiar with the map-matching and GIS techniques. (2) This method is easily transferred to other cities for the evaluation of traffic operation at most of the signalized intersections of the urban road networks. (3) Under the grid modelling, the intersections are transformed into discrete cells, and the FCD data are matched to the grids through a simple assignment process. 2.1. Data Collection and Cleaning 2.1. Data Collection and Cleaning The FCD data were collected by DiDi, a company offering ride-hailing services for 20 million registered taxi drivers in 380 cities in China [36]. DiDi is the largest on-demand ride service platform in China and one of the largest in the world [37]. In 2017, it served 25 million rides each day, with more than 20 billion path planning requests and data of more than 4500TB being processed. The data were sampled at a rate of 3 s (seconds) through the GPS sensors embedded on drivers’ mobile phones, and sent to the processing center of DiDi, where traffic information was extracted from the data. The FCD data mainly consist of two parts, including GPS log files and GPS trajectories defined as follows. Definition 1. A GPS log file Tri represents a data record (i.e., a GPS point) transmitted from GPS sensors. The format of the log file is < Id, t, lng, lat, v >, where Id refers to the Id of the vehicle, t is the timestamp, lng and lat are the longitude and latitude, v denotes the speed of the vehicle. Definition 2. A GPS trajectory Trak is a sequence of GPS log files, i.e., Trak = Tr1 →Tr2 →· · · Trm →· · · Trn, with Trm =< Idm, tm, lngm, latm, vm >, where the time stamps in the sequence strictly increase. The obtained GPS trajectory data were saved in txt files, and the variables and their attributes are listed in Table 1. Table 1. The detailed data attributes of the FCD system. Characteristic Field Name Field Type Field Description Id Terminal ID String 6 bytes characters, marking each vehicle t GPS time stamp Timestamp Accurate to second lng Longitude Floating Accurate to six decimal places lat Latitude Floating Accurate to six decimal places v Vehicle Speed Integer Kilometer per hour Table 1. The detailed data attributes of the FCD system. Table 1. The detailed data attributes of the FCD system. Raw GPS data may contain errors caused either by blockage of GPS signals or by hardware/software bugs during the data collection and transmission process [38]. In order to provide good quality of data and ensure accuracy of the derived results, it is important to detect and remove the wrong records that are incompatible with the physical phenomena of traffic. 2.1. Data Collection and Cleaning The data cleaning process is carried out according to the following two steps. g g p Step1: Remove the data that are beyond the range of the traffic analysis region. Step2:Remove the data in which the corresponding vehicle speeds are out of the range of 0–90 km/h 1. Introduction (4) From the constructed cell-based intersections, key traffic parameters are extracted, and intersections with the longest delay are identified and the detailed problems at these intersections are further diagnosed. The simplicity of the grid model and the parameter extraction, along with the constant input of the GPS data, makes the proposed method highly efficient in evaluating and diagnosing intersection operation in real time. Using the central area of Beijing as the case study, the potential and feasibility of the proposed method are demonstrated. The rest of this paper is organized as follows. Section 2 describes the FCD data and details the proposed methodology. Section 3 conducts a case study and in-depth analyses on the experimental results. Finally, Section 4 ends this paper with major conclusions and discussions for future research. 3 of 20 Sensors 2019, 19, 2256 2 2 1 Identifying the Directions of Trajectories 2.2.1. Identifying the Directions of Trajectories 2.2.1. Identifying the Directions of Trajectories 2 2 1 Identifying the Directions of Trajectories 2.2.1. Identifying the Directions of Trajectories 2.2.1. Identifying the Directions of Trajectories 2.2.1. Identifying the Directions of Trajectories Once the intersections are split into grids, the points of each trajectory are then mapped to the grids (see Figure 2a). According to the specific order of the Grid ID (i.e., from 0 to 8) of the first entrygrid and that of the last exit grid that vehicles pass through, the direction of the trajectory is subsequently identified. Thus, intersections are reconstructed into grid-based ones, as displayed in Figure 2b. The trajectory data along with the identified directions can be utilized to extract the traffic performance parameters at the intersections Once the intersections are split into grids, the points of each trajectory are then mapped to the grids (see Figure 2a). According to the specific order of the Grid ID (i.e., from 0 to 8) of the first entrygrid and that of the last exit grid that vehicles pass through, the direction of the trajectory is subsequently identified. Thus, intersections are reconstructed into grid-based ones, as displayed in Figure 2b. The trajectory data along with the identified directions can be utilized to extract the traffic performance parameters at the intersections. Once the intersections are split into grids, the points of each trajectory are then mapped to the grids (see Figure 2a). According to the specific order of the Grid ID (i.e., from 0 to 8) of the first entrygrid and that of the last exit grid that vehicles pass through, the direction of the trajectory is subsequently identified. Thus, intersections are reconstructed into grid-based ones, as displayed in Figure 2b. The trajectory data along with the identified directions can be utilized to extract the traffic performance parameters at the intersections. 3 7 1 5 4 Through Turn Left Turn Right Entering Direction 6 8 7 3 5 4 Center grid 0 2 1 6 8 7 3 5 4 Center grid 0 2 1 6 8 7 3 5 4 Center grid 0 2 1 6 8 7 3 5 4 Center grid 0 2 1 (a) (b) Figure 2. A basic grid network: (a) searching direction in mapping an intersection to a grid network, (b) the result of the direction of trajectories identified. 2 2 1 Identifying the Directions of Trajectories 2.2.1. Identifying the Directions of Trajectories 2.2.1. Identifying the Directions of Trajectories 3 7 1 5 4 Through Turn Left Turn Right Entering Direction 6 8 7 3 5 4 Center grid 0 2 1 6 8 7 3 5 4 Center grid 0 2 1 6 8 7 3 5 4 Center grid 0 2 1 6 8 7 3 5 4 Center grid 0 2 1 (a) (b) Figure 2. A basic grid network: (a) searching direction in mapping an intersection to a grid network, (b) the result of the direction of trajectories identified. Figure 2. A basic grid network: (a) searching direction in mapping an intersection to a grid network, (b) the result of the direction of trajectories identified. (b) (b) (a) (a) (b) (b) Figure 2. A basic grid network: (a) searching direction in mapping an intersection to a grid network, (b) the result of the direction of trajectories identified. Figure 2. A basic grid network: (a) searching direction in mapping an intersection to a grid network, (b) the result of the direction of trajectories identified. Figure 2. A basic grid network: (a) searching direction in mapping an intersection to a grid network, (b) the result of the direction of trajectories identified. 2.2. The Grid Model The traffic grid model is based on the City Management Grid Modelling Theory [39]. In this technique, a management area can be divided into disjoint grids with the same or different sizes. The facilities within the grids are then managed and dispatched by different categories (such as different types of civil facilities) in order to improve the management effectiveness. Figure 1 shows the flowchart of the grid model method that we applied in this study. Firstly, the procedures of identifying the directions of trajectories and defining the influential regions of intersections are used to extract the key traffic parameters from floating car data, which are further used for the intersection traffic delay index calculation and intersection of traffic delay diagnosis. 4 of 20 4 of 20 4 of 20 Sensors 2019, 19, 2256 Sensors 2019, 19, x FOR Sensors 2019, 19, x FOR Figure 1. The flowchart of the grid model method. Figure 1. The flowchart of the grid model method. Figure 1. The flowchart of the grid model method. Figure 1. The flowchart of the grid model method. Figure 1. The flowchart of the grid model method. Figure 1. The flowchart of the grid model method. j 2.2.2. Defining the Influential Regions of Intersections In the current study, we divide the area of each road intersection into grids with an unfixed size. In constructing such grids, two important parameters need to be specified: one is the range of the 5 of 20 5 of 20 Sensors 2019, 19, 2256 Sensors 2019, 19, x FOR intersection area, and the other is the size of each grid. Different ranges of intersections have been adopted in previous studies. For instance, Sun investigated Beijing’s road networks and defined the range as 160 m [40]. Zhang et al. considered the dynamic spatial distribution characteristics of floating cars, and set the range as 200 m, which started at the center of an intersection and extended both upstream and downstream by 100 m [41]. Choosing an appropriate intersection range is essential to distinguish different intersections, and ensure enough data in each intersection as well as in each cell of the intersection. If the range is too large, it may contain multiple intersections, and the traffic conditions between different intersections cannot be differentiated. On the contrary, if the range is too small and cannot cover an intersection adequately, the FCD data samples would be insufficient for some cells of the intersection, leading to the inaccurate calculations of travel speed and vehicle positions. i te sectio a ea, a d t e ot e is t e si e o eac g id i e e t a ges o i te sectio s a e bee adopted in previous studies. For instance, Sun investigated Beijing’s road networks and defined the range as 160 m [40]. Zhang et al. considered the dynamic spatial distribution characteristics of floating cars, and set the range as 200 m, which started at the center of an intersection and extended both upstream and downstream by 100 m [41]. Choosing an appropriate intersection range is essential to distinguish different intersections, and ensure enough data in each intersection as well as in each cell of the intersection. If the range is too large, it may contain multiple intersections, and the traffic conditions between different intersections cannot be differentiated. On the contrary, if the range is too small and cannot cover an intersection adequately, the FCD data samples would be insufficient for some cells of the intersection, leading to the inaccurate calculations of travel speed and vehicle positions. j 2.2.2. Defining the Influential Regions of Intersections Given the above consideration, we empirically estimate the range of intersections using the FCD data, which should cover the upstream spaces of intersections from the center points of the corresponding intersections to furthest queuing points of vehicles at intersections. According to the spatial-temporal FCD’ distributions, there will be different affected areas in different directions of intersections: the defined region of straight-through traffic (i.e., DRTI), the defined region of left-turn traffic (i.e., DRLTI), as shown in Figure 3. It is noted that the width of the grid side (i.e., WGS) was influenced by DRLTI and DRTI, whose boundaries are the maximum stop distances for trajectories, where the velocity of the position is 0m/s (i.e., V = 0). Then, we choose the 95th percentile of the distance of vehicles at the intersection between the stop point and the center point of the intersection as the defined region of different directions. The length of the grid side (i.e., LGS) should make sure that the FCD data are fully matched to the grids. p Given the above consideration, we empirically estimate the range of intersections using the FCD data, which should cover the upstream spaces of intersections from the center points of the corresponding intersections to furthest queuing points of vehicles at intersections. According to the spatial-temporal FCD’ distributions, there will be different affected areas in different directions of intersections: the defined region of straight-through traffic (i.e., DRTI), the defined region of left-turn traffic (i.e., DRLTI), as shown in Figure 3. It is noted that the width of the grid side (i.e., WGS) was influenced by DRLTI and DRTI, whose boundaries are the maximum stop distances for trajectories, where the velocity of the position is 0m/s (i.e., V = 0). Then, we choose the 95th percentile of the distance of vehicles at the intersection between the stop point and the center point of the intersection as the defined region of different directions. The length of the grid side (i.e., LGS) should make sure that the FCD data are fully matched to the grids. V = 0 V=v1 V=v2 V=v3 V=v2 V = 0 V=v1 DRLTI DRTI LGS WGS Figure 3. The region of an intersection defined by the grid model. Figure 3. The region of an intersection defined by the grid model. Figure 3. The region of an intersection defined by the grid model. Figure 3. j 2.2.2. Defining the Influential Regions of Intersections The region of an intersection defined by the grid model. 2 2 3 Defining the Intersection Traffic Delay Indexes 2.2.3. Defining the Intersection Traffic Delay Indexes 2 2 3 Defining the Intersection Traffic Delay Indexes 2.2.3. Defining the Intersection Traffic Delay Indexes g y Each trajectory j of FCD passing the intersection of the process has two stages: waiting stage and passing stage; non-stop trajectories do not involve the waiting stage. For each intersection, the corresponding total delay (i.e., j TD ) can be divided into two aspects: delay of waiting time (i.e., Each trajectory j of FCD passing the intersection of the process has two stages: waiting stage and passing stage; non-stop trajectories do not involve the waiting stage. For each intersection, the corresponding total delay (i.e., TDj) can be divided into two aspects: delay of waiting time (i.e., DWTj) and delay of passing time (i.e., DPTj), respectively, as shown in Figure 4. j DWT ) and delay of passing time (i.e., j DPT ), respectively, as shown in Figure 4. For each intersection, two key traffic parameters are derived, including the vehicle passing time (i.e., PTi) through each direction i of the intersection and the waiting time delay (i.e., WTi) of the direction i. To this end, the following variables are defined in Table 2. 6 of 20 Sensors 2019, 19, 2256 nsors 2019, 19, x FOR PEER REVIEW Figure 4. The total delay framework. V = 0 V=v2 V=v1 V=v3 V=v4 1. Wait stage 2. Passing Intersection stage Delay of waitting time Passing time Free flow passing time Delay of passing time Total Delay Start Stopping time End of stopping time Unit : Velocity(m/s); Time(s); Figure 4. The total delay framework. End of stopping Free flow passing time Passing time Delay of waitting time Delay of passing time Unit : Velocity(m/s); Time(s); Total Delay Figure 4. The total delay framework. Figure 4. The total delay framework. ction, two key traffic parameters are derived, including the ch direction i of the intersection and the waiting time d Table 2. The parameter definition of intersection traffic delay. For each intersection, two key traffic parameters are derived, including the vehicle passing t , i PT ) through each direction i of the intersection and the waiting time delay (i.e., i WT ) of ection i . To this end, the following variables are defined in Table 2. Table 2. The parameter definition of intersection traffic delay. 2 2 3 Defining the Intersection Traffic Delay Indexes 2.2.3. Defining the Intersection Traffic Delay Indexes Parameter Notation Definition j st Time stamp of the point of the trajectory j starting stop j et Time stamp of the point of the trajectory j ending stop j int Time stamp of the point of the trajectory j enter intersection j out t Time stamp of the point of the trajectory j exit intersection j in T Time deviation of the trajectory j enter intersection j out T Time deviation of the trajectory j exit intersection m in p The last point outside the intersection whose coordinate is ( ) , m m in in Lat Lng , point m in the trajectory j Table 2. The parameter definition of intersection traffic delay. Parameter Notation Definition tj s Time stamp of the point of the trajectory j starting stop tj e Time stamp of the point of the trajectory j ending stop tj in Time stamp of the point of the trajectory j enter intersection tj out Time stamp of the point of the trajectory j exit intersection Tj in Time deviation of the trajectory j enter intersection Tj out Time deviation of the trajectory j exit intersection pm in The last point outside the intersection whose coordinate is  Latm in, Lngm in  , point m in the trajectory j ps in The entry point of intersection side whose coordinate is  Lats in, Lngs in  pm+1 c The inside point of intersection whose coordinate is  Latm+1 in , Lngm+1 in  , the point m + 1 in the trajectory j ps out The exit point of intersection side whose coordinate is  Lats out, Lngs out  pm+2 out The first point outside the intersection whose coordinate is  Latm+2 out , Lngm+2 out  , the point m + 2 in the trajectory j Nt The number of the trajectories in terms of 5 min time slices, t = 5min, 15min, 30min. rst point outside the intersection whose coordinate is ( , out Lat Lng oint m + 2 in the trajectory j b f h j i i f 5 i i li NtP j=1  tj out −tj in −Tj in −Tj out  2 2 3 Defining the Intersection Traffic Delay Indexes 2.2.3. Defining the Intersection Traffic Delay Indexes in p y p ( ) in in g 1 m cp + The inside point of intersection whose coordinate is ( ) 1 1 , m m in in Lat Lng + + , the point m+1 in the trajectory j s out p The exit point of intersection side whose coordinate is ( ) , s s out out Lat Lng Three time slices (i.e., 5 min, 15 min and 30 min), are considered for the computations of the traffic parameters. The delay of go-through the intersection direction i is computed based on Formula (1). The free flow passing time (i.e., TFi) in the direction i is assumed as the 5th percentile of the distribution of all the passing time derived from the FCD data, as suggested by Brown et al. [42]. 2 m out p + The first point outside the intersecti , the point m + 2 in the trajectory j t N The number of the trajectories in ter 5 15 30 t min min min = DPTi = PTi −TFi = NtP j=1  tj out −tj in −Tj in −Tj out  Nt − (1) ms of 5min time slices, −TFi, t = 5 min, 15 min, 30 min traje 0min Nt 7 of 20 Sensors 2019, 19, 2256 Because most of the trajectory points arriving at and leaving the intersection do not exactly match the boundary of the intersection, the time deviation is produced. Time deviations of the entry and exit intersection can be calculated by Dj in, Sj in, Dj out, Sj out and 3 s time interval. 2 2 3 Defining the Intersection Traffic Delay Indexes 2.2.3. Defining the Intersection Traffic Delay Indexes 8 8 The traffic delay (TDi) in direction i and the total delay (TD) over all the directions are computed as Formula (5). TD = 8 X i=1 TDi = 8 X i=1 (DWTi + DPTi) (5) (5) 2 2 3 Defining the Intersection Traffic Delay Indexes 2.2.3. Defining the Intersection Traffic Delay Indexes As is shown in Figure 4, Dj in is defined as the distance between ps in  Lats in, Lngs in  and pm in  Latm in, Lngm in  , Sj in is defined as the distance between pm in  Latm in, Lngm in  and pm+1 c  Latm+1 in , Lngm+1 in  , Dj out is defined as the distance between ps out  Lats out, Lngs out  and pm+2 out  Latm+2 out , Lngm+2 out  , and Sj out is defined as the distance between pm+2 out  Latm+2 out , Lngm+2 out  and pm+1 c  Latm+1 in , Lngm+1 in  , where the intersection point of the trajectory and boundary is s, and the points m, m + 1, m + 2 in trajectory j. Therefore, the time deviations of the entry intersection and exit intersection (Tj in, Tj out) can be obtained by Formula (2) and (3). Tj in = 3 Dj in Sj in = 3 Arccos(sin(Latm in) sin(Lats in) cos(Lngs in −Lngm in) + cos(Lats in) cos(Latm in)) Arccos(sin(Latm in) sin(Latm+1 in ) cos(Lngm+1 in −Lngm in) + cos(Latm+1 in ) cos(Latm in)) (2) Tj in = 3 Dj in Sj in = 3 Arccos(sin(Latm in) sin(Lats in) cos(Lngs in −Lngm in) + cos(Lats in) cos(Latm in)) Arccos(sin(Latm in) sin(Latm+1 in ) cos(Lngm+1 in −Lngm in) + cos(Latm+1 in ) cos(Latm in)) (2) j Dj Arccos(sin(Latm+2) sin(Lats ) cos(Lngs −Lngm+2) + cos(Lats ) cos(Latm+2)) Tj out = 3 Dj out Sj out = 3 Arccos(sin(Latm+2 out ) sin(Lats out) cos(Lngs out −Lngm+2 out ) + cos(Lats out) cos(Latm+2 out )) Arccos(sin(Latm+2 out ) sin(Latm+1 in ) cos(Lngm+2 out −Lngm+1 in ) + cos(Latm+1 in ) cos(Latm+2 out )) (3) (3) The delay of waiting time (DWT) can be computed by Formula (4), in which the non-stop trajectory j leads to ϕ = 0, otherwise ϕ = 1. The delay of waiting time (DWT) can be computed by Formula (4), in which the non-stop trajectory j leads to ϕ = 0, otherwise ϕ = 1. DWTi = NtP j=1 ϕ  tj e −tj s  Nt , t = 5min, 15min, 30min (4) (4) The traffic delay (TDi) in direction i and the total delay (TD) over all the directions are computed as Formula (5). 2.2.4. Diagnosing the Intersection Traffic Delay There are two main reasons leading to frequent delays at signalized intersections: one is the unreasonable timing of signal lights, and the other is the large traffic volume which exceeds the traffic capacity of the intersections [43,44]. The diagnosis method of traffic delay at intersections is comprised of three steps. In the first step, the delay of each single intersection is computed according to Formula (1), using the time slices of 5 min, 15 min and 30 min. Based on the obtained results, the pattern of the delay for each intersection throughout the day, particularly during the rush hours, can be analyzed. Furthermore, the delays across all the intersections are sorted, and the ones with the worst performance are identified. In the second step, detailed traffic operation at the intersections with the serious delay problems is examined. Variables, including the time delay in different directions, the average speed, the ratio between the number of non-stop trajectories and that of stop trajectories, are investigated. In the third step, the delay problems are further diagnosed based on the comparison between the flow ratio and the signal split ratio of the effective green time. According to the above diagnostic method, parameters are defined as shown in Table 3. It should be noted that Qi, the observed number of floating vehicles passing through the intersection, is used rather than the real traffic volume at the intersection. Although the sampling rate of FCD may change in each signal cycle, historical analyses with multiple days’ data can steadily reflect the traffic volume of direction i of intersections and their changing patterns. 8 of 20 Sensors 2019, 19, 2256 Table 3. The parameter definition of intersection traffic delay. Table 3. The parameter definition of intersection traffic delay. Parameter Notation Definition Ns The number of stops of FCD Nns The number of non-stops of FCD τi The ratio of non-stop FCD to stop FCD in the direction i of the intersection vij The speed of the trajectory j passing through the intersection in the direction i rf j,φ The time stamp of the first point of the trajectory j in phase φ whose speed is 0 m/s. rlj,φ The time stamp of the last point of the trajectory j in phase φ whose speed is 0 m/s. n The number of the phases RTφ The red time of phases φ. 2.2.4. Diagnosing the Intersection Traffic Delay ge,φ The effective green time in the phase φ, φ ranges from 1 to n. C A signal cycle λφ The green ratio time in the phase φ, φ ranges from 1 to n. Qi The traffic volume of the direction i of intersections ωi The flow ratio in direction i ηi,φ The ratio between the flow ratio and the green signal ratio in the direction i of the phase φ. Table 3. The parameter definition of intersection traffic delay. The ratio of non-stop to stop is defined as the ratio between the number of non-stop trajectories and that of stop trajectories in direction i (See Formula (6)). It reflects the traffic condition and the efficiency of signal timing in the corresponding direction of the intersection. τi = Ns Nns (6) τi = Ns Nns (6) The red light time per direction in a signal cycle is estimated using the first stop vehicles’ trajectories that experience only one cycle at the intersections. Then, we choose the 95th percentile of the stop times of the vehicles at the intersection as the red light time. The 95th percentile of the stop times is selected because the floating car driver may have an abnormally long reaction time when the light turns green. 3. The Empirical Case Study in Beijing φ The data used in this case study include 2,712,744 floating cars’ trajectories (i.e., 35.7 GB in size) collected from 1 to 15 in August 2017, in the central area of Beijing with longitudes and latitudes as follows: <116.399450, 116.431700> and <39.947934, 39.966344>. In the study area, eight intersections were selected, namely, NO.1–NO.8 (See Figure 5), and they cover various types of intersections, including the intersecting of both arterial and branch roads (i.e., NO.2, NO.3, NO.6, NO.7, NO.8), only arterial roads (i.e., NO.4 and NO.5), and only branch roads (i.e., NO.1). 3. The Empirical Case Study in Beijing The data used in this case study include 2,712,744 floating cars’ trajectories (i.e., 35.7 GB in size) collected from 1 to 15 in August 2017, in the central area of Beijing with longitudes and latitudes as follows: <116.399450, 116.431700> and <39.947934, 39.966344>. In the study area, eight intersections were selected, namely, NO.1–NO.8 (See Figure 5), and they cover various types of intersections, including the intersecting of both arterial and branch roads (i.e., NO.2, NO.3, NO.6, NO.7, NO.8), only arterial roads (i.e., NO.4 and NO.5), and only branch roads (i.e., NO.1). Figure 5. The Case of the Selected Area. Figure 6 shows the distributions of the averages of hourly and daily traffic flows across all the selected intersections. It was noted that the traffic flow in the evening peak hour (i.e., 17:00–19:00) was larger than in other periods, including the morning rush hour (i.e., 7:00–10:00) and the off-peak period (e.g., 14:00–16:00) (See Figure 6a); while during the week, the flow was larger on workdays than on non-workdays (See Figure 6b). Figure 5. The Case of the Selected Area. Figure 6 shows the distributions of the averages of hourly and daily traffic flows across all the selected intersections. It was noted that the traffic flow in the evening peak hour (i.e., 17:00–19:00) was larger than in other periods, including the morning rush hour (i.e., 7:00–10:00) and the off-peak period (e.g., 14:00–16:00) (See Figure 6a); while during the week, the flow was larger on workdays than on non-workdays (See Figure 6b). Sensors 2019, 19, x FOR PEER REVIEW 10 of 20 Figure 5. The Case of the Selected Area. Figure 5. The Case of the Selected Area. Figure 5. The Case of the Selected Area. Figure 5. The Case of the Selected Area. 2.2.4. Diagnosing the Intersection Traffic Delay The vector RTφ for the red light time of φ phase is defined as: The vector RTφ for the red light time of φ phase is defined as: RTφ = rlj,φ −rf j,ϕ (7) (7) The effective green light time is defined as: ge,φ = C −RTφ (8) (8) The green time ratio is defined as the ratio between the effective green light time and a signal cycle: The green time ratio is defined as the ratio between the effective green light time and a signal cycle: λφ = ge,φ C = 1 − (n −1)RTφ Pn k=1 RTφ (9) (9) The traffic flow in different directions varies; the weight of the traffic flow in the direction i is: The traffic flow in different directions varies; the weight of the traffic flow in the direction i is: ωi,φ = Qi,φ P8 i=1 Qi,φ (10) (10) ηi,φ is defined as the ratio between the flow ratio (i.e., ωi,φ) and the green signal ratio (i.e., λi,φ) in the direction i of each phase φ: ω ηi,φ is defined as the ratio between the flow ratio (i.e., ωi,φ) and the green signal ratio (i.e., λi,φ) in the direction i of each phase φ: ηi,φ = ωi,φ λi,φ (11) (11) Using ηi,φ, we can diagnose problems of intersection delay caused by signal timing design. If the ηi,φ > 1 (i.e., ωi,φ > λi,φ), the effective green time of the phase φ is not enough for vehicles in a signal cycle to pass the intersection. If ηi,φ < 1 (i.e., ωi,φ < λi,φ), the effective green time of the phase φ is underutilized. Sensors 2019, 19, 2256 the , 1 i φ η > (i.e signal cycle to 9 of 20 a se 3. The Empirical Case Study in Beijing φ Figure 6 shows the distributions of the averages of hourly and daily traffic flows across all the selected intersections. It was noted that the traffic flow in the evening peak hour (i.e., 17:00–19:00) was larger than in other periods, including the morning rush hour (i.e., 7:00–10:00) and the off-peak period (e.g., 14:00–16:00) (See Figure 6a); while during the week, the flow was larger on workdays than on non-workdays (See Figure 6b). Figure 6 shows the distributions of the averages of hourly and daily traffic flows across all the selected intersections. It was noted that the traffic flow in the evening peak hour (i.e., 17:00–19:00) was larger than in other periods, including the morning rush hour (i.e., 7:00–10:00) and the off-peak period (e.g., 14:00–16:00) (See Figure 6a); while during the week, the flow was larger on workdays than on non-workdays (See Figure 6b). Sensors 2019, 19, x FOR PEER REVIEW 10 of 20 (a) (b) Figure 6. (a) Hourly and (b) daily distributions of traffic flow at the selected intersections. The Morning Peak (7:00–10:00) The Evening Peak (17:00–19:00) The off Peak (14:00–16:00). Figure 6. (a) Hourly and (b) daily distributions of traffic flow at the selected intersections. The Morning Peak (7:00–10:00) The Evening Peak (17:00–19:00) The offPeak (14:00–16:00). (b) (a) (b) (a) Figure 6. (a) Hourly and (b) daily distributions of traffic flow at the selected intersections. The Morning Peak (7:00–10:00) The Evening Peak (17:00–19:00) The off Peak (14:00–16:00). Figure 6. (a) Hourly and (b) daily distributions of traffic flow at the selected intersections. The Morning Peak (7:00–10:00) The Evening Peak (17:00–19:00) The offPeak (14:00–16:00). 3.1. The Intersections’ Total Del 3.1. The Intersections’ Total Delay To analyze the total delay at each of the intersections, we construct the spatial-temporal diagrams with the time slices of 30 min (Figure 7a), 15 min (Figure 7b) and 5 min (Figure 7c), respectively. For each intersection (indicated by the y-axis), the different levels of delay along the different times of the day (indicated by the x-axis) are described using a color scheme, with the darkest blue referring to the shortest delay (i.e., Level A) and the darkest red to the longest one (i.e., To analyze the total delay at each of the intersections, we construct the spatial-temporal diagrams with the time slices of 30 min (Figure 7a), 15 min (Figure 7b) and 5 min (Figure 7c), respectively. For each intersection (indicated by the y-axis), the different levels of delay along the different times of the day (indicated by the x-axis) are described using a color scheme, with the darkest blue referring 10 of 20 e ., 10 of 20 he ., Sensors 2019, 19, 2256 different times darkest blue ref to the shortest delay (i.e., Level A) and the darkest red to the longest one (i.e., Level F). Moreover, all eight intersections are sorted in terms of their total delay, and this order is indicated with the arrow on the left side of these figures. It was observed that the delay at the NO.2 intersection was the longest, particularly during the morning and evening rush hours. However, the delay at the NO.7 intersection was the shortest, which could be due to the fact that it is forbidden to make a left turn at this intersection. indicated with the arrow on the left side of these figures. It was observed that the delay at the No. 2 intersection was the longest, particularly during the morning and evening rush hours. However, the delay at the No. 7 intersection was the shortest, which could be due to the fact that it is forbidden to make a left turn at this intersection. Figure 8 further details of the distribution of the delay with the slice of 5 min during the two peak periods. It shows that the delay at intersection No. 2 reached the longest at 8:00–9:30 in the morning, and at 17:00–19:45 in the evening. g g Sensors 2019, 19, x FOR PEER REVIEW 11 of 20 Figure 7. Intersection delay. Figure 7. Intersection delay. 3.2. The Traffic Operation States for Individual Intersections 3.2. The Traffic Operation States for Individual Intersections Apart from the total delay, other major traffic parameters including the traffic flow, average velocity and delay in each direction of each of the intersections are further investigated. In the Apart from the total delay, other major traffic parameters including the traffic flow, average velocity and delay in each direction of each of the intersections are further investigated. In the following, the intersection with the longest delay among these selected ones, i.e., NO.2. intersection, is analyzed. The same process can be applied to any of the remaining intersections. 3.1. The Intersections’ Total Del 3.1. The Intersections’ Total Delay Figure 8 further details of the distribution of the delay with the slice of 5 min during the two p eriods. It shows that the delay at intersection NO.2 reached the longest at 8:00–9:30 in the morn nd at 17:00–19:45 in the evening. Sensors 2019, 19, x FOR PEER REVIEW 11 of 20 Sensors 2019, 19, x FOR PEER REVIEW 11 of 2 Figure 7. Intersection delay. Figure 7. Intersection delay. Figure 7. Intersection delay. Figure 7. Intersection delay. Figure 7. Intersection delay. Figure 7. Intersection delay. Figure 8 further details of the distribution of the delay with the slice of 5 min during the two peak periods. It shows that the delay at intersection NO.2 reached the longest at 8:00–9:30 in the morning, and at 17:00–19:45 in the evening. Figure 8 further details of the distribution of the delay with the slice of 5 min during the two peak periods. It shows that the delay at intersection NO.2 reached the longest at 8:00–9:30 in the morning, and at 17:00–19:45 in the evening. 11 of 20 Sensors 2019, 19, 2256 Figure 7. Intersection delay. Figure 8. The intersection delay of the morning peak and evening peak. Figure 8. The intersection delay of the morning peak and evening peak. Figure 7. Intersection delay. Figure 8. The intersection delay of the morning peak and evening peak. Figure 8. The intersection delay of the morning peak and evening peak. Figure 8. The intersection delay of the morning peak and evening peak. Figure 8. The intersection delay of the morning peak and evening peak. 3.2.1. The Traffic Delay Figure 9 describes the distribution of these three major parameters across different times of working days at the intersection, using the time slice of 15 min. Each point depicts a vector of three values of these parameters, including the traffic volume (i.e., represented by the size of the point), the average velocity (m/s) (i.e., represented by the color of the point), and the delay level (i.e., represented by the radius of the point at the polar coordinates). A point that is further from the origin of the coordinates and has a larger size and red color refers to a traffic state featured with more traffic volume, lower speeds, and longer delay. From Figure 9, it was noted that the traffic flows in the south- and northbound straight directions (See Figure 9a,c) were higher than those of the east- and westbound straight directions (See Figure 9e,g). When the conditions between the south- and northbound directions were compared, the northbound traffic flow was lower, its traffic speed was slower, and therefore the corresponding traffic delay was shorter. From Figure 9b,d, it was found that the distributions of the traffic flow and average velocity for the southbound and northbound left turn directions are similar. However, the southbound left turn had the longest delay during 16:00–20:00, while the northbound left turn suffered the worst delay problem at 10:00–14:00. Figure 9e–h further show that the eastbound traffic average speeds for both the straight and left turn directions were much slower than the westbound average speed. Consequently, the delays in the eastbound directions were longer, particularly during the morning and evening rush hours. In summary, Table 4 lists all of the key traffic performance parameters of the eight critical directions for the intersection. 12 of 20 e traffic Sensors 2019, 19, 2256 y coordinates and h Table 4. Internal evaluation parameters. Parameter Notation Southbound Northbound Eastbound Westbound Through Left-Turn Through Left-Turn Through Left-Turn Through Left-Turn Traffic Flow 328,692 17,348 237,894 19,728 18,065 20,047 16,388 12,347 Average velocity 3.20 2.39 3.19 2.11 2.64 2.53 3.13 2.63 Free Flow travel time 15 24 18 24 24 22 17 24 Average Delay time 52.61 64.22 50.60 81.88 57.53 62.53 64.79 49.09 Unit: Average velocity (m/s); Free travel time (s); Average Delay time (s). volume, lower speeds, and longer delay. 3.2.1. The Traffic Delay From Figure 9, it was noted that the traffic flows in the south- and northbound straight direction (See Figure 9a,c) were higher than those of the east- and westbound straight directions (See Figure 9e,g). When the conditions between the south- and northbound directions were compared, the northbound traffic flow was lower, its traffic speed was slower, and therefore the corresponding traffic delay was shorter. From Figure 9b,d, it was found that the distributions of the traffic flow and average velocity fo the southbound and northbound left turn directions are similar. However, the southbound left turn had the longest delay during 16:00–20:00, while the northbound left turn suffered the worst delay problem at 10:00–14:00. (a) Southbound, Straight (b) Southbound, left turn (c) Northbound, Straight (d) Northbound, left turn Figure 9. Cont. Table 4. Internal evaluation parameters. d that the traffic flows in the south- and han those of the east- and westbound Table 4. Internal evaluation parameters. d that the traffic flows in the south- and han those of the east- and westbound Parameter Notation Southbound Northbound Eastbound Westbound Through Left-Turn Through Left-Turn Through Left-Turn Through Left-Turn Traffic Flow 328,692 17,348 237,894 19,728 18,065 20,047 16,388 12,347 Average velocity 3.20 2.39 3.19 2.11 2.64 2.53 3.13 2.63 Free Flow travel time 15 24 18 24 24 22 17 24 Average Delay time 52.61 64.22 50.60 81.88 57.53 62.53 64.79 49.09 Unit: Average velocity (m/s); Free travel time (s); Average Delay time (s). See Figure 9a,c) were higher than those of the east and westbound straight directions (See Figure e,g). When the conditions between the south- and northbound directions were compared, the orthbound traffic flow was lower, its traffic speed was slower, and therefore the corresponding raffic delay was shorter. From Figure 9b,d, it was found that the distributions of the traffic flow and average velocity fo he southbound and northbound left turn directions are similar. However, the southbound left turn ad the longest delay during 16:00–20:00, while the northbound left turn suffered the worst delay (a) Southbound Straight (b) Southbound left turn (b) Southbound, left turn (a) Southbound Straight (a) Southbound, Straight (a) Southbound, Straight (b) Southbound, left turn (a) Southbound, Straight (c) Northbound, Straight (a) Southbound, Straight (b) Southbound, left turn (c) Northbound, Straight (d) Northbound, left turn Figure 9. Cont. (b) Southbound, left turn (d) Northbound, left turn (c) Northbound, Straight (d) Northbound, left turn Figure 9. Cont. 3.2.1. The Traffic Delay Sensors 2019, 19, 2256 S O 13 of 20 13 of 20 (e) Eastbound, Straight (f) Eastbound, left turn (g) Westbound, Straight (h) Westbound, left turn Figure 9. Intersection internal evaluation. Figure 9. Intersection internal evaluation. (f) Eastbound left turn (e) Eastbound, Straight (f) Eastbound, left turn (f) Eastbound, left turn (h) Westbound, left turn (e) Eastbound, Straight (f) Eastbound, left turn (g) Westbound, Straight (h) Westbound, left turn Figure 9. Intersection internal evaluation. Figure 9. Intersection internal evaluation. (g) Westbound, Straight (g) Westbound, Straight (h) Westbound, left turn (h) Westbound, left turn Figure 9. Intersection internal evaluation. Figure 9. Intersection internal evaluation. Figure 9. Intersection internal evaluation. Figure 9. Intersection internal evaluation. Figures from 9e–h f 3.2.2. The Traffic Speed Figures from 9e–h f 3.2.2. The Traffic Speed and left turn directions were much slower than the westbound average speed. Consequently, the delays in the eastbound directions were longer, particularly during the morning and evening rush hours. In summary, Table 4 lists all of the key traffic performance parameters of the eight critical directions for the intersection. Table 4. Internal evaluation parameters. Parameter Notation Southbound Northbound Eastbound Westbound For further analysis of the average speed, we compared the distribution of the average speed among different directions of the intersection, as depicted in Figure 10. A clear distinction was observed in the speeds between the straight and left turn directions in each of the southern or northern routes (See Figure 10a). In contrast, there was no large difference between the speeds of the straight and left turn directions for each of the eastern or western routes (See Figure 10b). This suggests that there would be a protected left-turn phase for the south- and northbound flows, causing little interference to the go-straight traffic. However, there was no protected left-turn designed for the east- and westbound flows. Sensors 2019, 19, 2256 little interference t the east- and westb 14 of 20 ned for 14 of 20 ned for (a) (b) Figure 10. Average speed through the intersection (a) the direction of south and north (b) the direction of east and west. Figure 10. Average speed through the intersection (a) the direction of south and north (b) the direction of east and west. (a) (a) (b) Figure 10. Average speed through the intersection (a) the direction of south and north (b) the direction of east and west. Figure 10. Average speed through the intersection (a) the direction of south and north (b) the direction of east and west. 3.2.3. The Ratio of Non-Stop FCD to Stop FCD 3.2.3. The Ratio of Non-Stop FCD to Stop FCD 3.2.3. The Ratio of Non-Stop FCD to Stop FCD 3.2.3. The Ratio of Non-Stop FCD to Stop FCD Figure 11a shows the total number of stop trajectories over the entire day across different directions of the intersection, indicating that the numbers in the south- and northbound straight directions (i.e., 33% and 31%, respectively) were much higher than those in the east- and westbound straight directions (i.e., 3% and 2%, respectively). On the contrary, the numbers of stop trajectories in the left turn of all the north-, south-, east- and westbound directions were similar (i.e., 3.2%, 2.6%, 3.0% and 1.7%, respectively). However, according to the ratio ( i) between the number of non-stop trajectories and that of stop ones, as described in Figure 11b, i was much lower in the left turn of the north- and southbound directions (i.e., 10.11% and 16.18%, respectively) than that of the east- and Figure 11a shows the total number of stop trajectories over the entire day across different directions of the intersection, indicating that the numbers in the south- and northbound straight directions (i.e., 33% and 31%, respectively) were much higher than those in the east- and westbound straight directions (i.e., 3% and 2%, respectively). On the contrary, the numbers of stop trajectories in the left turn of all the north-, south-, east- and westbound directions were similar (i.e., 3.2%, 2.6%, 3.0% and 1.7%, respectively). However, according to the ratio (τi) between the number of non-stop trajectories and that of stop ones, as described in Figure 11b, τi was much lower in the left turn of the north- and southbound directions (i.e., 10.11% and 16.18%, respectively) than that of the east- and westbound directions (i.e., 66.08% and 76.18%, respectively). This indicates that the number of non-stop trajectories was smaller and the traffic conditions were worse in the left turn of the north- and southbound directions than in the east- and westbound directions. Sensors 2019, 19, 2256 the southbound evening (i e 90 15 of 20 ower in th etween th north and southbound straight directions. (a) (b) (c) (d) Figure 11. 3.2.3. The Ratio of Non-Stop FCD to Stop FCD 3.2.3. The Ratio of Non-Stop FCD to Stop FCD The ratio of the number of non-stop cars to the number of stops: (a) The percentage of the number of stops in different directions within NO.2 intersection; (b) the ratio of the number of non stop cars to the number of stops; (c) The i in different directions at NO.2 intersection during th morning peak (7:00–10:00); (d) The i in different directions at NO.2 intersection during the evening peak hours (17:00–19:00) Figure 11. The ratio of the number of non-stop cars to the number of stops: (a) The percentage of the number of stops in different directions within NO.2 intersection; (b) the ratio of the number of non-stop cars to the number of stops; (c) The τi in different directions at NO.2 intersection during the morning peak (7:00–10:00); (d) The τi in different directions at NO.2 intersection during the evening peak hours (17:00–19:00). (b) (a) ( ) (c) ( ) (d) (c) (d) Figure 11. The ratio of the number of non-stop cars to the number of stops: (a) The percentage of th number of stops in different directions within NO.2 intersection; (b) the ratio of the number of non stop cars to the number of stops; (c) The i in different directions at NO.2 intersection during th morning peak (7:00–10:00); (d) The i in different directions at NO.2 intersection during the evenin peak hours (17:00 19:00) Figure 11. The ratio of the number of non-stop cars to the number of stops: (a) The percentage of the number of stops in different directions within NO.2 intersection; (b) the ratio of the number of non-stop cars to the number of stops; (c) The τi in different directions at NO.2 intersection during the morning peak (7:00–10:00); (d) The τi in different directions at NO.2 intersection during the evening peak hours (17:00–19:00). In summary, Table 5 lists all of the number of stops and the ratio of the number of non-stop ca to the number of stop ones of the eight critical directions for the intersection. Figure 11c,d further depict the values of τi during the morning and evening rush hours, respectively. It was observed that τi in the left turn of the north- and southbound directions in these two periods (i.e., 7.25% and 7.48% in the morning and 8.76% and 7.31% in the evening) was lower than that for the entire days (i.e., 10.11% and 16.18% as shown in Figure 11b). 3.2.3. The Ratio of Non-Stop FCD to Stop FCD 3.2.3. The Ratio of Non-Stop FCD to Stop FCD This suggests that in the peak period, the unreasonable timing of signal lights would be unable to match the increasing of traffic flow. Moreover, τi in the northbound straight direction during the morning peak (i.e., 68%) was lower than that in the evening peak (i.e., 115%). Meanwhile, an opposite trend was observed in the southbound straight direction, in which τi is higher in the morning (i.e., 104%) and lower in the evening (i.e., 90%). This indicates the existence of a tidal phenomenon in the traffic flow between the north- and southbound straight directions. In summary, Table 5 lists all of the number of stops and the ratio of the number of non-stop cars to the number of stop ones of the eight critical directions for the intersection. 16 of 20 . d Sensors 2019, 19, 2256 Table 5. Numb 16 of 20 s. d Table 5. Number of stops and the ratio of the number of non-stop cars to the number of stop ones. Parameter Notation Northbound Southbound Eastbound Westbound Through Left Turn Through Left Turn Through Left Turn Through Left Turn NOSF 156,073 16,219 217,461 13,349 14,254 15,164 12,012 8441 Total τi 0.81 0.10 0.79 0.16 0.74 0.66 0.68 0.76 Evening peak τi 0.68 0.07 1.04 0.075 0.92 0.93 1.06 0.96 Morning peak τi 1.15 0.088 0.90 0.073 0.74 0.80 0.36 0.70 3 3 Diagnosis of the Delay Problems for Individual Intersections notation Through Left Turn Through Left Turn Through Left Turn Through Left Tur NOSF 156,073 16,219 217,461 13,349 14,254 15,164 12,012 8441 Total i 0.81 0.10 0.79 0.16 0.74 0.66 0.68 0.76 Evening peak i 0.68 0.07 1.04 0.075 0.92 0.93 1.06 0.96 Morning peak i 1.15 0.088 0.90 0.073 0.74 0.80 0.36 0.70 3.3. Diagnosis of the Delay Problems for Individual Intersections Table 5. Number of stops and the ratio of the number of non-stop cars to the number of stop ones. Through Left Turn Through Left Turn Through Left Turn Through Le NOSF 156,073 16,219 217,461 13,349 14,254 15,164 12,012 3.3. Diagnosis of the Delay Problems for Individual Intersections 3.3. Diagnosis of the Delay Problems for Individual Intersections Figure 12 characterizes the distribution of  (i e th Figure 12 characterizes the distribution of ηi,φ (i.e., the ratio between the flow ratio and the green time ratio) over different directions of the intersection in each of the phases. 3.2.3. The Ratio of Non-Stop FCD to Stop FCD 3.2.3. The Ratio of Non-Stop FCD to Stop FCD Comparison of the ,i   in each direction. Figure 12. Comparison of the ηi,φ in each direction. Figure 12. Comparison of the ,i   in each direction Figure 12. Comparison of the ηi,φ in each direction. Table 6. Relation between the green signal ratio and the flow ratio. Parameter Notation Northbound Southbound Eastbound Westbound Through Left Turn Through Left Turn Through Left Turn Through Left Tu Max Min Max Min Max Min Max Min Max Min Max Min Max Min Max M ,i   0.421 0.271 0.039 0.012 0.581 0.404 0.033 0.014 0.039 0.015 0.040 0.016 0.032 0.006 0.029 0. ,i   1.064 0.740 0.120 0.030 2.131 1.015 0.083 0.044 0.191 0.047 0.099 0.039 0.178 0.016 0.075 0. Mean Med Mean Med Mean Med Mean Med Mean Med Mean Med Mean Med Mean M  Table 6. Relation between the green signal ratio and the flow ratio. Parameter Notation Northbound Southbound Eastbound Westbound Through Left Turn Through Left Turn Through Left Turn Through Left Turn Max Min Max Min Max Min Max Min Max Min Max Min Max Min Max Min ωi,φ 0.421 0.271 0.039 0.012 0.581 0.404 0.033 0.014 0.039 0.015 0.040 0.016 0.032 0.006 0.029 0.010 ηi,φ 1.064 0.740 0.120 0.030 2.131 1.015 0.083 0.044 0.191 0.047 0.099 0.039 0.178 0.016 0.075 0.029 Mean Med Mean Med Mean Med Mean Med Mean Med Mean Med Mean Med Mean Med ωi,φ 0.344 0.357 0.029 0.031 0.497 0.487 0.025 0.025 0.028 0.029 0.030 0.032 0.024 0.025 0.019 0.020 ηi,φ 0.928 0.946 0.075 0.076 1.357 1.295 0.064 0.065 0.127 0.138 0.078 0.081 0.112 0.118 0.049 0.052 3.2.3. The Ratio of Non-Stop FCD to Stop FCD 3.2.3. The Ratio of Non-Stop FCD to Stop FCD It should be noted that ηi,φ values of the north- and southbound straight directions were greater than those in other directions. Table 6 further lists the specific parameter values of ωi,φ and ηi,φ in each phase of each time scheme. It indicates that the minimum ηi,φ values in north- and southbound straight directions phase were greater than that in the other phases in all the timing schemes. This implies that the effective green time of phase of the north- and southbound straight direction would be much shorter than it should be, which leads to the longer delay in the corresponding directions of the intersection. g ,i   ( , green time ratio) over different directions of the intersection in each of the phases. It should be noted that ,i   values of the north- and southbound straight directions were greater than those in other directions. Table 6 further lists the specific parameter values of ,i   and ,i   in each phase of each time scheme. It indicates that the minimum ,i   values in north- and southbound straight directions phase were greater than that in the other phases in all the timing schemes. This implies that the effective green time of phase of the north- and southbound straight direction would be much shorter than it should be, which leads to the longer delay in the corresponding directions of the intersection. Figure 12. Comparison of the ,i   in each direction. Table 6. Relation between the green signal ratio and the flow ratio. Parameter Notation Northbound Southbound Eastbound Westbound Through Left Turn Through Left Turn Through Left Turn Through Left Tur Max Min Max Min Max Min Max Min Max Min Max Min Max Min Max M ,i   0.421 0.271 0.039 0.012 0.581 0.404 0.033 0.014 0.039 0.015 0.040 0.016 0.032 0.006 0.029 0.0 ,i   1.064 0.740 0.120 0.030 2.131 1.015 0.083 0.044 0.191 0.047 0.099 0.039 0.178 0.016 0.075 0.0 Mean Med Mean Med Mean Med Mean Med Mean Med Mean Med Mean Med Mean M  0 344 0 357 0 029 0 031 0 497 0 487 0 025 0 025 0 028 0 029 0 030 0 032 0 024 0 025 0 019 0 0 Figure 12. Comparison of the ηi,φ in each direction. Table 6. 3.2.3. The Ratio of Non-Stop FCD to Stop FCD 3.2.3. The Ratio of Non-Stop FCD to Stop FCD Relation between the green signal ratio and the flow ratio. Parameter Notation Northbound Southbound Eastbound Westbound Through Left Turn Through Left Turn Through Left Turn Through Left Turn Max Min Max Min Max Min Max Min Max Min Max Min Max Min Max Min ωi,φ 0.421 0.271 0.039 0.012 0.581 0.404 0.033 0.014 0.039 0.015 0.040 0.016 0.032 0.006 0.029 0.010 ηi,φ 1.064 0.740 0.120 0.030 2.131 1.015 0.083 0.044 0.191 0.047 0.099 0.039 0.178 0.016 0.075 0.029 Mean Med Mean Med Mean Med Mean Med Mean Med Mean Med Mean Med Mean Med ωi,φ 0.344 0.357 0.029 0.031 0.497 0.487 0.025 0.025 0.028 0.029 0.030 0.032 0.024 0.025 0.019 0.020 ηi,φ 0.928 0.946 0.075 0.076 1.357 1.295 0.064 0.065 0.127 0.138 0.078 0.081 0.112 0.118 0.049 0.052 Figure 12. Comparison of the ,i   in each direction. Table 6. Relation between the green signal ratio and the flow ratio. Parameter Notation Northbound Southbound Eastbound Westbound Through Left Turn Through Left Turn Through Left Turn Through Left Tu Max Min Max Min Max Min Max Min Max Min Max Min Max Min Max M ,i   0.421 0.271 0.039 0.012 0.581 0.404 0.033 0.014 0.039 0.015 0.040 0.016 0.032 0.006 0.029 0. ,i   1.064 0.740 0.120 0.030 2.131 1.015 0.083 0.044 0.191 0.047 0.099 0.039 0.178 0.016 0.075 0. Mean Med Mean Med Mean Med Mean Med Mean Med Mean Med Mean Med Mean M i   0.344 0.357 0.029 0.031 0.497 0.487 0.025 0.025 0.028 0.029 0.030 0.032 0.024 0.025 0.019 0. Figure 12. Comparison of the ηi,φ in each direction. Table 6. Relation between the green signal ratio and the flow ratio. Parameter Notation Northbound Southbound Eastbound Westbound Through Left Turn Through Left Turn Through Left Turn Through Left Turn Max Min Max Min Max Min Max Min Max Min Max Min Max Min Max Min ωi,φ 0.421 0.271 0.039 0.012 0.581 0.404 0.033 0.014 0.039 0.015 0.040 0.016 0.032 0.006 0.029 0.010 ηi,φ 1.064 0.740 0.120 0.030 2.131 1.015 0.083 0.044 0.191 0.047 0.099 0.039 0.178 0.016 0.075 0.029 Mean Med Mean Med Mean Med Mean Med Mean Med Mean Med Mean Med Mean Med ωi,φ 0.344 0.357 0.029 0.031 0.497 0.487 0.025 0.025 0.028 0.029 0.030 0.032 0.024 0.025 0.019 0.020 ηi,φ 0.928 0.946 0.075 0.076 1.357 1.295 0.064 0.065 0.127 0.138 0.078 0.081 0.112 0.118 0.049 0.052 Figure 12. ,i  i   0.928 0.946 0.075 0. 4. Discussion and Conclusions ,i   4. Discussion and Conclusions In this study, a novel traffic grid algorithm was proposed, which is based on the FCD data but makes the map-matching process for signalized intersections more efficient. Furthermore, it provided an accurate estimation of traffic operation states at the intersections and an efficient traffic performance evaluation for signalized intersections at the network level, and can be applied to diagnose the specific problems for individual intersections. Sensors 2019, 19, 2256 17 of 20 17 of 20 The traditional data-collecting devices cover only fixed points of the traffic network, such as loop detectors, which are impossible to produce reliable information about travel time within the network [45]. In comparison, based on the massive amount of FCD data that are highly spatial-temporal detailed, their 3s sampling rate guarantees the accurate estimation of these intersections’ operation parameters. This study selected three kinds of time dimensions (i.e., 5 min, 15 min, 30 min) as the time slices to describe the intersections’ operation states. As for the traffic analysis region of defined intersection, Zhang et al. defined a fixed one with a 200 m length from the center of an intersection to a 100 m distance from upstream and downstream directions [41]. The fixed analysis region may cause an inaccurate estimation of the traffic parameters because the upstream queue lengths might be frequently beyond the 100 m limit, especially for large-scale intersections. In this study, the differences of intersections’ configurations and the spatial-temporal FCD’ distributions were considered, and the region sizes are variable corresponding to the actual traffic performance, which guaranteed the assurance of the veracity of the arithmetic. g y In previous studies, only one or two traffic parameters (e.g., delay and stop rate) were used for intersection performance analyses, which were insufficient to explore the traffic operation performance deeply [46]. Using the radar figures (e.g., Figure 9), we combined the three traffic parameters (i.e., time delay, traffic flow, average speed) to estimate the traffic intersection operation state and evaluate the intersections’ levels of service. Thus, the intersections’ operation performances in the network can be sorted, and the ones with the worst performance can be identified. The intersections with the serious delay problems can be further investigated in terms of the traffic condition in different directions. Author Contributions: Conceptualization, X.Y.; methodology, D.C., X.Y. and J.Z.; software, D.C. and L.W.; validation, D.C., X.Y., F.L., X.L., L.W. and J.Z.; formal analysis, D.C.; investigation, X.L.; resources, X.Y.; data curation, D.C.; writing—original draft preparation, D.C., X.Y. and F.L.; writing—review and editing, D.C., X.Y., F.L., X.L. and L.W.; visualization, D.C.; supervision, X.Y.; project administration, D.C.; funding acquisition, X.Y. Acknowledgments: This work is financially supported by Fundamental Research Funds for Beijing Jiaotong University (2019JBZ003). Many thanks are extended to the Institute of Policy Studies, DiDi Company for providing the data in this study. Conflicts of Interest: The authors declare that there is no conflict of interest in any aspect of the data collection, analysis, or the funding received regarding the publication of this paper. ,i  i   0.928 0.946 0.075 0. 4. Discussion and Conclusions 18 of 20 18 of 20 Sensors 2019, 19, 2256 References 1. Nielsen, O.A.; Frederiksen, R.D.; Simonsen, N. Using expert system rules to establish data for intersections and turns in road networks. Int. Trans. Oper. Res. 1998, 5, 569–581. [CrossRef] 2. Homburger, W.S.; Hall, J.W.; William, R.R.; Edward, C.S.; Michelle, D.; Loretta, H.; John, J.L.; Matthew, R.; Vernon, H.W. Fundamentals of Traffic Engineering; Institute of Transportation Studies, University of California: Berkeley, CA, USA, 2007. 3. Wang, Y.; Zheng, Y.; Xue, Y. Travel time estimation of a path using sparse trajectories. In Proceedings of the 20th ACM SIGKDD International Conference on Knowledge Discovery and Data Mining, San Francisco, CA, USA, 24–27 August 2014; ACM: New York, NY, USA, 2014. . Heidemann, D. Queue length and delay distributions at traffic signals. Transp. Res. Part B Methodol. 1994 377–389. [CrossRef] . Denney, R.W., Jr.; Curtis, E.; Olson, P. The national traffic signal report card. ITE J. 2012, 82, 22–26. 6. Liu, X.; Lu, F.; Zhang, H.; Qiu, P. Intersection delay estimation from floating car data via principal curves: A case study on Beijing’s road network. Front. Earth Sci. 2013, 7, 206–216. [CrossRef] 7. Xiang, J.; Chen, Z. An adaptive traffic signal coordination optimization method based on vehicle-to-infrastructure communication. Clust. Comput. 2016, 19, 1503–1514. [CrossRef] 8. Cheng, J.; Wu, W.; Cao, J.; Li, K. Fuzzy group-based intersection contr transportations. IEEE Trans. Ind. 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Vehicular ad hoc networks enabled traffic controller for removing traffic lights in isolated intersections based on integer linear programming. IET Intell. Transp. Syst. 2012, 6, 115–123. [CrossRef] 13. Younes, M.B.; Boukerche, A. Intelligent traffic light controlling algorithms using vehicular networks. IEEE Trans. Veh. Technol. 2016, 65, 5887–5899. [CrossRef] 14. Hu, J.; Park, B.B.; Lee, Y. References Coordinated transit signal priority supporting transit progression under connected vehicle technology. Transp. Res. Part C Emerg. Technol. 2015, 55, 393–408. [CrossRef] 15. He, Q.; Head, K.L.; Ding, J. Multi-modal traffic signal control with priority, signal actuation and coordination. Transp. Res. Part C Emerg. Technol. 2014, 46, 65–82. [CrossRef] 6. Tomescu, O.; Moise, I.M.; Alina, S.E.; Bă¸tro¸s, I. Adaptive traffic light control system using ad-hoc vehic communications network. Upb Sci. Bull. Ser. D 2012, 74, 67–78. 17. Chou, L.-D.; Deng, B.-T.; Li, D.C.; Kuo, K.-W. A passenger-based adaptive traffic signal control mechanism in Intelligent Transportation Systems. In Proceedings of the 2012 IEEE 12th International Conference on ITS Telecommunications, Taipei, Taiwan, 5–8 November 2012. 18. Zhang, W.; Tan, G.; Ding, N.; Wang, G. Traffic Congestion Evaluation and Signal Timing Optimization Based on Wireless Sensor Networks: Issues, Approaches and Simulation. Math. Probl. Eng. 2012, 2012, 573171. [CrossRef] 18. Zhang, W.; Tan, G.; Ding, N.; Wang, G. Traffic Congestion Evaluation and Signal Timing Optimization Based on Wireless Sensor Networks: Issues, Approaches and Simulation. Math. Probl. Eng. 2012, 2012, 573171. [CrossRef] 19. Yildirimoglu, M.; Geroliminis, N. Experienced travel time prediction for congested freeways. Transp. Res. 19. Yildirimoglu, M.; Geroliminis, N. Experienced travel time prediction for congested freeways. Transp. Res. Part B Methodol. 2013, 53, 45–63. [CrossRef] 20. Laval, J.; Chen, D.; Amer, K.B.; Guin, A. Evolution of oscillations in congested traffic: Improved estimation method and additional empirical evidence. Transp. Res. Rec. 2009, 2124, 194–202. [CrossRef] 20. Laval, J.; Chen, D.; Amer, K.B.; Guin, A. Evolution of oscillations in congested traffic: Improved estimation method and additional empirical evidence. Transp. Res. Rec. 2009, 2124, 194–202. [CrossRef] 21. Wieczorek, J.; Fernández-Moctezuma, R.J.; Bertini, R.L. Techniques for validating an automatic bottleneck 21. Wieczorek, J.; Fernández-Moctezuma, R.J.; Bertini, R.L. Techniques for validating an automatic bo detection tool using archived freeway sensor data. Transp. Res. Rec. 2010, 2160, 87–95. [CrossRef] 21. Wieczorek, J.; Fernández-Moctezuma, R.J.; Bertini, R.L. Techniques for validating an automatic bottleneck detection tool using archived freeway sensor data. Transp. Res. Rec. 2010, 2160, 87–95. [CrossRef] 22. Cui, J.; Liu, F.; Janssens, D.; An, S.; Wets, G.; Cools, M. Detecting urban road network accessibility problems 22. Cui, J.; Liu, F.; Janssens, D.; An, S.; Wets, G.; Cools, M. Detecting urban road network accessibility p using taxi GPS data. J. Transp. Geogr. 2016, 51, 147–157. [CrossRef] 23. Cui, J.; Liu, F.; Hu, J.; Janssens, D.; Wets, G.; Cools, M. ,i  i   0.928 0.946 0.075 0. 4. Discussion and Conclusions It was found that there was a significant difference in the average speed between the straight and the left turn directions when unprotected left-turn phases were applied, which is consistent with the studies of Angell et al. and Wang et al. [47,48]. In addition, the ratio of non-stop to stop FCD (τi) also exhibits variations across different times of the day and in different directions. Particularly, a tidal phenomenon of traffic flow during the morning and evening peak hours was discovered. Traffic engineers should take the tidal phenomenon into consideration when designing traffic signal control strategies. For the individual intersections, the traffic delay problem can be further diagnosed based on the parameter ηi,φ. The value of the parameter is either larger or smaller than one, indicating imbalanced signal phasing design issues for the intersections, which can be utilized as an efficient tool for individual intersections’ signal phase readjustment. Owing to unavailability of loop detector data for the case study, we could not examine the floating cars’ penetration ratio in the total traffic. However, this study focuses on the traffic signal intersection performance evaluation. The huge numbers of FCD can sufficiently reflect the historical traffic conditions and accurately provide travel speed information. Therefore, the comparative results of levels of service between the intersections in the traffic networks and the diagnoses of signal phasing design issues for individual intersections should be reliable. In future work, if the loop detector data are available, it is suggested to consider the floating cars’ penetration ratio in total traffic in order to provide more detailed methods for the real-time cases that the samples of FCD are insufficient during some cycles. Author Contributions: Conceptualization, X.Y.; methodology, D.C., X.Y. and J.Z.; software, D.C. and L.W.; validation, D.C., X.Y., F.L., X.L., L.W. and J.Z.; formal analysis, D.C.; investigation, X.L.; resources, X.Y.; data curation, D.C.; writing—original draft preparation, D.C., X.Y. and F.L.; writing—review and editing, D.C., X.Y., F.L., X.L. and L.W.; visualization, D.C.; supervision, X.Y.; project administration, D.C.; funding acquisition, X.Y. Acknowledgments: This work is financially supported by Fundamental Research Funds for Beijing Jiaotong University (2019JBZ003). Many thanks are extended to the Institute of Policy Studies, DiDi Company for providing the data in this study. Conflicts of Interest: The authors declare that there is no conflict of interest in any aspect of the data collection, analysis, or the funding received regarding the publication of this paper. References Identifying mismatch between urban travel demand and transport network services using GPS data: A case study in the fast growing Chinese city of Harbin. Neurocomputing 2016, 181, 4–18. [CrossRef] Sensors 2019, 19, 2256 19 of 20 19 of 20 24. Bauza, R.; Gozálvez, J. Traffic congestion detection in large-scale scenarios using vehicle-to-vehicle communications. J. Netw. Comput. Appl. 2013, 36, 1295–1307. [CrossRef] 25. Turksma, S. The Various Uses of Floating Car Data, Road transport information and control. In Proceedings of the Tenth International Conference on (Conf. Publ. No. 472), London, UK, 24–27 July 2000. 26. Miwa, T.; Tawada, Y.; Yamamoto, T.; Morikawa, T. En-route updating methodology of travel time prediction using accumulated probe-car data. 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An Exploration of Driver Behavior During Turns at Intersections (for Drivers in Different Age Groups); National Surface Transportation Safety Center for Excellence (NSTSCE, VTTI): Blacksburg, VA, USA, 2015. © 2019 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/). 48. Wang, X.; Zhao, D.; Peng, H.; LeBlanc, D.J. Analysis of unprotected intersection left-turn conflicts based on naturalistic driving data. In Proceedings of the 2017 IEEE Intelligent Vehicles Symposium (IV), Redondo Beach, CA, USA, 11–14 June 2017. References In Proceedings of the 2013 IEEE International Conference on Green Computing and Communications and IEEE Internet of Things and IEEE Cyber, Physical and Social Computing, Beijing, China, 20–23 August 2013; pp. 512–517. 6. DiDi. 2017. Available online: http://www.xiaojukeji.com/en/company.html (accessed on 30 August 201 37. Shih, G. China taxi apps Didi Dache and Kuaidi Dache announce $6 billion tie-up. Reuters, 14 February 2015. 38. Deng, B.; Steve, D.; Vassilis, Z.; Jin, Y. Estimating traffic delays and network speeds from lowfrequency GPS 7. Shih, G. China taxi apps Didi Dache and Kuaidi Dache announce $6 billion tie-up. Reuters, 14 February 2 Deng, B.; Steve, D.; Vassilis, Z.; Jin, Y. Estimating traffic delays and network speeds from lowfrequency GPS taxis traces for urban transport modelling. Eur. J. Transp. Infrast. Res. 2015, 15, 639–661. 39. Zhao, X.-J.; Zhao, J.-Y. Research on model of resource management for traffic grid. Procedia Eng. 2011, 15, 1476–1480. [CrossRef] 40. Sun, L. An approach for Intersection Delay Estimate Based on Floating Vehicles. Master’s Thesis, Beijing University of Technology, Beijing, China, 2007. (In Chinese). 41. Zhang, H.; Lu, F.; Zhou, L.; Duan, Y. 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Kong, X.; Xu, Z.; Shen, G.; Wang, J.; Yang, Q.; Zhang, B. Urban traffic congestion estimation and prediction based on floating car trajectory data. Future Gener. Comput. Syst. 2016, 61, 97–107. [CrossRef] Sensors 2019, 19, 2256 20 of 20 20 of 20 47. Angell, L.; Aitch, S.; Antin, J.; Wotring, B. References An Exploration of Driver Behavior During Turns at Intersections (for Drivers in Different Age Groups); National Surface Transportation Safety Center for Excellence (NSTSCE, VTTI): Blacksburg, VA, USA, 2015. 47. Angell, L.; Aitch, S.; Antin, J.; Wotring, B. An Exploration of Driver Behavior During Turns at Intersections (for Drivers in Different Age Groups); National Surface Transportation Safety Center for Excellence (NSTSCE, VTTI): Blacksburg, VA, USA, 2015. 48. Wang, X.; Zhao, D.; Peng, H.; LeBlanc, D.J. Analysis of unprotected intersection left-turn conflicts based on naturalistic driving data. In Proceedings of the 2017 IEEE Intelligent Vehicles Symposium (IV), Redondo Beach, CA, USA, 11–14 June 2017. 48. Wang, X.; Zhao, D.; Peng, H.; LeBlanc, D.J. Analysis of unprotected intersection left-turn conflicts based on naturalistic driving data. In Proceedings of the 2017 IEEE Intelligent Vehicles Symposium (IV), Redondo Beach, CA, USA, 11–14 June 2017. © 2019 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/W4386768295
https://link.springer.com/content/pdf/10.1140/epjs/s11734-023-00978-3.pdf
English
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Thought experiments in electromagnetic theory and the ordinary Hall effect
˜The œEuropean physical journal. Special topics
2,023
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a e-mail: maresjj@fzu.cz b e-mail: spicka@fzu.cz (corresponding author) c e-mail: hubik@fzu.cz Thought experiments in electromagnetic theory and the ordinary Hall effect te of Physics of the Czech Academy of Sciences, Cukrovarnick´a 10, 162 00 Prague 6, Czech Repub Received 13 March 2023 / Accepted 22 August 2023 / Published online 15 September 2023 © The Author(s) 2023 Received 13 March 2023 / Accepted 22 August 2023 / Published online 15 September 2023 © The Author(s) 2023 Abstract Thought experiments are effective tools of theoretical physics, and historically they have led to the discovery of many useful ideas and relations. Yet they also give rise to paradoxes that persist for long periods of time despite enormous efforts to resolve them. A problem par excellence, one that falls within the realm of classical electrodynamics and has been investigated exclusively by means of thought experiments, is the famous problem of hidden momentum. It concerns the conversion of the electromagnetic momentum generated by static electromagnetic fields into momentum of a non-electromagnetic nature and the resulting momentum balance. As a rule, this effect, which takes place in ponderable systems, is very subtle, of the order of ∼1/c2; thus, until now it has only been studied theoretically and has never been demonstrated experimentally. The main subject of this paper is the analysis of a robust and experimentally well-established phenomenon operating in static electromagnetic fields—the ordinary Hall effect—which has been interpreted anew in terms of the Poynting vector and proven to be sufficiently sensitive for direct comparison with typical quantitative estimates encountered in the hidden momentum problem. Confrontation of hypothetical models with a real experiment enables us to formulate the general conditions and rules that should be observed when designing thought experiments in classical electrodynamics. THE EUROPEAN PHYSICAL JOURNAL SPECIAL TOPICS THE EUROPEAN PHYSICAL JOURNAL SPECIAL TOPICS Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 https://doi.org/10.1140/epjs/s11734-023-00978-3 Review a e-mail: maresjj@fzu.cz b 1 Introduction arguments by Bartoli [4]; nonetheless, obtaining exper- imental proof of their existence was no easy task. The first serious attempts to experimentally prove the exis- tence of the pressure of light, treated as an electromag- netic wave, were made at the beginning of the twentieth century by Lebedew [5] and, somewhat later, by Nichols and Hull [6]. However, the first credible evidence would not appear until 1924 and is attributed to Golsen [7]. Today, after numerous experiments [8] (and the inven- tion of optical tweezers [9] in the 1980s, for example), there is no question about the existence of the pondero- motive effect of electromagnetic waves. Classical electrodynamics is traditionally regarded as a well-established and complete area of physics, yet there are some difficult questions that have persisted unanswered for more than ∼120 years and have thus far been the subject of much controversy and discus- sion. One such class of problems involves the trans- fer of energy and momentum by electromagnetic field. The formal description of these processes was essen- tially given by Poynting [1], while the importance of electromagnetic momentum for the comprehension of various complex effects was emphasized by Thomson [2]. g An essentially different and much more difficult sit- uation, however, occurs in cases where the electromag- netic fields are static. The behavior of the Poynting vector and related electromagnetic momentum is quite counterintuitive and reveals almost mysterious features. For example, a battery and a piece of permanent mag- net placed together in a plastic jar [10] should gener- ate a permanent energy flow in the surrounding space with uncompensated momentum. In another example, we can estimate the flow of electromagnetic energy aris- ing from static electric and magnetic fields existing near the surface of the earth [11]. The mean value of the vertical fair-weather electric field is approximately E = −120 V/m, and the mean value of the horizontal Among such effects, the mechanical forces arising from the interaction of light with opaque surfaces play an important role. They are now viewed as evidence that quickly oscillating electromagnetic waves carry momentum. The existence of such forces was previously predicted within the framework of electrodynamics by Maxwell ([3], sec. 792) and based on thermodynamic 3 12 3 3530 Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 Fig. 1 Illustration of the problem of a charged magnetized sphere—a model of the earth’s globe [12]. 1 Introduction Among other things it is due to an enormously small factor ∼ 1/c2 (≈1.1 × 10−17 s2/m2, c is the speed of light, see Eq. (5) below) controlling the relation between the elec- tromagnetic energy flow and the corresponding electro- magnetic momentum representing possible observable forces. Thus, we are dealing with very tiny effects or, in the words of some authors, relativistic effects that are extremely difficult to demonstrate experimentally. As far as we know, the only credible observation of an exchange between angular electromagnetic momentum generated by independent quasi-static electric and mag- netic fields and a ponderable system is attributed to Graham and Lahoz of the University of Toronto (see [13] and references therein). There is, however, a com- plete lack of experimental evidence for a similar inter- action between linear electromagnetic momentum and ponderable matter. That is why in this area of elec- trodynamics we must turn to thought experiments and theoretical speculations with varying degrees of reliabil- ity. Of course, such a state of the art provides a fertile substrate for many controversies, arbitrary constructs, and the introduction of rather peculiar concepts as well. T li i hi i fl hi h i id l d 1 Introduction Using formula (1) below, we determine that near the surface of the earth there must be a massive permanent east- ward energy flow with a density of S = 3.8 kW/m2 and having no apparent effect. In theoretically address- ing the similar problem of a charged magnetized sphere (see Fig. 1), Heaviside [12] commented on the existence of such a flow with the words: “What is the use of it? On the other hand, what harm does it do?” What is the main reason that such a seemingly significant phenomenon is not observed experimentally? Among other things it is due to an enormously small factor ∼ 1/c2 (≈1.1 × 10−17 s2/m2, c is the speed of light, see Eq. (5) below) controlling the relation between the elec- tromagnetic energy flow and the corresponding electro- magnetic momentum representing possible observable forces. Thus, we are dealing with very tiny effects or, in the words of some authors, relativistic effects that are extremely difficult to demonstrate experimentally. As far as we know, the only credible observation of an exchange between angular electromagnetic momentum generated by independent quasi-static electric and mag- netic fields and a ponderable system is attributed to Graham and Lahoz of the University of Toronto (see [13] and references therein). There is, however, a com- plete lack of experimental evidence for a similar inter- action between linear electromagnetic momentum and ponderable matter. That is why in this area of elec- trodynamics we must turn to thought experiments and theoretical speculations with varying degrees of reliabil- ity. Of course, such a state of the art provides a fertile substrate for many controversies, arbitrary constructs, and the introduction of rather peculiar concepts as well. component of the magnetic field is B = 40 μT. Using formula (1) below, we determine that near the surface of the earth there must be a massive permanent east- ward energy flow with a density of S = 3.8 kW/m2 and having no apparent effect. In theoretically address- ing the similar problem of a charged magnetized sphere (see Fig. 1), Heaviside [12] commented on the existence of such a flow with the words: “What is the use of it? On the other hand, what harm does it do?” What is the main reason that such a seemingly significant phenomenon is not observed experimentally? 1 Introduction B is the horizontal component of the terrestrial magnetic field and E the vector of the fair-weather electric field field, we have been looking for effects operating in quasi- static EM fields that are sensitive enough to reveal essential features of momentum exchanges. Therefore, in this paper we reanalyze the ordinary Hall effect, a well-known and robust phenomenon oper- ating nominally in static electromagnetic fields, where the conversion of electromagnetic energy and momen- tum can be quantitatively evaluated and compared with experimental data. We are convinced that a detailed study of this phenomenon can be very helpful in the resolution of some ambiguities in contemporary electro- dynamics and may also contribute to the elucidation of the experimentally poorly manageable problem of so- called hidden momentum. The present paper uses SI units throughout and is organized as follows: we begin by providing a brief for- mal description of flows of electromagnetic power and momentum as well as a concise overview of the prob- lem of hidden momentum in electromechanical systems within a static electromagnetic field; next, we review the basics of the phenomenology of the Hall effect applied to the simplest possible metallic system with electrons as exclusive charge carriers, followed by a short critical discussion on the traditional interpreta- tion of the ordinary Hall effect in terms of the Lorentz force; we then move on to a rather uncommon analysis of the Hall effect using concepts of the Poynting vector and linear electromagnetic momentum of static elec- tromagnetic fields. In this section, which represents the core of the paper, we use quantitative estimates to show that the conversion of electromagnetic momentum into mechanical momentum and vice versa can be depend- ably traced. In Sect. 7, the general concepts of thought experiment and isolated system are discussed in connec- tion with real Hall effect measurements. In addition, the significance of the time factor represented by the period of system assembly is also addressed. Our conclusions are then briefly summarized in the last section. For the sake of convenience and the flow of the text, some data and quantitative estimates appear in the Appendix. Fig. 1 Illustration of the problem of a charged magnetized sphere—a model of the earth’s globe [12]. B is the horizontal component of the terrestrial magnetic field and E the vector of the fair-weather electric field component of the magnetic field is B = 40 μT. 123 Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 3531 is very useful for describing quickly oscillating electro- magnetic waves. Application of formula (1) to static fields is sometimes considered to be even unphysical [16]. Namely it may happen that S is generally non- zero, while its divergence identically vanishes (S ̸= 0, div S = 0). In such a case we can thus imagine that the electromagnetic energy circulates persistently in a closed path without any apparent effect. According to Jeans, [16] “it is difficult to believe that this predicted circulation of energy can have any physical reality”. Of a somewhat different opinion, however, was Feynman [17], who claimed that such a “mystic circulating flow of energy, which is seemed ridiculous, is absolutely nec- essary. There is really momentum flow needed to main- tain the (angular) momentum conservation of the whole world”. Considering formula (1), this quantity may be alter- natively expressed as. g = ε0(E × B), (6) (6) where ε0 is the permittivity of vacuum (ε0 = 8.85 × 10−12 F/m). Applying Eq. (2) to the case of purely static electro- magnetic fields, e.g. to a crossed electric and magnetic field, the time derivative of the Poynting vector ˙S will be identically zero, meaning that there will be no force exerted by these fields on, and no momentum imparted to, the electronic subsystem. However trivial this con- clusion may sound, it is crucial for our further consid- erations. For example, the macroscopic systems at rest submitted to the static electromagnetic fields, where ˙S = 0 identically, does not, according to Eq. (2), interact with these fields, although its parts, i.e. electrons, may “feel” these fields. For the same reason, there is also no integral electromagnetic momentum imparted to the system or stored within it in some form. Incidentally, this fact fully accounts for the absence of mechanical effects induced by the electromagnetic energy flow in Heaviside’s problem mentioned above. Since the inter- actions may take place only if the Poynting vector enter- ing the system is changing in time ( ˙S ̸= 0), it is only the period during which the external fields are set into their final static state that is decisive for correct analysis of systems in static electromagnetic fields. This period (or process) will be tentatively called the period of system assembly (cf. [22]). Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 The key significance of this time factor for correct interpretation of systems submitted to static electromagnetic fields is discussed in detail in Sect. 7. To resolve problems of this type satisfactorily, one has to be more specific about relations between electromag- netic forces and electromagnetic energy fluxes existing under various conditions. Surely there is an intimate connection between the volume force F exerted by an electromagnetic field on a system of charged particles, e.g. electrons (also, of course, on ponderable matter containing these electrons) and the flow of electromag- netic energy penetrating this system. According to H. A. Lorentz [18, 19], putting aside for a while the contact stresses mediated by the surface of the system, force F is given by the following integral taken over the volume V of the whole system: F = −1 c2  V ˙S dV. (2) (2) Here, ˙S is the time derivative of the Poynting vector incident to the volume element dV , (cf. also [20]). This fundamental equation may be rewritten using a concept of quantity introduced by Abraham [21] under the name of electromagnetic momentum G: As will be further shown, the simple fact that an electromagnetic field may carry momentum, inevitably entering into the momentum balance of electromechan- ical systems, frequently generates problems and para- doxes that have yet to be satisfactorily resolved, includ- ing the problem of hidden momentum; therefore, the following section looks at some leading opinions con- cerning this puzzling entity and related concepts. G = −1 c2  V S dV. (3) (3) The law of a purely electromagnetic force exerted on a ponderable electronic system then reads. The law of a purely electromagnetic force exerted on a ponderable electronic system then reads. 2 Description of static electromagnetic fields The conventional physical meaning of the Poynting vec- tor, which in media with no intrinsic magnetization is defined by the formula [1, 14, 15] S = 1 μ0 (E × B), (1) (1) where E and B are vectors of an electric and a magnetic field, respectively, in a given point and μ0 is the perme- ability of vacuum (μ0 = 4π × 10−7 H/m), is the den- sity of electromagnetic power flow measured in W/m2. As mentioned above, the Poynting vector is only sel- domly applied to static electromagnetic fields, but it To eliminate this serious flaw, which is evidently due to the absence of relevant experimenta crucis in this 123 123 Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 3 Hidden momentum F = −˙G, (4) (4) In his estimable papers [2, 23], Thomson investigated the behavior of the momentum of an electric (i.e. elec- tromagnetic) field corresponding to various configura- tions of electric charges. Studying the momentum bal- ance of these systems, he produced a remarkable para- doxical result, namely that an electromagnetic system at rest may contain non-zero electromagnetic momen- tum, and in doing so launched a lively debate that con- tinues in the present day. His further computations give rise to the possibility of constructing an electromagnetic (bootstrap) spaceship, a contrivance that would propel itself in some direction without transferring momen- tum to its surroundings [24]. He also discovered that where ˙G again means the time derivative of electro- magnetic momentum. This equation is, however, in full accord with the law of force known from classical mechanics, thus it can be inferred that the electromag- netic momentum is of the same nature as the mechan- ical one. Equation (3) can also be reformulated locally. For the volume density of electromagnetic momentum g we obtain the following simple relation: g = S c2 . (5) (5) 12 3 3 Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 3532 charges and currents and corresponding static electro- magnetic fields are credited to Calkin [30, 31]. Con- sequently, separating field net momentum and source net momentum requires a description of electromag- netic momenta that is slightly different than the one used in Sect. 2. While electrostatic fields are as usu- ally described by the Coulomb law in terms of charge density ρ, the static magnetic field generated by the stationary distribution of electric currents is defined by vector potential Λ: the angular electromagnetic momentum of a magnetic monopole plus an electric point-charge is independent of the separation between the two, anticipating in this way Dirac’s model of electron spin [25]. Essentially the same conclusion was later arrived at by Page [26], who in connection with Gunn’s theory of double star forma- tion, which was popular at the time, investigated the behavior of ionic atmospheres in static electromagnetic fields. The subject experienced a boom in the 1960s when Shockley and James [27] interpreted their highly ide- alized conceptual thought experiment using a special model device involving a combination of electrostatic forces and momenta induced by magnetic dipoles of either a Gilbertian or Amperian nature. 3 Hidden momentum Among other things, they showed that an electrically neutral current loop in an electric field experiences a special force, and that a body whose center is at rest can carry a non- zero mechanical momentum. These surprising discov- eries compelled them to coin the term hidden momen- tum, which they associated with the lacking mechan- ical momentum balancing electromagnetic momentum in systems at rest. They conjectured that the effect was due to the presence of reactive forces, likely of a rel- ativistic origin, acting on the sources of the external fields. Λ = μ0 4π  V i r dV. (7) (7) Since qΛ(x) physically represents the linear momen- tum acquired by the electromagnetic field when charge q is brought from infinity to point x, we can write the linear electromagnetic momentum within a certain vol- ume ν as. G =  V ρΛdV. (8) (8) Nonetheless, this formula, which provides an alter- native to (3), directly connects the electromagnetic momentum with characteristics of the sources. If we now apply the center-of-mass/energy theorem, we find that the absolute magnitude of linear electromagnetic momentum (8) and that of the mechanical momen- tum of the sources must be equal and opposite to one another; however, when the source current realized by a circulating charge (i.e. an Amperian loop) is treated not classically but in the framework of special relativ- ity, this argument is shown to be not exactly valid. This is because in relativistic mechanics, the mass of a charge carrier varies with velocity, so the momentum is no longer directly proportional to it. Their pioneering paper was followed by one from Coleman and Van Vleck [28], in which the authors investigated a similar hypothetic system consisting of an electric point charge and a magnet. Their computa- tions, which were model independent, showed that if the magnetic moment is changed, the magnet exerts a force on the distant electric charge without apparent back action. Since evidence for this was prevailingly given in terms of the relativistic center-of-mass/energy theo- rem, they considered such a discrepancy in the action- reaction balance, which was in full agreement with Shockley and James, a relativistic correction. It should be stressed that the relativistic center-of-mass/energy theorem traditionally provides a basis for many argu- mentations concerning the problem of hidden momen- tum. Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 A key point of his argumen- tation was a special lemma derived from the center-of- mass/energy theorem given above, namely: / Any finite stationary distribution of matter has zero total momentum. In stationary situations, where the Amperian loop carrying constant current is placed in a static external electric field E, the momentum of the resulting electro- magnetic field of the whole system can be computed by formula Gem = −ε0μ0(m × E), (10) (10) where m is the magnetic dipole moment of the loop. Since the system is finite and stationary with respect to the laboratory frame, the above lemma can be used, and consequently the total momentum of the system must be zero, i.e. Gh + Gem = 0. (11) (11) Therefore, the current loop necessarily contains inter- nal hidden mechanical momentum Gh, which pre- cisely compensates the electromagnetic momentum of the surrounding field. Yet because this argumentation implicitly depends on the application of the center-of- mass/energy theorem, caution towards such a deriva- tion is in order. A valuable contribution to the problem of hidden momentum was made by Hnizdo [44], who carefully analyzed stationary systems carrying both charges and currents. In his paper, he demonstrates that the exis- tence of hidden momentum in such systems is sine qua non for maintaining correct relativistic transforma- tion properties of total energy and momentum. Conse- quently, hidden momentum is not an omittable con- stituent in relativistic momentum analysis of station- ary systems; moreover, for ponderable bodies, the con- stituents of which move stationarily, formula (9) was derived independently. Essential points of this approach are the assumption of validity of Vaidman’s lemma, i.e. particularly the validity of formula (11) and also the natural assumption that the magnetic fields involved are generated dynamically by stationary movements of charged elements of the system (Amperian loops) and not by an arrangement of relativistic invariant mag- netic charges (Gilbert’s dipoles). Interestingly enough [43], the force of density f V does not necessarily have to be of an electromagnetic origin—the formula remains valid for gravitational forces as well. This fact sheds new light on the concept of hidden momentum, which is obviously more general than is usually thought. In addition to analysis of various idealized thought models, there have been efforts to quantify hidden momentum by using a single compact formula to sim- ply identify hidden momentum in any arbitrary system. Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 A rather discordant contribution to the problem of electromagnetic forces and hidden momentum was pub- lished by Mansuripur [36, 37]. Analyzing anew the behavior of electromagnetic forces exerted by a mag- netic dipole on an electric charge in a magnetically polarizable medium, he inferred that the Lorentz force law is incompatible with special relativity and conser- vation laws. Therefore, he formulated the radical claim that the Lorentz force law should be abandoned in favor of the one proposed by Einstein and Laub [20, 38], which is in conformity with these conditions. What is the essence of this alternative law completing the sys- tem of Maxwell’s equations? The main difference of this approach, with respect to the Lorentzian one, lies in the introduction of electric and magnetic polarization vectors as independent variables in addition to usual bound electric charges and currents. Consequently, in formulae for electromagnetic energy density and the Poynting vector (see formula (1)), quantity H (mag- netic field strength) must appear in place of magnetic vector B. In polarizable materials, there are also dis- tributions of local electromagnetic forces, and momen- tum densities change correspondingly. Nevertheless, the integral end formulae for finite systems are identical for both approaches. For example, the last equation for the electromagnetic volume force in the Einstein–Laub paper [20] coincides with Lorentz’s relation (2). Quite naturally, both approaches must remain equivalent for materials without intrinsic polarizations, e.g. metallic systems. Considering then the aforesaid differences in the distribution of momentum densities, Mansuripur claimed that if one uses the Einstein–Laub formalism, the introduction of the concept of hidden momentum into problems of the Shockley–James type is redundant. This claim, however, was rebutted by Griffiths and Hnizdo [39, 40], who, referring to an older paper [41], have shown that the solution to the problem is depen- dent on which magnetic dipole model is used (i.e. the Gilbertian or the Amperian). Moreover, they showed that the Lorentz force law is consistent with special relativity. flow of electromagnetic power mediating their mechani- cal interactions, and Eq. (9) then represents the hidden mechanical momentum stored in the stationary struc- tural patterns within the system. The main persisting flaw of this definition, however, is the uncertainty as to which subsystem Eq. (9) actually applies. Torque and forces affecting stationary magnetic dipoles of a various physical constitution were investi- gated by Vaidman in [33]. 3 Hidden momentum The theorem as proposed by Taylor [29] reads: g y p p A good illustration of this is provided by the following instructive but somewhat non-realistic model (ignoring the unavoidable existence of surface charges on the con- ductor [32, 33]) suggested by Babson et al. [34]: a rect- angular current loop made of a hypothetical conduc- tor having no screening ability and no internal scatter- ing, and which is subjected to an external electric field, was treated either classically or relativistically. It was shown that in the latter case, due to the difference of charge carrier speeds in different parts of the loop, an extra relativistic correction to the momentum occurs that is not associated with the macroscopic movement of the loop as a whole. Evidently, then, the momentum has the character not of overt momentum but of hid- den momentum. Moreover, the value of this relativis- tic correction makes it possible to distinguish whether the involved charged particles have a dynamical char- acter as Amperian current loops or are pairs of static Gilbertian magnetic charges. In this way, for example, hyperfine splitting of the hydrogen ground state con- firms that the magnetic momentum of the proton is due to an Amperian current loop [35]. Center-of-mass/energy theorem: The total momen- tum of an isolated system is zero in that inertial frame in which the center of mass/energy is at rest. / Unfortunately, the straightforward application of this theorem to various hypothetical electromechani- cal models is not always correct (cf. [22]). Namely in cases where a part of the electromagnetic momentum imparted to the system, which was originally at rest, is transformed into some hidden form and not an overt form, the momentum balance is uncertain, and the cen- ter of mass/energy of the system will no longer be at rest. Thus, the conditions of the theorem are violated, making its usage questionable. This very fact is often ignored in the literature or left without an adequate explanation. General considerations concerning the linear elec- tromagnetic momentum of stationary distributions of 123 3 3533 Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 Of interest, then, is a definition of hidden momentum formulated by Vanzella [42], cf. [43]: if we consider the electromagnetic force of density f V , which exerts upon the volume element dV of a certain specified subsystem having the relative velocity v, the hidden momentum Gh will be defined by the volume integral over this sub- system, namely, Gh = −1 c2  V r(vf V )dV , (9) (9) where r is the position vector of a given volume body element with respect to the center of mass/energy of the entire system. Where the movements of body elements are stationary, the product r (v f V ) corresponds to the 123 3 Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 3534 Previous opinions in the literature concerning the existence of hidden momentum were reexamined by Redfern [45], who turned his attention to a fact of primary importance, namely, that one often tac- itly assumes the possibility of making electromagnetic experiments in absolute isolation from the surround- ings. Of course, an ad hoc system consisting of, say, a current loop and a point charge can be considered (without concern for how the system came to be) at rest in an arbitrary frame of reference. This approach makes it possible to apply the relativistic center-of- mass/energy theorem without problems; it does, how- ever, ignore the Lorentz forces inevitably involved when the system is built up [22]. Computed examples have shown that when the momentum imparted to the sys- tem during its assembly is taken into account, the total momentum is conserved without the need to postulate a hidden form of momentum at all. supposition and believed that a magnet acts upon the current and not primarily on the conductor. Since his lecturer, Rowland, had a similar view, Hall undertook a series of experiments as part of his Ph.D. disserta- tion intended to confirm an opinion that was precisely opposite to that of Maxwell. Under the supervision of Rowland, who provided him with unusually accurate instrumentation and support, Hall did a series of inter- esting experiments, though the results were, unfortu- nately, only negative. In the end, Hall carefully repeated an experiment that had previously been carried out by Rowland, and although he had no foreknowledge of Rowland’s result, he proved the direct influence of an external magnetic field on a current inside a conductor [51]. Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 [ ] The original layout of Hall’s successful 1879 experi- ment was as follows (cf. [51, 52, 55], see Fig. 2): the ends of a strip of thin gold leaf mounted on a glass plate were fitted with massive contacts as wide as the strip itself, forming part of an electric circuit through which a cur- rent was passing. The sample was placed between the poles of an electromagnet, with the plane of the strip perpendicular to the lines of magnetic force. A sensi- tive galvanometer was connected at different points on opposite edges of the strip, and the positions of these points were then changed until they were found to be at the same potential. When the magnetic field was cre- ated or destroyed, a permanent deflection of the gal- vanometer needle was observed, indicating a change in the relative potential of the two points at opposite edges. It was thus shown that the magnetic field pro- duced a new electromotive force in the strip of gold leaf at right angles to the primary electromotive force as well as to the magnetic force and also proportional to the product of these forces. In contemporary notation, the corresponding empirical relation for the observed Although no more than a concise digest of elementary facts related to hidden momentum, this brief review clearly demonstrates that in spite of the enormous attention devoted to the subject, it remains a vague and controversial entity, something of a fa¸con de parler about perfectly genuine non-zero momentum (largely of a non-electromagnetic nature) that occurs or can be stored in a system whose center-of-mass remains at rest. On the other hand, it convincingly shows that this seemingly marginal problem reaches to the more distant parts of classical electrodynamics and physics. As such, it deserves a satisfactory theoretical resolution supported (in the best-case scenario) by experimental evidence. 4 Phenomenology of the ordinary Hall effect Fig. 2 A sketch of Hall’s original experimental arrange- ment (according to [52], g—glass substrate, m—metal (gold leaf), b—copper blocks) Let us now recall and summarize the basic experimental facts concerning the ordinary Hall effect, a promising candidate for a future case study on momentum bal- ance in electromechanical systems, which also has very interesting historical and epistemological connotations. g p g The effect is generally known as a useful practical tool for the study and characterization of electronic conduc- tion processes in metals and semiconductors [46–50]. Its discovery in 1879 [51, 52], however, was closely related to somewhat general questions concerning the physical nature of electric currents [53]. After experimental con- firmation of the equivalence between an electric current and a moving charged body (an idea originally proposed by Faraday) by means of Rowland’s famous experiment [54], pressing new questions arose. For Hall, the fol- lowing passage in Maxwell’s Treatise [3] (section 501) was the most challenging: “It must be carefully remem- bered, that the mechanical force which urges a con- ductor carrying a current across the lines of magnetic force, acts, not on the electric current, but on the con- ductor, which carries it.” Hall disagreed. He regarded the statement to be in conflict with the most natural Fig. 2 A sketch of Hall’s original experimental arrange- ment (according to [52], g—glass substrate, m—metal (gold leaf), b—copper blocks) 123 123 123 3535 Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 Hall voltage V H based on these measurements reads which explicitly gives the correction term in (13), thus making (15) the most important empirical relationship describing the ordinary Hall effect. VH = RH IB d , (12) (12) where I is the primary current, B the applied magnetic field, d the thickness of the gold strip, and RH the constant factor (with a dimension of m3/C), now called the Hall coefficient. Interestingly enough, Hall believed that RH should be a universal constant independent of the size and material of the sample, thus in some way anticipating the quantum Hall effect [56]. 5 The Hall effect as a manifestation of the Lorentz force We can thus express this effect as a small correction (E ≫E H ) to Ohm’s law [55, 58], namely, i = γ(E + EH), (13) (13) q f = q{E + (v × B)}, (16) (16) where, as usual,i is current density, E the external field, and γ conductivity. The correction means, how- ever, that the current and the conjugate equipotential lines can no longer be rectangular, and the equipo- tential lines must be slightly skewed. In cases where the voltage drop V H is measured by means of a high- impedance device, it can be assumed that the normal current component along the sample edges is identi- cally zero. Then the currents driven by EH will charge the strip edges until it is completely canceled by the induced electrostatic field. The result of this process, which is quite analogous to the formation of the flow tube (sphondyloid) structure in conductors [32, 59], will be the reestablishment of a system of current lines in bulk parallel to the sample edges. Absolute values of the vector quantities involved can then be expressed using (12) and other obvious relations as where, as usual,i is current density, E the external field, and γ conductivity. The correction means, how- ever, that the current and the conjugate equipotential lines can no longer be rectangular, and the equipo- tential lines must be slightly skewed. In cases where the voltage drop V H is measured by means of a high- impedance device, it can be assumed that the normal current component along the sample edges is identi- cally zero. Then the currents driven by EH will charge the strip edges until it is completely canceled by the induced electrostatic field. The result of this process, which is quite analogous to the formation of the flow tube (sphondyloid) structure in conductors [32, 59], will be the reestablishment of a system of current lines in bulk parallel to the sample edges. Absolute values of the vector quantities involved can then be expressed using (12) and other obvious relations as where f is the Lorentz force per unit charge, E and B are local electric and magnetic fields, respectively, and v is the velocity of exposed charge element q. All quan- tities are related to the same inertial frame, and the charge element is assumed to be infinitesimally small, q →0. 5 The Hall effect as a manifestation of the Lorentz force This somewhat specific form of Eq. (15) describing the Hall effect immediately gave rise to fundamental ques- tions, such as whether the magnetism is of a rotary or linear nature [60] and how the system of Maxwell’s equations should be completed to involve the forces exerted by a magnetic field on electric charges and currents [18]. One of the results of this effort was the most common explanation of the Hall effect based on the Lorentz electromagnetic force [61], which affects the moving charges and electric current inside the sample. [ ] In Hall’s experiment, the length of strip L was much greater than its width w (w ≪L), which corresponds to the arrangement now known as the long-sample con- figuration. Since the influence of the current-supplying contacts can be neglected in this configuration [48], which significantly simplifies theoretical considerations and analysis of the experiments, we will use it in this paper exclusively. Moreover, for the sake of definite- ness, a gold sheet (Au) with thickness d, length L, and width w is used as a model device. For interpretation and quantitative estimates, we can thus use the simplest version of transport theory, the free electron gas model [57] (see also Appendix) with appreciable credibility. The position of the Lorentz force within the system of classical electrodynamics is somewhat solitary [14] and reveals ambiguous aspects [36]. While Maxwell’s equa- tions describe how moving charged particles, or equiv- alently macroscopic currents, give rise to surrounding electromagnetic fields, the Lorentz force law describes the influence of an electromagnetic field on moving charges. Since Maxwell’s equations do not contain any direct information about the effect of fields on charges, it is impossible (without strong additional assumptions) [62] to derive the Lorentz force law from Maxwell’s equations alone. Rather it is a separate physical law that was independently established by generalizing cer- tain experimental results [18]. The corresponding for- mula can be written as The phenomenology of the ordinary Hall effect can be summarized as follows: in the experimental arrange- ment described above, the turning-on of the magnetic field B gives rise to an extra electromotive force, which can be represented by a certain vector EH (Hall field) perpendicular to both—the ohmic current and mag- netic field. 5 The Hall effect as a manifestation of the Lorentz force Formula (16) also enables one to unambiguously decompose the observed force density f into an elec- tric and a magnetic field, provided that the kinematic parameters of the test charge path are known and the Bremsstrahlung for this path is negligible. where f is the Lorentz force per unit charge, E and B are local electric and magnetic fields, respectively, and v is the velocity of exposed charge element q. All quan- tities are related to the same inertial frame, and the charge element is assumed to be infinitesimally small, q →0. Formula (16) also enables one to unambiguously decompose the observed force density f into an elec- tric and a magnetic field, provided that the kinematic parameters of the test charge path are known and the Bremsstrahlung for this path is negligible. g g g The traditional textbook Lorentz force-based deriva- tion of the formula describing the Hall effect is then astonishingly concise. (Throughout this paper the fol- lowing convention is used: charge of electron q = −e, where elementary charge 0 < e = 1.602 × 10−19 C). Charge carriers—electrons traveling through a conduc- tor with drift velocity vD—are deflected by the mag- netic component of the Lorentz force (16) toward one edge of the sample, which is enriched by charge carri- ers while the opposite one is depleted. Then, across the sample, this charge redistribution generates the Hall electric field, which in turn counterbalances the mag- netic component of the Lorentz force so that the charge EH ≈VH w = RH IB dw = RHiB = RHγEB. (14) (14) Considering the relative positions of the correspond- ing vectors, we obtain the 3-vector formula EH = RHγ (E × B), (15) (15) 12 3 3 3 Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 3536 Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 equations for the cyclotron (Larmor) frequency ω and cyclotron radius r, carriers in the bulk continue to flow as before. This idea of a dynamical equilibrium between the Hall field EH and magnetic part of the Lorentz force affecting an elec- tron with drift velocity vD (velocity of electricity [51]), can be expressed as ω = eB m = v r , (23) (23) eEH + e (vD × B) = 0. (17) (17) where m is the electron mass and v is the component of electron velocity perpendicular to the magnetic field. 5 The Hall effect as a manifestation of the Lorentz force The radius of the cyclotron orbit at B = 0.2 T cor- responding to the typical drift velocity (see Appendix) vD ≈2.1 × 10−5 m/s is r ≈6.0 × 10−16 m, so that it is even smaller than the classical radius of the electron ≈ 2.8 × 10−15 m [66]. The idea that such an effect can be responsible for some observable deflection of individual electrons is questionable at best. On the other hand, the use of a seemingly more plausible mean electron velocity ⟨v⟩≤vF ≈1.4 × 106 m/s in (16) will result in a non-realistically huge estimate for the Hall field. Taking into account conventional forms of Ohm’s law and Drude’s relations [63], between current density i and drift velocity vD of electrons, i.e. i = −ne vD, (18) (18) we immediately obtain a relationship that is identical to formula (15) above: y g To resolve this problem, a slightly modified strategy must be used. The central concept of this approach is relaxation time τ, which is assumed to be independent of the energy of the charge carriers freely moving in the conductor. This quantity, which primarily measures the mean time interval between subsequent collisions of a charge carrier with scattering centers in the conduc- tor (see Appendix), also provides an estimate for the characteristic time of charge redistributions within the conductor. This time scale τ may also be used for inde- pendent definition of quantities known from Drude’s transport theory [64], i.e. of mobility μ and drift veloc- ity vD, EH = 1 ne(i × B) = RHγ (E × B), (19) (19) where the Hall coefficient RH clearly equates to RH = 1 ne. (20) (20) The most straightforward derivation of the Hall effect formula is, nevertheless, to simply identify correction term EH in (13) with the magnetic term of the Lorentz force density f in (16) (cf. e.g. [64]). The empirical rela- tionship (15) then follows immediately, provided that v, the velocity of electricity, again has the meaning of drift velocity vD. Indeed, let us start with the original form of Ohm’s constitutive relation for electric current densityi in terms of generalized force density f (= Elek- troskopische Kraft [65]), which assumes the existence of constant proportionality factor γ. 123 5 The Hall effect as a manifestation of the Lorentz force It reads simply μ = e τ m = −vD E , (24) (24) where E is again an electric field causing a coordinated drift of the system of electrons. Another time scale brought about in the problem by a magnetic field is the inverted Larmor frequency 1/ω (23), which is independent of the velocity with which an electron enters the domain of a magnetic field. The ratio of these two time scales, then, is decisive for the behavior of an electronic system in a magnetic field. Of physical importance is the limiting case where i = γ f. (21) (21) By inserting f from (16), we obtain the formula i = γ {E + (vD × B)}, (22) (22) τ ≪1 ω . (25) (25) which is fully congruent with (13). Somewhat formal as it may be, this approach makes clear the nature of the Hall effect as a local magnetically induced correction to linear transport. By using (23) and (24), this condition can also be rewritten in the equivalent form Despite the fact that both derivations above provide us with the “correct” result, they have some serious flaws, which in the current literature are either com- pletely ignored or not discussed properly. Quite incom- prehensible is the rather teleological use of drift velocity vD instead of, for example, the actual electron veloc- ity v ≤vF (Fermi velocity) in formulae (17) and (22), which traditionally accounts for deflection of drifting charge carriers. Granted the electrons are deflected by the magnetic part of the Lorentz force, the path of every moving electron must be adequately described by μB ≪1. (26) (26) Relations (25) and (26) define an analytically treat- able weak magnetic field limit and also provide the characteristic condition defining the ordinary Hall effect [67]. For samples having medium mobility μ (in our case is μ = 5.2 × 10−3 m2/Vs, see Appendix), condition (26) is fulfilled practically for all magnetic fields accessible in the laboratory. What is the physical meaning of con- dition (25)? 123 The angle θ corresponding to the circumscribed part of the cyclotron orbit and simultaneously characteriz- ing the deflection of the electron velocity vector can be expressed as θ ≈ωτ. (27) (27) u = E × B B2 . (29) Thus, regardless of their actual velocities, the paths of all the electrons are rotated within time τ in the plane perpendicular to the vector of the magnetic field, through about angle θ. Since on average the chaoti- cally moving electrons constitute an ordered drift along current lines, it is evident that the current lines them- selves must be simultaneously tilted through the same angle θ. These current lines then inevitably collide with the sample boundaries, instantaneously creating surface charges there. Deposited charges induce electric field EH (i.e. the Hall field), which forces the current to flow in parallel with the edges again. These considerations (among others) are the basis for the very fact that the entire process of deviation of electrons or, equivalently, of current paths by the Hall angle θ, takes place dur- ing the relaxation time τ; in the opposite case, namely, would be Eq. (27) inconsistent with observations. We can formulate this important piece of knowledge in the following lemma: (29) Vector u, being collinear with the corresponding Poynting vector (1), is perpendicular to both vectors E and B, meaning there is no mechanical work car- ried out by these forces. The side progress of circu- lar orbits thus takes place only at the expense of the original kinetic energy of the particles. Due to such a decrease in energy, the cyclotron radius exposed to the electric field is diminished by ratio η = √ v2 −2uv + u2 v . (30) (30) Using this scenario, the Hall effect can be explained as follows: free electron orbits start to shift with speed u in the direction of the Poynting vector until they reach the sample edge. The charge deposited there then induces the field EH , which stops the process and reestablishes the original distribution of current lines within the sample. A serious flaw in such an explana- tion, however, is the interesting fact that vector u has, according to (29), the same direction and value for all charged particles regardless of their masses, charges, and sign of charges. 123 123 123 Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 3537 Fig. 3 Projection of the path passed between two subse- quent collisions by an electron having velocity v on a plane perpendicular to the vector of the external magnetic field. Insert depicts the configuration of vectors of the primary and the Hall electric field. θ is the Hall angle The success of the use of collective drift velocity vD in the derivation of formulae (19) or (22) for the Hall field EH may be now explained as follows: when the mag- netic field appears, all paths of free electrons, regard- less of their velocities, simultaneously rotate through the same angle θ. If the distribution of electron veloc- ities were isotropic, there would be no change in the frequency with which the electrons collide with the sample edges; however, in cases where all the electrons have, on average, a common velocity component, i.e. the drift velocity vD, the collisions with one edge will be more frequent than with the other. Consequently, in the vicinity of the sample edges, thin space charge lay- ers appear bearing opposite net charges, inducing the compensating Hall field EH . Fig. 3 Projection of the path passed between two subse- quent collisions by an electron having velocity v on a plane perpendicular to the vector of the external magnetic field. Insert depicts the configuration of vectors of the primary and the Hall electric field. θ is the Hall angle For the sake of the completeness of this section, let us give one more potential explanation, also using the Lorentz force, of the ordinary Hall effect (cf. [48]). It is based on analysis of the so-called gyroscopic effect, which was studied, for example, by Page [68]. Accord- ingly, a free particle of mass m and charge q, passing across a magnetic field with velocity v, starts to rotate like a mechanical flywheel about vector B on a circular or helical path with a cyclotron radius r. The electrical part of the Lorentz force (16), i.e. the term qE, then causes uniform movement of these circular paths with constant velocity u, which is given by formula Using the classical description, it can be said that between subsequent collisions, the electron travels only a small segment of the cyclotron orbit (see Fig. 3). 6 The Hall effect and the Poynting vector by direct application of formula (1) (see Fig. 5), i.e. SH = 1 μ0 (E × B). (31) This section attempts to homologize the general con- ceptual basis currently used for describing electromag- netic fields (see Sect. 2) with the description of our con- crete case, i.e. the ordinary Hall effect. To interpret this effect in terms of the flow of energy or electromagnetic momentum, it is first necessary to separate the com- ponents of the Poynting vector belonging to the ohmic transport and the Hall effect, respectively. If the con- tacts are perfectly conducting equipotential surfaces, Ohm’s component SΩ of the Poynting vector incident to the sample surface can be visualized immediately (see Fig. 4). Near its surface, the primary current I flowing through the sample generates electric field E parallel to the current lines and, simultaneously, proper magnetic field BΩ closely circumventing the sample in the planes, which are perpendicular to these lines. (31) Obviously, because of the Hall experimental arrange- ment, the vectorSH enters the sample through one edge and leaves it unchanged through the opposite one (see Fig. 5). At first glance, a striking congruence between for- mula (31) for the Poynting vector SH and empirical relation (15) for the Hall field EH is apparent. These formulae, differing only in a certain pre-factor, can be recast into an interesting relationship with the help of (19) and (24): EH = (μ0SH)μ = e mτ (μ0SH). (32) (32) Considering general formula (1), we can conclude that the vector SΩ enters the interior of the sample through its whole surface with the exception of the con- tacts. This component of the Poynting vector, for which obviously div SΩ < 0, provides the continuous energy supply from the voltage source, which within the bulk of the sample is then converted to Joule’s heat. The continuous production of this heat is compensated by enhanced thermal radiation represented by the Poynt- ing vector component SR. In addition to these two com- ponents ensuring the dynamical energy balance of the sample, there is another independent one, which we ten- tatively call the Hall component of the Poynting vector SH . This vector SH can be easily constructed from lon- gitudinal electric field E and external magnetic field B Accordingly, the vector of Hall field EH and inci- dent Hall component of the Poynting vector SH are collinear and proportional to one another. 123 Moreover, this gyroscopic model is seriously disturbed by frequent scattering events cor- responding to the weak magnetic field limit (25). For these reasons, the gyroscopic effect does not provide any plausible explanation of the ordinary Hall effect. It can be taken as an empirical fact that the Hall field is, after application of an external magnetic field, fully established during the relaxation time τ. The validity of Eq. (27) also enables one to define the Hall angle in a more convenient way, i.e. directly in terms of the electric fields, or in terms of mobility and the magnetic field, as (cf. Fig. 3)  EH E  = μB = tg θ ≈θ, (28) (28) where the last approximation takes place in very weak magnetic fields. 123 123 3 Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 3538 by direct application of formula (1) (see Fig. 5), i.e. 6 The Hall effect and the Poynting vector This formula also reflects a remarkable fact, i.e., that the Hall field EH provides a direct measure of the relaxation time τ. It should be emphasized here, however, that formula (31) is not exactly valid. Its right side is related to the moment of the turning-on of external fields (i.e. specif- ically of magnetic field B in the case where a steady- state of ohmic flow through the sample was already established), while its left side corresponds to the delay time τ. For the Hall Poynting vector at time τ after the turning-on of the magnetic field, it is more correct to write SH(τ) = SH + ΔSH, (33) Fig. 4 Schematic view of the ohmic components of the steady-state Poynting energy flows. Notice the inward ohmic energy flow SΩ and total outward thermal electromagnetic radiation flow SR SH(τ) = SH + ΔSH, (33) Fig. 5 Construction of the Hall component of the Poynting vector SH , generated by ohmic electric field E and external magnetic field B (33) Fig. 4 Schematic view of the ohmic components of the steady-state Poynting energy flows. Notice the inward ohmic energy flow SΩ and total outward thermal electromagnetic radiation flow SR Fig. 5 Construction of the Hall component of the Poynting vector SH , generated by ohmic electric field E and external magnetic field B Fig. 4 Schematic view of the ohmic components of the steady-state Poynting energy flows. Notice the inward ohmic energy flow SΩ and total outward thermal electromagnetic radiation flow SR Fig. 5 Construction of the Hall component of the Poynting vector SH , generated by ohmic electric field E and external magnetic field B 3 3 3539 Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 where the correction ΔSH can be estimated from the Hall correction EH to the overall electric field vector appearing at time τ. Thus, taking into account (31) and (32), we immediately obtain effectively transferred during the relaxation time τ from one edge of the sample to the other is, evidently, (m/e) ε0 EH A (A is oriented edge area). If we compare this quantity with the electromagnetic energy influx SH entering the sample through one edge during the estab- lishment of the Hall field and expressed in mass units, we find that both these quantities are exactly equal, namely, μ0 ΔSH = μ { (E × B) × B} = μ { B (BE) −E(B2)}. (34) )}. (34) Because throughout the process the vector of the total electric field in the plane remains perpendicular to the vector of the magnetic field (i.e. E⊥B), the first term in the last brackets disappears, and for the change of the Poynting vector we arrive at the following for- mula: m e ε0 EH A = SH τ A c2 . (37) (37) However, this direct consequence of Eq. (32), which in fact bears a characteristic of tautology, is very difficult to interpret convincingly. One is tempted to consider relationship (37) as a manifestation of the preservation of the center of mass/energy mentioned above. Indeed, considering the left side of (37), the shift δ of the center- of-mass of the sample, which is due to the transfer of ponderable matter (i.e. electrons), can be computed by the following formula: μ0 ΔSH = −μ E (B2). (35) (35) In analogy with Eq. (28), for the ratio of quantities on the right side of Eq. (33) we can write  ΔSH SH  =  μ E (B2) E × B  = μB = tg θ. (36) (36) δ = ε0EH ne . (38) (38) This means that during time τ the Hall Poynting vec- tor is also deviated by the Hall angle θ, precisely like pri- mary electric field vector E. There is, however, a differ- ence in their layout. Whereas in the case of the electric fields the angle θ should be measured anticlockwise (cf. Fig. 3 3), in the case of the Poynting vectors it increases in the clockwise direction; moreover, the vectors E and SH are orthogonal. Since the Poynting vector must continu- ously rotate from its initial position to the end position at time τ, the Hall electric field EH must also simultane- ously appear following Larmor rotation of the electron subsystem. From this point of view, EH is not a con- sequence of charges deposited on the sample edges but is created locally by the deformation of the Poynting vector induced by the Larmor rotation. Our approach to the Hall effect using the Poynting energy flows thus makes clear one persisting logical paradox. As we have already seen above, in standard treatment of the Hall effect, the charging of the edges generates an electric field, which within time τ compensates the action of the magnetic field, thus stopping further deviation of the electron paths. This consideration is based on the tacit assumption that the information about the charg- ing of the edges is spread throughout the sample practi- cally instantaneously. Nevertheless, taking into account the fact that the maximal speed of any signal is ≤c, we find that in most cases cτ < w (= sample width, see Appendix), so no such coherent compensation of magnetic action is possible. Such a shift requires an action of electromagnetic forces characterized by the electromagnetic momentum “consumed” within the sample during time τ. This loss of electromagnetic momentum realized in the vicin- ity of the enriched edge, however, shifts the center-of- mass/energy back about the length ≈−μ RH (SH /c2), just opposite to the length given by formula (38). When the stationary state of the Poynting flow through the sample is reestablished after time τ, the center- of-mass/energy is set into its original position. Even though the deeper insight into the problem makes such an explanation seem somewhat naive, the above quan- titative estimates supporting this scenario are correct. Knowing now the rough features of the time evolu- tion of the Poynting vector, we can immediately deter- mine the electromagnetic volume forces active during the establishment of the Hall field. For the time deriva- tive of the Poynting vector, we can use (33) and (35) to obtain estimate ˙S →ΔSH(τ) τ = − e mμ0 E (B2), (39) (39) which can be inserted into the Lorentz Eq. 3 (2) for the electromagnetic volume force, namely, F = e m  V ε0E (B2) dV. (40) (40) The formulae presented in this section allow one to make some interesting estimates or conclusions con- cerning spatial redistributions of electrons within the system and formulate hypotheses concerning the depo- sition of electromagnetic momentum as well. First of all, let us evaluate the net mass transfer due to the rise of the Hall field. Working with simple electrostatic considerations, the total ponderable mass of electrons Operating for time τ along the bulk of the sample, this force results in the Hall field, which manifests itself by permanent surface forces seated at the edges of the sample. The electric component of the surface forces, which until now has been ignored and should be added 123 12 3 3540 Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 as an extra term to formula (2), can be expressed by an integral [19], i.e. Accordingly, we can distinguish two essentially different interpretations of the thought experiment. The first one works with an idealized system and imaginary scenario with the aim of obtaining (through speculations which are not at odds with known laws of nature) answers to some hypothetical questions. The main difficulty of this approach is the arbitrariness of starting assump- tions, such as artificial initial and boundary conditions, which are often decisive for the resulting conclusions. The second interpretation, proposed by Mach, views the thought experiment as a necessary precondition for the realization of any physical experiment; it is, in fact, a detailed technical plan, the goal of which is to eliminate weak points in the future execution of the experiment. It may happen, however, that such a thought experi- ment solves the posed problem even without the actual experiment being performed. FSE =  2A  ε0(n EH) EH −1 2ε0 n |EH|2  dA, (41) (41) taken over the sample edges only; n is the unit nor- mal vector to the sample surface. This formula can be reduced to a simpler relation for the electrostatic stress P (i.e. pressure) imposed on the sample edges by the Hall field, namely P = 1 2ε0(EH)2. (42) (42) This stress of electrostatic origin is inevitably relax- ing into the mechanical strain of the edges, cumulat- ing in this way some potential energy. 3 One can thus be sure that at least one part of the electromagnetic momentum entering the sample is deposited there with- out affecting the movement of the system. It should be noted that the expression on the right side of (42) is also exactly equal to the energy density of the elec- trostatic Hall field inside the sample (for the fringing effect see [69]). After the external magnetic field has been turned off, the capacitor consisting of the sam- ple edges will be discharged and the mechanical strain localized at the edges during the assembly period in the form of electromagnetic momentum will be released. While the discharge of said capacitor may, with some proviso, be treated as a reversible process, the mechan- ical part of the Hall field decay has features typical for temporal reservoirs of hidden momentum, as discussed above. Stress related phenomena of this type thus pro- vide a “traditional” account of the existence of hidden momentum. Importantly, these effects do always exist, and as such they should be carefully identified among other possible sources of hidden momentum. When it comes to problems relating to hidden momentum, clearly, we must work with the first of the two approaches mentioned above. There is no intent to perform the suggested experiments. We dare to claim that most of the authors of such experiments are con- vinced there is no hope of actually conducting them and that, therefore, there will never be any quantita- tive results to compare with their expectations. This is why some important or even unrealistic aspects of their conceptual models can be safely overlooked. Since our ultimate aim is to implement the real Hall effect experiment into considerations concerning problems of momentum balance in electromechanical systems, we incline rather to Mach’s (second) conception of the thought experiment. The discussion, then, must begin with an assessment of the range of quantities involved and with detailed specification of the experimental con- ditions, including the preparation phase. Firstly, to have some idea of the order of the quanti- ties we are talking about, let us make some quantitative estimates of the forces and effects involved. Using our model of the gold leaf Hall structure (described in full in the Appendix), we determine from Eq. 3 (37) that the excess (or lack) of electron density on the edges is ≈ ± 230 electrons/m2 and the total mass transferred is approximately 2.1 × 10−36 kg. The volume force oper- ating in the bulk of the sample during establishment of the Hall field may then be determined from Eq. (40), F ≈1.27 × 10−14 N. For the surface stress, we use (42) to arrive at an estimate of P ≈7.73 × 10−23 N/m2, the same as for the volume energy density of the Hall field (≈7.73 × 10−23 J/m3). The center-of- mass shift δ ≈3.9 × 10−27 m, computed by means of (38), also seems unrealistically small. Obviously, we are dealing with very tiny quantities and extremely sub- tle effects, though these quantities were derived not from some abstract theoretical considerations but are computed from realistic values currently observed in the laboratory using the Hall measurements. Such mea- surements, then, are sensitive enough to “weigh” small fractions of electron masses and indicate force densi- ties of the order of μN/m3, so the ordinary Hall effect evidently belongs to the same realm of electromagnetic We have shown the usefulness of interpretation of the ordinary Hall effect in terms of electromagnetic energy flows represented by the Poynting vector. This approach, among others, has revealed some new aspects of this interesting effect and also generated formulae enabling deeper insight into its mechanism. 7 Discussion If we provisionally label the component of the experimental setup that is effectively decoupled from the surrounding as milieu-independent, we can conclude that the part of the electromagnetic energy and momentum fluxes needed for the establish- ment of the Hall field, which are fully converted and stored by the electrons redistributed inside the sample, are the milieu-independent ones. Furthermore, there are inevitably momenta compensating the reaction of the electromagnet during application of the magnetic field and mechanical stresses due to the charging of sample surfaces, which are fully dependent on the concrete con- figuration of the experiment. Since the sample reveals no overt motion, these latter momenta entering the momentum balance might, in principle, possess a char- acteristic of hidden momentum. Yet this is inadmissi- ble, because in an open system these momenta cannot be identified and localized unambiguously. B = β t, (43) (43) where the sweep rate β is a constant vector collinear with the magnetic field and t ∈(0, T) is the time mea- sured from the beginning of application of the field. Keeping in mind that τ < < T, we can rewrite (32) as EH(t) = e m τ (E × β t). (44) (44) This equation describes the progression of generation of the Hall field in real time. Similarly, the time evolu- tion of the corresponding Poynting vector can be char- acterized by ˙S →SH(t + τ) −SH(t) τ = − e mμ0 E (β t)2. (45) (45) The time derivative of SH (t), namely ˙S, then con- tinuously increases to its ultimate value −(e/mμ0)E (β2T 2), which is in fact identical to that given by (39). Therefore, curiously enough, the stepwise increasing of the magnetic field for time T should have the same effect as instantaneous application of the magnetic field, B = β T. In light of this fact, one can conclude that our previous estimates remain essentially correct quite inde- pendently of the particular way in which the stationary end state of the system was achieved. In this connec- tion, simultaneously, the key significance of relaxation time τ for the establishment of any stationary end state emerges. Indeed, τ seconds after the achievement of a certain magnetic fieldB, the Hall field EH given by (15) appears and the resulting system starts to be station- ary. 7 Discussion Let us assume that the external electric field E and corresponding electric current flowing through the sam- ple are already established and stationary. The follow- ing turning-on of a magnetic field can be then satis- factorily modelled by a magnetic field linearly increas- ing from zero to some required end value. A realistic approximation of such a process is given by formula As we have already seen in Sect. 6 above, in principle the Poynting–Abraham formalism allows one to follow the fate of the energy and momentum fluxes responsible for the establishment of the Hall field within the sample. Inspection of the experimental arrangement and allied conditions, however, clearly reveals another quite seri- ous difference between most of the hypothetical mod- els and real experiments: while hypothetical systems in which the momentum balance is studied are consid- ered to be isolated, i.e. completely decoupled from the rest of the Universe, any real experimental apparatus is inevitably open. Let us assume that the external electric field E and corresponding electric current flowing through the sam- ple are already established and stationary. The follow- ing turning-on of a magnetic field can be then satis- factorily modelled by a magnetic field linearly increas- ing from zero to some required end value. A realistic approximation of such a process is given by formula y p For example, in the real Hall measurement, where the sample is firmly fixed in its position with respect to the sample holder, the momenta and the forces appearing during application of the external fields are completely compensated by the mechanical stresses both inside and outside the sample. The decomposition of momenta of differing natures among the parts of such an open sys- tem are obviously far from unambiguous. At the same time, the generated Hall field EH does not depend on the details of the experimental arrangement but only on the quantities explicitly entering into formulae (15), (32), and (37). It is, however, quite a general and essen- tial property of any experimental method striving for a sense of objectivity. It necessarily has a component that (in order to provide reproducible results) is decoupled as far as possible from random external influences; on the other hand, it also has the components controlled mainly by the surrounding. 7 Discussion As we have already seen, arguments concerning linear hidden momentum are exclusively confined to analy- sis of thought experiments using abstract models of various kinds. The thought experiment, though, tradi- tionally a mighty tool of theoretical physics, does have its limitations. A similar demonstrative method, one that has been widely used since antiquity, is known for generating many valuable yet also quite mislead- ing ideas. The term thought experiment, which was first coined by Oersted (Gedankenexperiment or Gedanken- versuch), was later systematically treated by Mach [70]. 123 Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 3541 masses and momenta as those encountered in hypothet- ical problems relating to hidden momentum and other electromagnetic thought experiments. the relaxation time. We can claim that this parame- ter, measuring the smallest time in which the transi- tion between two stationary states of the system can be finalized, plays the role of assembly period, cf. [22], which simultaneously represents the unique dynamical aspect of any stationary system. Until now, we have investigated the processes taking place in the Hall structure under rather abstract condi- tions, in particular after the instantaneous application of finite external magnetic field B. The practical labo- ratory time limit for setting up a typical magnetic field of the order of ∼10−1 Tesla is, however, T ∼10−2 s and greater. As relaxation time τ in the current materials is much shorter (i.e. τ ≪T), the rise of the Hall field EH is in fact controlled by relatively slow changes of B; EH follows these changes with a negligible delay equal to τ ≈10−12–10−14 s; therefore, it is necessary to investigate how these facts may influence our considerations above. Until now, we have investigated the processes taking place in the Hall structure under rather abstract condi- tions, in particular after the instantaneous application of finite external magnetic field B. The practical labo- ratory time limit for setting up a typical magnetic field of the order of ∼10−1 Tesla is, however, T ∼10−2 s and greater. As relaxation time τ in the current materials is much shorter (i.e. τ ≪T), the rise of the Hall field EH is in fact controlled by relatively slow changes of B; EH follows these changes with a negligible delay equal to τ ≈10−12–10−14 s; therefore, it is necessary to investigate how these facts may influence our considerations above. 7 Discussion Notice that without such a prelude of duration τ, no stationary state of the system and no Hall effect at all would be possible. This idea can also be natu- rally generalized to other similar systems controlled by Let us now return to the entity known as an iso- lated system. It is specified by the property that it can- not exchange either energy or matter with the envi- ronment beyond the system boundary. The boundary, which does not belong to the isolated system, can be realized either by an enclosure that is non-penetrable to matter and external forces or by maintaining suffi- cient (ideally infinite) distance from the other systems. 123 12 3 Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 3542 An example of the first approach, frequently used in electrical measurements, is electrostatic shielding [64, 71], where the regions inside and outside of the con- ductive enclosure are electrically isolated from each other and electric fields from one region cannot pen- etrate or affect the other. On the other hand, the pos- sibility of using some artificial means to eliminate the influence of long-range gravitational forces is still only hypothetical [72], thus we must rely on the method of maintaining a large distance from other bodies. Clearly these means of isolating are not perfect, and each has its own limitations. As a rule, they are sufficient with required accuracy to realize the milieu-independent sys- tem but not the ideally isolated system. Despite the fact that milieu-independent systems have much in common with isolated systems (and are in fact weak versions of such systems), these two concepts are fundamen- tally different. A real milieu-independent system can be approximated by a certain isolated system, but the success of such an approximation can never be ensured a priori. This circumstance, which is closely related to the non-critical use of infinity in physics [73], can be illustrated by an example mentioned earlier in Sect. 3. The angular electromagnetic momentum of a magnetic monopole plus an electric point charge (independent of their separation) does not converge in infinity; thus, attempts to isolate this system by increasing the rele- vant distances must totally fail. It becomes apparent, then, that the gap between isolated systems and milieu- independent systems, which are crucial for real exper- iments, is in principle hardly surmountable. 7 Discussion Identification of this time scale, the so-called period of system assembly, which rep- resents the dynamical aspect of the studied station- ary system, is a crucial condition for correct com- prehension of a real experiment as well as for the non-contradictory design of thought experiments. • In a real experiment or any thought experiment, one must take into account that every stationary situa- tion is the result of a certain development. As we have seen in the case of the ordinary Hall effect, the preparational phase may be controlled by a cou- ple of different characteristic time scales, but only the shortest one is relevant for final establishment of the stationary state. Identification of this time scale, the so-called period of system assembly, which rep- resents the dynamical aspect of the studied station- ary system, is a crucial condition for correct com- prehension of a real experiment as well as for the non-contradictory design of thought experiments. • Further important differences between real and thought experiments are firmly rooted in the fact that any real experiment is inevitably made with open sys- tems, while in thought experiments systems are fre- quently used that are assumed to be strictly isolated. Since the paradoxes are symptoms of non-justified assumptions, it is no wonder that incompetent use of isolated systems in the construction of thought experiments could generate such paradoxes. There- fore, possible consequences of any implementation of an isolated system into the thought experiment should be considered individually and very critically. • Further important differences between real and thought experiments are firmly rooted in the fact that any real experiment is inevitably made with open sys- tems, while in thought experiments systems are fre- quently used that are assumed to be strictly isolated. Since the paradoxes are symptoms of non-justified assumptions, it is no wonder that incompetent use of isolated systems in the construction of thought experiments could generate such paradoxes. There- fore, possible consequences of any implementation of an isolated system into the thought experiment should be considered individually and very critically. 7 Discussion Deciding whether the approximation of an investigated system by a given isolated system is right requires solving dif- ficult convergence-divergence problems, among others. The devil is in the details, and since, as we have seen, any neglect of the smallest detail in system specifica- tion can lead to unexpected consequences, we must keep in mind that a single assumption in a thought exper- iment, e.g. that the system under consideration is iso- lated, could lead to either incorrect or correct results. effect can also be alternatively treated in the framework of the Poynting-Abraham formalism based on electro- magnetic energy and momentum flows. This approach provided us with relationships that allowed us to trace with sufficient credibility the evolution of an electronic subsystem and pertaining electromagnetic fields. Another key result of this work is that it represents a comparison of various features intrinsic either to real experiments or to thought experiments. This allows us to formulate the following important pieces of knowl- edge; they are seemingly trivial and often ignored, but we are convinced that they are deserving of respect: • The relations based on the Poynting-Abraham for- malism made it possible to show that the ordinary Hall effect, which is commonly regarded as quite robust, is in fact, as concerns momentum-related effects, also a very tiny effect of the same rank as those studied by thought experiments relating, for example, to the problem of hidden momentum. • The relations based on the Poynting-Abraham for- malism made it possible to show that the ordinary Hall effect, which is commonly regarded as quite robust, is in fact, as concerns momentum-related effects, also a very tiny effect of the same rank as those studied by thought experiments relating, for example, to the problem of hidden momentum. • In a real experiment or any thought experiment, one must take into account that every stationary situa- tion is the result of a certain development. As we have seen in the case of the ordinary Hall effect, the preparational phase may be controlled by a cou- ple of different characteristic time scales, but only the shortest one is relevant for final establishment of the stationary state. Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which per- mits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creative commons.org/licenses/by/4.0/. 3. J.C. Maxwell, A Treatise on Electricity and Magnetism. (Vol. II. Clarendon Press, Oxford, 1873) p. 144, p. 391 4. A. Bartoli, Il calorico raggiante e il secondo principio di termodinamica. Nuovo Cimento 15, 193–202 (1884) ermodinamica. Nuovo Cimento 15, 193–202 (1884 5. P. Lebedew, Untrsuchungen ¨uber die Druckkr¨afte des Lichtes. Ann. d. Phys. 311, 433–458 (1901) 6. E.F. Nichols, G.H. Hull, The pressure due to radiation. Astrophys. J. 17, 315–351 (1903) 7. A. Golsen, ¨Uber eine neue Messung des Strahlungs- drucks. Ann. Phys. 378, 624–642 (1924) 8. M.L. Schagrin, Early observations and calculations of light pressure. Am J. Phys. 42, 927–940 (1974) 9. C.N. 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Larmor frequency (at 0.2 T): ω = 3.52 × 1010 s−1. Larmor frequency (at 0.2 T): ω = 3.52 × 1010 s−1. 3 ( ) Tangent of Hall angle: tgθ = 1.03 × 10−3. Tangent of Hall angle: tgθ = 1.03 × 10−3. 24. K.T. McDonald, No Bootstrap Spaceships via Mag- nets in Electric Fields (2018). http://kirkmcd.prince ton.edu/examples/redfern.pdf. Accessed 11 Sept 2018 Hall Poynting flux: S H (0) = 649 W/m2. Hall Poynting flux: S H (0) = 649 W/m2. Correction to Hall Poynting flux: ΔS H (τ) = 0.67 W/m2. Correction to Hall Poynting flux: ΔS H (τ) = 0.67 W/m2. 25. P.A.M. Dirac, Quantised singularities in the electromag- netic field. Proc. R. Soc. Lond. A 133, 60–72 (1931) 26. L. Page, Momentum relations in crossed fields. Phys. Rev. 42, 101–107 (1932) Appendix A 12. O. Heaviside, Electrical Papers, (Vol. II., Sec. 35, Macmillan and Co., London, 1894) For the sake of the accuracy of our estimates, we have chosen a model that, similar to the one used in Hall’s original experiment [51], is as simple as possible. 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Primary electric field E = 4.08 × 10−3 V/m. 8 Conclusions In this paper, we have made an attempt to confront the thought experiment methodology traditionally used to identify a solution to the puzzling problem of hid- den momentum, addressing momentum balance in elec- tromechanical systems submitted to static electromag- netic fields, with an actual experiment belonging to the same class of phenomena. As we have suggested, the ordinary Hall effect is a good candidate for such an experimentally treatable phenomenon operating in static electromagnetic fields. It is simultaneously sen- sitive enough to have the potential to effectively visu- alize some discrepancies persisting in classical electro- dynamics. In order to explore this possibility, the phe- nomenology and standard theory of this effect in terms of the Lorentz force has been critically reviewed; fur- thermore, it has been shown that the ordinary Hall Acknowledgements J. J. M. would like to acknowl- edge funding support from the Operational Programme Research, Development and Education financed by Euro- pean Structural and Investment Funds and the Czech Min- istry of Education, Youth and Sports (SOLID21—Project No. CZ.02.1.01/0.0/0.0/16_019/0000760). Funding Open access publishing supported by the National Technical Library in Prague. Data availability statement No data associated in the manuscript. 123 123 3543 Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 Eur. Phys. J. Spec. Top. (2023) 232:3529–3544 ( ) 2. J.J. Thomson, On momentum in the electric field. Phi- los. Mag. 8, 331–356 (1904) References 291, 503–506 (1895) ( ) 37. A. Cho, Textbook electrodynamics may contradict rel- ativity. Sci. New Ser. 336, 4041205.4646v1 (2012) 38. A. Einstein, J. Laub, ¨Uber die elektromagnetischen Grundgleichungen f¨ur bewegte K¨orper. Ann. Physik 331, 532–540 (1908) 61. J.D. Jackson, Classical electrodynamics, 3rd edn (Wiley, 1999) 62. V.P. 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Theoretical Interpretation of Polarized Light-Induced Supramolecular Orientation on the Basis of Normal Mode Analysis of Azobenzene as Hybrid Materials in PMMA with Chiral Schiff Base Ni(II), Cu(II), and Zn(II) Complexes
Journal of applied solution chemistry and modeling
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Journal of Applied Solution Chemistry and Modeling, 2016, 5, 30-47 Journal of Applied Solution Chemistry and Modeling, 2016, 5, 30-47 30 Maiko Ito1, Takashiro Akitsu1,* and Mauricio A. Palafox2,3 1Department of Chemistry, Faculty of Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan 1Department of Chemistry, Faculty of Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan 2Departamento de Química-Física I, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Madrid-28040, Spain 2Departamento de Química-Física I, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Madrid-28040, Spain 3Nofima AS, Norwegian Institute of Food, Fisheries and Aquaculture Research, Osloveien 1, 1430 Ås, Norway 3Nofima AS, Norwegian Institute of Food, Fisheries and Aquaculture Research, Osloveien 1, 1430 Ås, Norway 3Nofima AS, Norwegian Institute of Food, Fisheries and Aquaculture Research, Osloveien 1, 1430 Ås, Norway Abstract: We have prepared hybrid materials of azobenzene and chiral Schiff base Ni(II), Cu(II), and Zn(II) complexes and investigated their linearly or circularly polarized UV (ultraviolet) light-induced supramolecular orientation with polarized electronic and IR spectra or CD (circular dichroism) spectra. The experimental FT-IR (Fourier transfer-infrared) spectra of azobenzene molecules were recorded at room temperature, and the results were compared with quantum chemical theoretical values using B3LYP, M052X, and M062X DFT (density functional theory) methods. The interaction of azobenzene with PMMA was simulated. Molecular geometry, vibrational wavenumbers, and thermodynamic parameters were calculated in all these systems. With the help of specific scaling procedures for the computed wavenumbers, the experimentally observed FT-IR bands were analyzed and assigned to different normal modes of the molecule. Most modes had wavenumbers in the expected range, and the error obtained was in general very low. Several general conclusions were deduced. Keywords: Chirality, azobenzene, polarized light, IR spectra, DFT. *Address correspondence to this author at the Department of Chemistry, Faculty of Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan; Tel: +81-3-5228-8271; E-mail: akitsu@rs.kagu.tus.ac.jp Preparation of bis(N-2-hydroxy-1-phenyl-3,5- dibromosalicydenaminato)zinc(II) (Zn) Preparation of bis(N-2-hydroxy-1-phenyl-3,5- dibromosalicydenaminato)zinc(II) (Zn) To a solution of 3,5-dibromosalicylaldehyde (0.2799 g, 1.000 mmol) dissolved in methanol (50 mL), (R)-()- 2-phenylglycinol (0.1372 g, 1.000 mmol) was added and stirred at 313 K for 2 h to give a yellow solution of the ligand. Zinc(II) acetate dihydrate (0.1097 g, 0.5000 mmol) was added to the resulting solution to give a yellow solution of the complex. After stirring for 4 h, this crude yellow compound was filtered and recrystallized from methanol. Yield: 0.3284 g (77.1%). Anal. Found: C, 41.58; H, 2.46; N, 3.06%. Calcd. for C30H24ZnBr4N2O4 C, 41.82; H, 2.81; N, 3.25%. IR [Nujol mull (cm1)]: 702 (w), 1028 (w), 1052 (w), 1146 (w), 1212 (w), 1301 (w), 1377 (m), 1461 (m), 1610 (w) (C=N), 2854 (s), 2923 (s). Electronic spectra (nm): 387 (acetone); 240, 385 (methanol); 248, 388 (chloroform). Scheme 1: Structures of chiral Schiff base complexes (abbreviated as Ni, Cu, and Zn) [left] and AZ (trans-form) [right]. Preparation of Hybrid Materials, Ni + AZ + PMMA, Cu + PMMA, and Zn + AZ + PMMA To a solution of 3,5-dibromosalicylaldehyde (0.2799 g, 1.000 mmol) dissolved in methanol (50 mL), (R)-()- 2-phenylglycinol (0.1372 g, 1.000 mmol) was added and stirred at 313 K for 2 h to give a yellow solution of the ligand. Nickel(II) acetate tetrahydrate (0.1246 g, 0.5000 mmol) was added to the resulting solution to give a green solution of the complex. After stirring for 4 h, this crude green compound was filtered and recrystallized from methanol. Yield: 0.2689 g (62.9%). Anal. Found: C, 42.22; H, 2.46; N, 3.08%. Calcd. for C30H24NiBr4N2O4 C, 42.15; H, 2.83; N, 3.28%. IR [Nujol mull (cm1)]: 721 (w), 1144 (w), 1211 (w), 1304 (w), 1377 (m), 1462 (s), 1629 (w) (C=N), 2853 (s), 2925 (s). Electronic spectra (nm): 394, 595 (acetone); 238, 249, 392, 596 (methanol); 250, 393, 608 (chloroform). For measurements of UV-vis spectra, acetone solutions of AZ (1.83  103 g, 1.00  105 mol) with PMMA and acetone solutions of Ni (4.27  103 g, 5.0 106 mol), Cu (4.29  103 g, 5.0  106 mol), or Zn (4.26  103 g, 5.0  106 mol) with PMMA were mixed and cast onto a glass slide at room temperature. General Procedures Chemicals of the highest commercial grade available (solvents from Kanto Chemical, organic compounds from Tokyo Chemical Industry, and metal sources from Wako) were used as received. Preparation of bis(N-2-hydroxy-1-phenyl-3,5- dibromosalicydenaminato) copper(II) (Cu) Preparation of bis(N-2-hydroxy-1-phenyl-3,5- dibromosalicydenaminato) copper(II) (Cu) groups results in enhanced intermolecular interaction with the PMMA matrix [38, 39]. The molecular design aims to induce supramolecular orientation by linearly or circularly polarized UV light irradiation. In addition, using normal mode AZ analysis in PMMA film (not in the gas phase) and simulated IR spectra of the complexes by TD-DFT calculation, we could individually observe components in the hybrid material. To the best of our knowledge, this is the first attempt at selective interpretation of induced molecular orientation after polarized UV light irradiation. To a solution of 3,5-dibromosalicylaldehyde (0.2799 g, 1.000 mmol) dissolved in methanol (50 mL), (R)-()- 2-phenylglycinol (0.1372 g, 1.000 mmol) was added and stirred at 313 K for 2 h to give a yellow solution of the ligand. Copper(II) acetate monohydrate (0.0998 g, 0.500 mmol) was added to the resulting solution to give a green solution of the complex. After stirring for 4 h, this crude green compound was filtered and recrystallized from methanol. Yield: 0.2123 g (49.4%). Anal. Found: C, 39.22; H, 2.31; N, 2.70%. Calcd. for C30H24CuBr4N2O4; C, 39.00; H, 2.48; N, 2.46%. IR [Nujol mull (cm1)]: 721 (w), 1151 (w), 1376 (m), 1462 (s), 1628 (w) (C=N), 2854 (s), 2925 (s). Electronic spectra (nm): 386, 625 (acetone); 249, 384, 613 (methanol); 249, 389, 607 (chloroform). N O Br Br M N O Br Br OH HO M=Ni,Cu,Zn (R) (R) * * N N Scheme 1: Structures of chiral Schiff base complexes (abbreviated as Ni, Cu, and Zn) [left] and AZ (trans-form) [right]. N O Br Br M N O Br Br OH HO M=Ni,Cu,Zn (R) (R) * * N N N O Br Br M N O Br Br OH HO M=Ni,Cu,Zn (R) (R) * * N N INTRODUCTION Cu(II), or Zn(II) complexes and AZ in polymethyl methacrylate (PMMA) cast films and observed linearly or circularly polarized light-induced molecular orientation [33] by means of polarized electronic (UV- vis) or IR spectroscopy [34]. By carefully neglecting solid-state artifacts, CD spectroscopy may be effective in observing chiral components separately [35-37]. Thus, if it is possible to create functional materials that can control the various functions of inorganic metal complexes via light or their physical (spatial) information, such materials will be desirable for the production of new devices. As a component of photofunctional organic/ inorganic hybrid materials, azobenzene (AZ), a typical photochromic dye, is a molecule of great interest at present. Therefore, many theoretical studies using ab inito, DFT, and molecular dynamic methods have been reported, together with spectroscopic studies of the ground and excited states [1-18]. One of the advantages of these hybrid materials may be that it is possible to introduce the optical function of photofunctional organic compounds to non-photoactive inorganic metal complexes showing attractive electronic properties or functions [19-22]. However, polarized UV-vis spectra are not able to individually observe each component for the following reasons: (1) there is overlap of the -* and n-* bands of the AZ and complexes and (2) the Zn(II) complex has no d-d- band. Therefore, IR spectroscopy was employed for the selective observation of C=N bands of complexes and N=N bands of AZ as composites in PMMA films. Using TD-DFT calculations, theoretical interpretation of IR spectra on the basis of normal vibration is required for reliable assignment of the IR spectra. In this context, we have developed light-induced molecular orientation and supramolecular chiral ordering by irradiating polarized UV light to photofunctional hybrid materials containing the photoresponsive molecules and metal complexes in polymer film [23-32]. We have tested various conditions of complexes, azo-dyes, and light irradiation. For example, we synthesized organic/inorganic hybrid materials composed of chiral Schiff base Co(II), Ni(II), Here we have synthesized new chiral Schiff base Ni(II), Cu(II), and Zn(II) complexes. Incorporating -OH E-ISSN: 1929-5030/16 © 2016 Lifescience Global © 2016 Lifescience Global Theoretical Interpretation of Polarized Light-Induced Supramolecular Journal of Applied Solution Chemistry and Modeling, 2016, Volume 5, No. 1 31 31 Computational Details Calculations were performed using the B3LYP, M052X, and M062X density functional (DFT) methods implemented in the GAUSSIAN 09 [40] program package using the Quipu computer of the Computational Center at the Complutense University of Madrid. The standard parameters of the UNIX version of this package were used. The DFT methods are the most adequate because they provide a compromise between the desired chemical accuracy and the heavy demands placed on the computer time and power. In addition, DFT methods have been used satisfactorily in many studies [41-45]. The B3LYP DFT method was chosen because several studies have shown that the data obtained using this method are in good agreement with those obtained using other costly computational methods (such as MP2), and it predicts vibrational wavenumbers better than HF and MP2 methods [46- 50]. The M052X and M062X methods are members of the M05 and M06 family of DFT methods recently developed by Zhao, Truhlar, and Schultz, which were designed to yield broad applicability in the field of chemistry [51]. The optimized bond lengths, bond angles, and torsional angles calculated in the cis and trans orientation of AZ at the B3LYP/6-31G(d,p) and B3lYP/6-311+G(2d,p) levels are listed in Table 1, while the labeling of the atoms is plotted in Figure 1. Because of the symmetry of the molecule, only the values for one side of the structure are provided in Table 1. The computed values with the two basis sets were, in general, very close and were in accordance with the data reported for related molecules. The differences were in accordance with the average error of this method. These values for the isolated state are in agreement with previous theoretical [54] and experimental results obtained by electron diffraction [55]. In the trans orientation, the structure is fully planar, and all the torsional angles are 0° or 180°. Both benzene rings are independent, and one ring has no influence on the other in the planarity of the structure. However, the nitrogen atom attached to C6 has a noticeable influence on the aromaticity of the ring. The nitrogen atom withdraws negative charge on the C6 atom and thus changes it to a positive value, 0.093 e (where e is the charge of an electron) with the 6- 311+G(2d,p) basis set (on C3, it is 0.185 e) (Table 2). This leads to a shortening of C1-C2 and C4-C5 e.g. Physical Measurements Elemental analyses for C, H, and N were conducted using a Perkin-Elmer 2400II CHNS/O analyzer at Tokyo University of Science. Infrared (IR) spectra were recorded as KBr on a JASCO FT-IR 4200 and spectrophotometer equipped with a polarizer with a range of 4000–400 cm1 at 298 K. Absorption Journal of Applied Solution Chemistry and Modeling, 2016, Volume 5, No. 1 Ito et al. 32 optimization process and by analytical evaluation of the second derivative of the energy with respect to nuclear displacement. The absence of imaginary wavenumbers confirmed the optimized conformations to be local minima. Relative energies were obtained by adding zero-point vibrational energies (ZPEs) to the total energy. For the calculation of ZPEs, the wavenumbers were retained unscaled. electronic (UV-vis) spectra were measured on a JASCO V-570 UV/VIS/NIR spectrophotometer or a JASCO V-650 UV/VIS spectrophotometer equipped with a polarizer with a range of 800–220 nm at 298 K. Photo-illumination was performed using a lamp (1.0 mW/cm2) with optical filters (UV  = 200–400 nm). Thus, a sample was produced using optical fibers and a polarizer through optical filters. Angles of polarizer denote the direction of electric vector of linearly polarized light from vertical line. RESULTS AND DISCUSSION Geometry Optimization of AZ Molecule. Isolated State Geometry Optimization of AZ Molecule. Isolated State Computational Details a slight quinonoided characteristic on the benzene ring. This effect has also been observed in the aniline molecule [56]. The nitrogen atom also affects the C1- C6 length, which is slightly longer (0.005 Å) than the C5-C6 length. Because of the short N=N bond, the C-N bond length is slightly longer than that found in the aniline molecule: at the B3LYP/6-31G(d,p) level, 1.419 Å vs. 1.398 Å in aniline [56]. With a longer C-N bond, the effect of the nitrogen atom on the ipso angle C-C2- C is smaller than that in the aniline molecule: 120.4° vs. 118.6° in aniline. Because of the symmetry of the structure, the dipole moment is 0.0 (Table 3). Several basis sets were used, starting from 6- 31G(d,p) to 6-311++G(3df,pd). The 6-31G(d,p) set represents a compromise between the accuracy and computational cost; thus, it was the base set selected as the reference for all of the calculations. The Berny optimization algorithm was used under the TIGHT convergence criterion. Conformational equilibrium at 298.15 K was evaluated by means of the Boltzmann distribution. The natural NBO procedure was employed for the determination of the atomic charges [52, 53]. Wavenumber calculations were performed on all optimized conformers to confirm the stationary points as local minima on the potential energy landscape and to calculate Gibbs energies G as the sum of the electronic and thermal free energies. These were performed at the same level as the respective Theoretical Interpretation of Polarized Light-Induced Supramolecular Journal of Applied Solution Chemistry and Modeling, 2016, Volume 5, No. 1 33 Journal of Applied Solution Chemistry and Modeling, 2016, Volume 5, No. PMMA Films However, in the cis orientation, there is a strong repulsion of both the rings. Thus, the structure is not planar, with one ring being almost perpendicular to the other. The repulsion is so strong that the atoms of both benzene rings appear with slight out-of-planarity (Table 1). It is noted that the torsional angles of the ring appear to be more easily deformed by this repulsion than the CCC angles, which are very close to those in the trans orientation. The C1-C2 and C4-C5 bond lengths are also shorter than the remaining C-C bonds; however, the difference is very small e.g. the aromaticity of the benzene ring is very small. The nitrogen atom withdraws less negative charge on C6 e.g. the C-N length appears to be longer than that in the trans form: 1.435 Å vs. 1.419 Å for the trans form. Because of this lengthening, the nitrogen atoms can be more strongly bonded: 1.241 Å vs. 1.251 Å for the trans form. All these features indicate the high flexibility of the molecule. Because of the asymmetry of the structure, the dipole moment is markedly increased, by up to 3.215 D, from the trans  cis isomerization (Table 3). We also simulated the interaction of trans-AZ in PMMA films using a simplified model with two sets of PMMA (n = 5) (Figure 1b) and its full optimization. (n = 5) (Figure 1b) and its full optimization. Only two very weak C-HO H-bonds and a C-HN interaction appear between PMMA and AZ, leading to the rotation of both benzene ring planes to an angle of 37.6°. The effect of PMMA in the bond lengths of AZ is small, with changes lower than 0.005 Å. However, it is very large in the C1-C6-N angle (Table 1). Computational Details 1 3 Theoretical Interpretation of Polarized Light-Induced Supramolecular 33 Table 1: Optimized Geometrical Parameters (Å, °) at B3LYP Level of AZ cis Trans Trans in PMMA Parameters 6-31G (d,p) 6-311+G (2d,p) 6-31G (d,p) 6-311+G (2d,p) 6-31G (d,p) Bond lengths C1-C2 C2-C3 C3-C4 C4-C5 C5-C6 C1-C6 C6-N N=N Bond angles C2-C1-C6 C-C2-C C-C3-C C-C4-C C-C5-C C-C6-C C1-C6-N C-N=N Torsion angles C1-C2-C3-C4 C6-C1-C2-C3 C2-C1-C6-N C1-C6-N=N C6-N=N-C 1.393 1.397 1.397 1.393 1.401 1.403 1.436 1.249 119.6 120.5 119.8 120.1 120.0 119.9 123.0 124.0 -1.5 0.8 172.1 50.5 9.8 1.389 1.392 1.392 1.388 1.395 1.397 1.435 1.241 119.6 120.4 119.7 120.2 119.9 120.0 122.7 124.1 -1.6 0.9 172.4 52.1 9.1 1.389 1.401 1.396 1.394 1.401 1.406 1.419 1.261 119.6 120.4 120.0 119.8 120.2 119.9 124.8 114.8 0.0 0.0 180.0 0.0 0.0 1.385 1.396 1.391 1.389 1.396 1.401 1.419 1.251 119.6 120.5 120.0 119.9 120.2 120.0 124.7 115.4 0.0 0.0 180.0 0.0 0.0 1.393 1.397 1.400 1.390 1.405 1.403 1.416 1.261 120.2 119.7 120.2 120.4 119.6 119.9 115.3 115.8 -0.2 0.7 -179.0 -170.6 -176.4 Table 1: Optimized Geometrical Parameters (Å, °) at B3LYP Level of AZ PMMA Films (B3LYP/6-31G(d,p)) Figures 2 and 3 show the simulated scaled IR spectra. Other scaling procedures can be performed, but they appear less accurate than LSEP [48, 49]. Table S1 gives the theoretical and scaled wavenumbers obtained by IR and Raman (calculated for comparison) spectroscopy for the trans form, while Table S2 refers to the cis form. The first column lists, in increasing order, the calculated wavenumbers at the B3LYP/6-31G(d,p) level, while the second column shows their respective relative (%) IR intensities (A), and the third column shows their relative Raman scattering activities (S). The relative IR and Raman intensities were obtained by normalizing the computed values to the intensity of the strongest line. The fourth to tenth columns show the calculated values at the B3LYP/6-311+G(2d,p) level. The reduced masses (μ) Wavenumber Calculation of AZ Molecule The AZ molecules and their derivatives have been the subject of many experimental studies using IR and Journal of Applied Solution Chemistry and Modeling, 2016, Volume 5, No. 1 Ito et al. (a) (b) ure 1: (a) Top and side views of optimized molecular structures of cis- and trans-AZ. (b) Top and side views of optimized ecular system of trans-AZ and two sets in PMMA (n = 5). 34 Journal of Applied Solution Chemistry and Modeling, 2016, Volume 5, No. 1 Ito et al. (a) (b) Figure 1: (a) Top and side views of optimized molecular structures of cis- and trans-AZ. (b) Top and side views of optimized molecular system of trans-AZ and two sets in PMMA (n = 5). Figure 1: (a) Top and side views of optimized molecular structures of cis- and trans-AZ. (b) Top and side views of optimized molecular system of trans-AZ and two sets in PMMA (n = 5). Table 2: Calculated Natural NBO Atomic Charges with the B3LYP Method in AZ cis trans Atom 6-31G (d,p) 6-311+G (2d,p) 6-31G (d,p) 6-311+G (2d,p) C1 C2 C3 C4 C5 C6 N -0.242 -0.231 -0.236 -0.233 -0.233 0.069 -0.128 -0.209 -0.192 -0.202 -0.195 -0.200 0.077 -0.138 -0.223 -0.237 -0.222 -0.242 -0.211 0.097 -0.197 -0.189 -0.199 -0.185 -0.206 -0.173 0.093 -0.205 Table 2: Calculated Natural NBO Atomic Charges with the B3LYP Method in AZ Journal of Applied Solution Chemistry and Modeling, 2016, Volume 5, No. 1 Theoretical Interpretation of Polarized Light-Induced Supramolecular Journal of Applied Solution Chemistry and Modeling, 2016, Volume 5, No. 1 3 oretical Interpretation of Polarized Light-Induced Supramole 35 Table 3: Theoretical Computed Total Energies and Gibbs Free Energy (A.U.), Rotational Constants (GHz), Entropies (calmol-1K-1) and Dipole Moments (Debyes) at the B3LYP Level in AZ Molecule cis trans Trans in PMMA Parameters 6-31G (d,p) 6-311+G (2d,p) 6-31G (d,p) 6-311+G (2d,p) 6-31G (d,p) Total energy+ZPE Gibbs Free energy Rotational constants Entropy Total Translational Rotational Vibrational Dipole moments .563267 a .601152 a 1.446 0.455 0.408 104.4 41.5 31.5 31.5 3.197 .709860 a .747804 a 1.438 0.460 0.414 104.6 41.5 31.4 31.7 3.215 .585832 a .625272 a 2.733 0.292 0.264 105.7 41.5 31.7 32.5 0.0 .733520 a .772352 a 2.753 0.292 0.264 106.7 41.5 31.7 33.5 0.0 .006332 b 0.023 0.009 0.008 12.315 a-572. b-4112. Wavenumber Calculation of AZ Molecule Table 3: Theoretical Computed Total Energies and Gibbs Free Energy (A.U.), Rotational Constants (GHz), Entropies (calmol-1K-1) and Dipole Moments (Debyes) at the B3LYP Level in AZ Molecule ed Total Energies and Gibbs Free Energy (A.U.), Rotational Constants (GHz), Entropies pole Moments (Debyes) at the B3LYP Level in AZ Molecule (seventh column), force constant of each vibration (f) (eighth column), Raman depolarization ratios for plane (P) (ninth column), and unpolarized incident light (U) (tenth column) are also shown. Columns 11 and 12 correspond to the scaled values from the scaling equation with the two basis sets. Two wavenumbers have been characterized from each ring mode corresponding to the two phenyl rings (with the exception of modes 1 and 10b). These values can be directly compared to the experimental IR bands. In the last column, the characterization for each calculated wavenumber determined in the ring and substituent modes is shown. Assignment of the ring modes followed the Varsanyi notation [61] for a mono- substituted benzene. Raman spectroscopic techniques [54, 57-60]. However, they have not been as accurately assigned as in the present study (Table S1). Moreover, we have reassigned several bands. In addition, the analysis of AZ in PMMA films has not yet been reported; thus, we present a discussion of such an analysis. The simulation of the vibrational spectra is based on DFT calculations and accurate scaling. The calculated wave numbers in the AZ molecule were improved using the linear scaling equation procedure (LSEP). For this purpose, a scaling equation was determined in the benzene molecule at the same level as that in the AZ: scaled = 22.1 + 0.9543 · calculated (B3LYP/6-31G(d,p)) For our simulated model, analysis and assignment of the computed values of AZ in PMMA films were also performed. Table S1 shows the vibrations corresponding to AZ in this system. These were identified from the 546 frequencies calculated for the system. The simulated scaled IR spectrum with these frequencies is included in Figures 2 and 3, in addition to others in which there appears to be a noticeable % contribution of the modes of AZ. The last column of Table S1 shows the calculated frequency shift of AZ in the simulated PMMA film. As observed, few modes appear to be coupled between AZ and PMMA i.e. the interaction between them is weak and has little influence on the frequency values. (B3LYP/6-31G(d,p)) Metal Complexes (with AZ in PMMA Films) Electron-withdrawing Br atoms are bonded as substituents in two phenyl rings. Figures 4-6 show the 36 Journal of Applied Solution Chemistry and Modeling, 2016, Volume 5, No. 1 Ito et al. Ito et al. 36 36 Journal of Applied Solution Chemistry and Modeling, 2016, Volume 5, No. 1 Ito et al Figure 2: Theoretical scaled IR spectra in the 3300–2900 cm1 range in the cis and trans isomers and at the B3LYP/6-31G(d,p) d B3LYP/6 311 G(2d ) l l i th l ti d bt i d f th b l l d th t i l t d Journal of Applied Solution Chemistry and Modeling, 2016, Volume 5, No. 1 Ito et al. ure 2: Theoretical scaled IR spectra in the 3300–2900 cm1 range in the cis and trans isomers and at the B3LYP/6-31G(d,p) d B3LYP/6-311+G(2d,p) levels using the scale equation procedure obtained from the benzene molecule and that simulated h t AZ i PMMA Figure 2: Theoretical scaled IR spectra in the 3300–2900 cm1 range in the cis and trans isomers and at the B3LYP/6-31G(d,p) and B3LYP/6-311+G(2d,p) levels using the scale equation procedure obtained from the benzene molecule and that simulated with trans-AZ in PMMA. optimized structure obtained using the B3LYP, M052X, and M062X DFT methods and the 6-31G(d,p) basis set. Conformer 1 corresponds to the most stable case, although the difference in energy is very small in the Ni complex (0.98 kcal mol1 with B3LYP) and high in the Cu complex (2.83 kcal mol1). In these structures, all the phenyl rings are out-of-planarity to avoid steric interactions. The calculated wavenumbers were scaled following LSEP equations: The experimental and scaled IR spectra are shown in Figures S6-S8. Because of the IR intensity depending of many factors not considered in the theory, this comparison must be performed on the frequency values. A summary of the most intense IR bands is provided in Tables S3-S5. In general, the scaled wavenumbers appear very close to the experimental data, with errors less than 2%. Moreover, the three DFT methods show similar results i.e. the simulated structure and the assignment seem to be correct. Small differences are observed in the experimental IR spectra of the complexes with Ni, Cu, and Zn e.g. the influence of the metal in the structure of each individual fragment is small. Metal Complexes (with AZ in PMMA Films) The differences optimized structure obtained using the B3LYP, M052X, and M062X DFT methods and the 6-31G(d,p) basis set. Conformer 1 corresponds to the most stable case, although the difference in energy is very small in the Ni complex (0.98 kcal mol1 with B3LYP) and high in the Cu complex (2.83 kcal mol1). In these structures, all the phenyl rings are out-of-planarity to avoid steric interactions. The calculated wavenumbers were scaled following LSEP equations: scaled = 22.1 + 0.9543 · calculated (B3LYP/6-31G(d,p)) scaled = 24.5 + 0.9366 · calculated (M052X/6-31G(d,p)) scaled = 22.7 + 0.9417 · calculated (M062X/6-31G(d,p)) scaled = 22.1 + 0.9543 · calculated (B3LYP/6-31G(d,p)) scaled = 24.5 + 0.9366 · calculated (M052X/6-31G(d,p)) scaled = 22.7 + 0.9417 · calculated (M062X/6-31G(d,p)) Theoretical Interpretation of Polarized Light-Induced Supramolecular Journal of Applied Solution Chemistry and Modeling, 2016, Volume 5, No. 1 37 Light-Induced Supramolecular Journal of Applied Solution Chemistry and Modeling, 2016, Volume 5, No. 1 37 of Polarized Light-Induced Supramolecular Journal of Applied Solution Chemistry and Modeling, heoretical scaled IR spectrum in the 1700–50 cm1 range using the scale equation procedure from th d experimental IR spectra in the 1700–400 cm1 range. Figure 3: Theoretical scaled IR spectrum in the 1700–50 cm1 range using the scale equation procedure from the benzene molecule and experimental IR spectra in the 1700–400 cm1 range. Journal of Applied Solution Chemistry and Modeling, 2016, Volume 5, No. 1 Ito et al. 38 Figure 4: Two views of the optimized molecular structure with the B3LYP, M052X, and M062X DFT methods in conformer 1 of the azobenzene complex with Ni. All the bond lengths values are in Å. At the bottom is the total energy of the complex (+ ZPE) in AU. Figure 4: Two views of the optimized molecular structure with the B3LYP, M052X, and M062X DFT methods in conformer 1 of the azobenzene complex with Ni. All the bond lengths values are in Å. At the bottom is the total energy of the complex (+ ZPE) in AU. observed experimentally are markedly smaller than those determined theoretically in the isolated state, indicating that the Nujol solution perhaps reduces the molecular flexibility and special distribution of the fragments in these complexes to a simpler form. Only the spectrum of the complex with Zn shows some significant difference. Metal Complexes (with AZ in PMMA Films) This is confirmed by the similar wavenumber observed for the (C-N) mode, corresponding to the complexes with Ni (1629 cm1), Cu (1628 cm1), and Zn (1610 cm1). respectively, and very strong IR intensity in accordance with the experimental band, showing strong intensity at 1629 cm1. The bands corresponding to C45-N44 and C2-N1 appear at lower wavenumbers (in accordance with their longer bond lengths) and with weak IR intensity. In the Cu complex (Figure 5), the carbon and nitrogen atoms bonded to the Cu atom are not in-plane, with values of O-Cu-O = 154.7° and N-Cu-N = 152.9° with B3LYP. Similar values were obtained by M052X and M062X. Similar to the Ni complex, this molecule is asymmetric with three weak contacts, as shown in Figure 5. It should be noted that the removal of one hydrogen atom was necessary (Figure 5) to produce a stable structure. The (C=N) stretchings are predicted with very strong IR intensity at 1851 cm1 (scaled at 1788 cm1) and at 1676 cm1 (scaled at 1621 cm1) by B3LYP. This is also in very good accordance with the M052X and M062X values and with the experimental band detected at 1628 cm1. With regard to the molecular structure of the Ni complex (Figures 4 and S3), all the carbon and nitrogen atoms bonded to the Ni atom are in-plane, with values of O-Ni-O = 177.4° and N-Ni-N = 178.9° Contributing to the asymmetry of the molecule are three weak contacts/H-bonds: H8Br (2.546 Å with B3LYP), O33H5 (2.053 Å), and O32H58 (2.068 Å), observed as a violet color at the B3LYP/6-31G(d,p) level in Figure 4. Consequently, the O33-Ni bond is slightly shorter than the O32-Ni bond, and the N44-Ni bond is slightly longer than the N1-Ni bond. In the middle IR spectrum, the bands with the highest intensity correspond to C-N stretching. The shortest bonds are C=N, and these correspond to C64-N44 (1.293 Å with B3LYP) and C20-N1 (1.301 Å). They have calculated wavenumbers at 1684 cm1 (scaled at 1629 cm1) and at 1669 cm1 (scaled at 1615 cm1), The Zn complex shows the greatest degree of out- of-planarity of the carbon and nitrogen atoms bonded to the Zn atom, with values of O-Zn-O = 133.4° and N- Zn-N = 136.7° by B3LYP. This molecule is also stabilized by weak contacts, as shown in Figure 6. Linearly Polarized UV Light-Induced Optical Anisotropy To allow for selective observation of contained components (AZ or complexes) in hybrid materials (in other words, direct proof of supramolecular interaction from the Weigert effect to the optical anisotropy of complexes), polarized IR spectra (Figure 8) were measured, in which C=N bands of metal complexes were used as a probe to detect each component separately. Tables 6 and 7 summarize the optical anisotropic R and S values from the polarized IR spectra of the hybrid materials Ni + AZ + PMMA, Cu + AZ + PMMA, and Zn + AZ + PMMA after linearly polarized UV light irradiation. The 10-min R values due to C=N bands of Ni (1629 cm1), Cu (1628 cm1), and Zn (1610 cm1) are 1.1770, 1.0058, and 1.0509 for Ni + AZ + PMMA, Cu + AZ + PMMA, and Zn + AZ + PMMA, respectively. Figure 7 shows polarized UV-vis spectra before and after linearly polarized UV light irradiation for 10 min at 45° and circular diagrams of the angular dependence of absorbance at -* (318 nm), n-* (440 nm), and d- d bands in polarized UV-vis spectra before and after linearly polarized UV light irradiation (0.5, 1, 3, 5, and 10 min). At the d-d band (Ni: 595 nm, Cu: 625 nm), increases in optical anisotropy were observed. These results suggest that both AZ and the complexes were oriented by linearly polarized UV light irradiation. Tables 4 and 5 summarize optical anisotropic parameters (R and S values) of the hybrid materials Ni + AZ + PMMA, Cu + AZ + PMMA, and Zn + AZ + PMMA. To allow for discussion of the Weigert effect, the degree of photo-induced optical anisotropy (in other words, spectral dichroism) of polarized UV-vis spectra can be commonly described by two parameters: The R and S values at the -* bands of the polarized UV-vis spectra provided information on molecular orientation and conformation. Meanwhile, the R and S values at the d-d band of the polarized UV-vis spectra and the C=N bands of the polarized IR spectra provide information such as the coordination environment. Tables 4 and 6 indicate the following mechanism. Metal Complexes (with AZ in PMMA Films) In the middle IR-spectrum, the bands with the highest IR Theoretical Interpretation of Polarized Light-Induced Supramolecular Journal of Applied Solution Chemistry and Modeling, 2016, Volume 5, No. 1 39 Light-Induced Supramolecular Journal of Applied Solution Chemistry and Modeling, 2016, Volume 5, No. 1 39 39 Figure 5: Two views of the optimized molecular structure with the B3LYP, M052X, and M062X DFT methods in conformer 1 of the azobenzene complex with Cu. Figure 5: Two views of the optimized molecular structure with the B3LYP, M052X, and M062X DFT methods in conformer 1 of the azobenzene complex with Cu. Figure 5: Two views of the optimized molecular structure with the B3LYP, M052X, and M062X DFT methods in conformer 1 of the azobenzene complex with Cu. Figure 6: Two views of the optimized molecular structure with the B3LYP, M052X, and M062X DFT methods in conformer 1 of the azobenzene complex with Zn. e optimized molecular structure with the B3LYP, M052X, and M062X DFT methods in conformer 1 with Zn Figure 6: Two views of the optimized molecular structure with the B3LYP, M052X, and M062X DFT methods in conformer 1 of the azobenzene complex with Zn. Figure 6: Two views of the optimized molecular structure with the B3LYP, M052X, and M062X DFT methods in conformer 1 of the azobenzene complex with Zn. Through the analysis of the molecular structure of these complexes in the isolated state, we have not observed any particular difference that would give rise to a flexible structure for Ni and Cu and a rigid complex for Zn. The only special characteristic of the Zn complex is the high out-of-planarity of the structure. intensity also correspond with the (C=N) stretchings calculated at 1680 cm1 (scaled at 1625 cm1) and at 1667 cm1 (scaled at 1613 cm1). They are also in very good accordance with the M052X and M062X values and with the weak band observed in the experimental spectrum at 1610 cm1. Journal of Applied Solution Chemistry and Modeling, 2016, Volume 5, No. 1 Ito et al. 40 Perhaps, this out-of-planarity leads to high molecular interactions (H-bonds) with neighboring molecules, leading to a rigid complex. This complex with Zn is also more open, with a larger Br30…Br43 distance (14.8 Å by B3LYP) than that in the Ni complex (14.4 Å) and in the Cu complex (13.9 Å). Metal Complexes (with AZ in PMMA Films) Assignment of spectral peaks was in agreement with the analogous compounds [26, 28, 38, 39]. As listed in Tables 4 and 5, the -* and n-* bands of AZ were directly changed by the Weigert effect, while the d-d bands of Ni or Cu were changed by supramolecular interaction in the PMMA matrix. However, Zn did not exhibit d-d bands because of d10 configuration of the Zn(II) center [22]. Low-lying molecular vibrations have been studied by several authors [63-65], and their presence indicates a high flexibility in the molecular structure, which may be caused by various factors. Twenty-nine frequencies below 200 cm1 were found in the complex with Cu, corroborating its high flexibility, while in the complex with Zn, 28 were found and in the complex with Ni, 26 were found. A first group of these vibrations includes those that represent the motion of the different rings with respect to each other as almost rigid subsystems, while a second group involves their intramolecular deformation. In the PMMA hybrid materials, the 10-min R values due to the d-d bands of Ni and Cu are 5.6838 and 1.5286 for Ni + AZ + PMMA and Cu + AZ + PMMA, respectively. Focusing on the common -* bands of AZ around 318 nm, the 10-min R values are 1.0139, 1.0037, and 0.9338 for Ni, Cu, and Zn + AZ + PMMA, respectively. According to Table 6, the degree of freedom may be caused by intermolecular interaction of non-contacted molecules of AZ and Ni, Cu, or Zn. Similar behavior was observed for the S values, as summarized in Table 5. In general, looseness of data is mainly attributed to AZ degradation [23]. Linearly Polarized UV Light-Induced Optical Anisotropy Increases in optical anisotropy after linear UV light irradiation for 10 min followed the following order: Zn + AZ + PMMA > Ni + AZ + PMMA > Cu + AZ + PMMA at -*, Ni + AZ + PMMA > Cu + AZ + PMMA > Zn + AZ + PMMA at n-*, and Ni + AZ + PMMA > Cu + AZ + PMMA at d-d. The reason for maximum -* values for Zn + AZ + PMMA may be R = Aperpendicular/Aparrallel where Aperpendicular and Aparrallel values denote absorbance measured with the polarizer perpendicular or parallel, respectively, to the electric vector of irradiation polarized light. The ideal isotropic systems of S = 0 and R = 1, and both S and R parameters, are changed with increasing dichroism by molecular alignment. where Aperpendicular and Aparrallel values denote absorbance measured with the polarizer perpendicular or parallel, respectively, to the electric vector of irradiation polarized light. The ideal isotropic systems of S = 0 and R = 1, and both S and R parameters, are changed with increasing dichroism by molecular alignment. Theoretical Interpretation of Polarized Light-Induced Supramolecular Journal of Applied Solution Chemistry and Modeling, 2016, Volume 5, No. 1 41 Light-Induced Supramolecular Journal of Applied Solution Chemistry and Modeling, 2016, Volume 5, No. 1 41 Theoretical Interpretation of Polarized Light-Induced Supramolecular 41 Figure 7: Polarized UV-vis spectra at 45° [above] and angular dependence of absorbance at 318, 440, and 595 nm [below] for Ni + AZ + PMMA after linearly polarized UV light irradiation for 0–10 min. Figure 7: Polarized UV-vis spectra at 45° [above] and angular dependence of absorbance at 3 Ni + AZ + PMMA after linearly polarized UV light irradiation for 0–10 min. pectra at 45° [above] and angular dependence of absorbance at 318, 440, and 595 nm [below] for y polarized UV light irradiation for 0–10 min. 90° direction became stronger because of an allowed transition, which is parallel to the transition dipole moment (long molecular axis) of cis-AZ. The N=N bonds of cis-AZ are expected to be oriented parallel to the 90° polarized IR by the vibration mode of the N=N stretching vibration; this has been discussed on the basis of TD-DFT theoretical calculations. that distorted Zn become to exhibit small contribution and mainly observed intense absorption from orientated AZ. The order of C=N (complexes) and N=N (AZ) bands from the polarized IR spectra is Ni + AZ + PMMA > Zn + AZ + PMMA > Cu + AZ + PMMA. The results indicate that Ni + AZ + PMMA may induce optical anisotropy most effectively. This also suggests that re-orientation of the complexes due to the Weigert effect of AZ depends on the flexibility of the coordination environment produced by the metal ions [28] and the overall planarity of the ligands or molecules. Next, we discuss orientation of the complexes. R = Aperpendicular/Aparrallel Because the C=N bond also has a strong absorption at 90° in the polarized IR spectra, this bond is considered to be oriented parallel to 90°. In other words, the C=N bond of complexes and N=N bond of AZ are aligned in a parallel fashion after linearly polarized light irradiation (Figure 11). Interpretation of the Molecular Orientation According to our findings, we can examine the orientation of each molecule. Initially, we discuss the orientation of AZ on the basis of the results of the polarized UV-vis spectra. As shown in Figure 9, when linearly polarized UV light vibrating (having an electric vector) in the 0° direction was irradiated onto the hybrid materials, the intensity of the -* band in the 90° direction became stronger because of the Weigert effect, which is parallel to the transition dipole moment (long molecular axis) of cis-AZ [34, 38, 39]. Therefore, N=N bonds of AZ are expected to be oriented perpendicular to the irradiated polarized light. Here we consider the driving force that leads to complexes becoming oriented. If complexes are directly oriented because of the Weigert effect by polarized UV light irradiation, the dipole moment of the complexes should be perpendicular to the irradiated polarized UV light (electric vector). According to the molecular shape (long molecular axis) of the complexes, the dipole moment of the complex must be vertical to the electric vector; in other words, the C=N bonds of the complex are not parallel to the N=N bond in AZ. Consequently, the mechanism of light-induced molecular orientation of the complexes was not directly because of the Weigert effect but because of supramolecular transmission from the re-orientation of AZ [33]. As shown in Figure 10, when polarized UV light vibrating in the 0° direction was irradiated onto the hybrid materials, the IR intensity of the N=N band in the 42 Journal of Applied Solution Chemistry and Modeling, 2016, Volume 5, No. 1 Ito et al. Ito et al. 42 42 42 Journal of Applied Solution Chemistry and Modeling, 2016, Volume 5, No. 1 Ito et al. Interpretation of the Molecular Orientation Table 4: The R Values from UV-vis Spectra after Linearly Polarized UV light irradiation UV radiation minutes initial UV0.5 UV1 UV3 UV5 UV10 318 nm, -* 1.0079 1.0134 1.0100 1.0075 1.0069 1.0139 440 nm, n-* 1.1817 1.2466 1.1736 1.2046 1.2056 1.2646 Ni+AZ+ PMMA 595 nm, d-d (Ni) 1.8098 3.2780 4.1863 -173.79 -4.7056 -5.6838 318 nm, -* 0.9775 1.0163 1.0154 1.0534 1.0247 1.0037 440 nm, n-* 0.8465 1.2605 1.2092 1.2938 1.2207 1.2335 Cu+AZ+ PMMA 625 nm, d-d(Cu) 1.2043 1.4599 1.4123 1.6162 1.4930 1.5286 318 nm, -* 0.9499 0.9680 0.9706 0.9453 0.9439 0.9338 Zn+AZ+ PMMA 440 nm, n-* 0.7825 1.1476 1.2053 1.1661 1.1658 1.1797 Table 5: The S Values from UV-vis Spectra after Linearly Polarized UV Light Irradiation UV radiation minutes initial UV0.5 UV1 UV3 UV5 UV10 318 nm, -* 0.0026 0.0045 0.0033 0.0025 0.0023 0.0046 440 nm, n-* 0.0571 0.0760 0.0547 0.0638 0.0641 0.0811 Ni+AZ+ PMMA 595 nm, d-d (Ni) 0.2125 0.4316 0.5151 1.0175 2.1088 1.8144 318 nm, -* -0.0075 0.0054 0.0051 0.0175 0.0082 0.0012 440 nm, n-* -0.0539 0.0799 0.0652 0.0892 0.0685 0.0722 Cu+AZ+ PMMA 625 nm, d-d(Cu) 0.0637 0.1329 0.1208 0.1704 0.1411 0.1498 318 nm, -* -0.0170 -0.0108 -0.0099 -0.0186 -0.0191 -0.0226 Zn+AZ+ PMMA 440 nm, n-* -0.0782 0.0469 0.0640 0.0525 0.0524 0.0565 Table 6: The R Values from IR Spectra after Linearly Polarized UV Light Irradiation Table 4: The R Values from UV-vis Spectra after Linearly Polarized UV light irradiation UV radiation minutes initial UV0.5 UV1 UV3 UV5 UV10 318 nm, -* 1.0079 1.0134 1.0100 1.0075 1.0069 1.0139 440 nm, n-* 1.1817 1.2466 1.1736 1.2046 1.2056 1.2646 Ni+AZ+ PMMA 595 nm, d-d (Ni) 1.8098 3.2780 4.1863 -173.79 -4.7056 -5.6838 318 nm, -* 0.9775 1.0163 1.0154 1.0534 1.0247 1.0037 440 nm, n-* 0.8465 1.2605 1.2092 1.2938 1.2207 1.2335 Cu+AZ+ PMMA 625 nm, d-d(Cu) 1.2043 1.4599 1.4123 1.6162 1.4930 1.5286 318 nm, -* 0.9499 0.9680 0.9706 0.9453 0.9439 0.9338 Zn+AZ+ PMMA 440 nm, n-* 0.7825 1.1476 1.2053 1.1661 1.1658 1.1797 4: The R Values from UV-vis Spectra after Linearly Polarized UV light irradiation Table 5: The S Values from UV-vis Spectra after Linearly Polarized UV Light Irradiation UV radiation minutes initial UV0.5 UV1 UV3 UV5 UV10 318 nm, -* 0.0026 0.0045 0.0033 0.0025 0.0023 0.0046 440 nm, n-* 0.0571 0.0760 0.0547 0.0638 0.0641 0.0811 Ni+AZ+ PMMA 595 nm, d-d (Ni) 0.2125 0.4316 0.5151 1.0175 2.1088 1.8144 318 nm, -* -0.0075 0.0054 0.0051 0.0175 0.0082 0.0012 440 nm, n-* -0.0539 0.0799 0.0652 0.0892 0.0685 0.0722 Cu+AZ+ PMMA 625 nm, d-d(Cu) 0.0637 0.1329 0.1208 0.1704 0.1411 0.1498 318 nm, -* -0.0170 -0.0108 -0.0099 -0.0186 -0.0191 -0.0226 Zn+AZ+ PMMA 440 nm, n-* -0.0782 0.0469 0.0640 0.0525 0.0524 0.0565 Table 5: The S Values from UV-vis Spectra after Linearly Polarized UV Light Irradiation Table 6: The R Values from IR Spectra after Linearly Polarized UV Light Irradiation UV radiation minutes initial UV0.5 UV1 UV3 UV5 UV10 Ni+AZ+ PMMA 1629 cm -1 C=N(Ni) 1.0079 1.0134 1.0100 1.0075 1.0069 1.0139 Cu+AZ+ PMMA 1628 cm -1 C=N(Cu) 0.9775 1.0163 1.0154 1.0534 1.0247 1.0037 Zn+AZ+ PMMA 1610 cm -1 C=N(Zn) 0.9499 0.9680 0.9706 0.9453 0.9439 0.9338 Table 6: The R Values from IR Spectra after Linearly Polarized UV Light Irradiation Theoretical Interpretation of Polarized Light-Induced Supramolecular Journal of Applied Solution Chemistry and Modeling, 2016, Volume 5, No. CONCLUSION We have prepared organic/inorganic hybrid materials (Ni + AZ + PMMA, Cu + AZ + PMMA, and Zn + AZ + PMMA) and investigated their light-induced supramolecular orientation using experiments and theoretical calculations. Irradiation of the hybrid materials with linearly polarized light resulted in increased optical anisotropy in each case. Anisotropy of Ni + AZ + PMMA was increased most markedly. After irradiation with circularly polarized light, new CD peaks emerged, and these were attributed to an induced helical orientation. We could individually observe the molecular orientation of AZ and complexes by polarized IR measurement. We found a parallel arrangement of N=N (AZ) and C=N (complexes) bands, which is perpendicular to the electric vector of the irradiated light. This is proof of supramolecular orientation of complexes with transmission from the re- orientation of AZ, which is directly rotated by the Weigert effect. The development of other systems for polymer matrices such as proteins [67] for new photofunctional materials is in progress. Figure 11: Expected supramolecular orientation of a complex with AZ from polarized IR spectra: (a) irradiated polarized light (electric vector). Circularly Polarized UV Light-Induced Chiral Supramolecular Arrangement We also attempted to induce helical orientation by the irradiation of circularly polarized light onto the hybrid materials. Figure 12 depicts the CD spectra before and after circularly polarized UV light irradiation and their difference spectra. After irradiation for 10 min, new CD peaks appeared at 330 nm. Because complexes do not have peaks in the CD spectra (or UV-vis spectra) in this region [38, 39], this suggests that supramolecular chiral arrangements were induced [66]. Interpretation of the Molecular Orientation 1 43 Light-Induced Supramolecular Journal of Applied Solution Chemistry and Modeling, 2016, Volume 5, No. 1 43 Theoretical Interpretation of Polarized Light-Induced Supramolecular Journal of Applied Solution Chemistry and M Theoretical Interpretation of Polarized Light-Induced Supramolecular 43 Figure 8: Polarized IR spectra at 45° [above] and angular dependence of transmittance at 1629 cm1 (C=N) and 1558 cm1 (N=N) [below] for Ni + AZ + PMMA after linearly polarized UV light irradiation for 0–10 min. Figure 8: Polarized IR spectra at 45° [above] and angular dependence of transmittance at 1629 cm1 (C=N) and 1558 cm1 (N=N) [below] for Ni + AZ + PMMA after linearly polarized UV light irradiation for 0–10 min. Table 7: The S Values from IR Spectra after Linearly Polarized UV Light Irradiation UV radiation minutes initial UV0.5 UV1 UV3 UV5 UV10 Ni+AZ+ PMMA 1629 cm -1 C=N(Ni) 0.0422 0.0586 0.0682 0.0753 0.0783 0.0557 Cu+AZ+ PMMA 1628 cm -1 C=N(Cu) 0.0019 0.0026 0.0020 -0.0034 0.0039 0.0019 Zn+AZ+ PMMA 1610 cm -1 C=N(Zn) 0.0209 0.0115 0.0202 0.0113 0.0140 0.0167 Figure 9: Expected molecular orientation of AZ on the basis of polarized UV-vis spectra: (a) irradiated polarized light (electric vector), (b) transition dipole moment (AZ), and (c) strong absorption (experimental result). Figure 10: Expected molecular orientation of AZ from polarized IR spectra: (a) irradiated polarized light (electric vector), (b) N=N stretching vibration, and (c) strong absorption (experimental results). Table 7: The S Values from IR Spectra after Linearly Polarized UV Light Irradiation Figure 10: Expected molecular orientation of AZ from polarized IR spectra: (a) irradiated polarized light (electric vector), (b) N=N stretching vibration, and (c) strong absorption (experimental results). Figure 10: Expected molecular orientation of AZ from polarized IR spectra: (a) irradiated polarized light (electric vector), (b) N=N stretching vibration, and (c) strong absorption (experimental results). Figure 9: Expected molecular orientation of AZ on the basis of polarized UV-vis spectra: (a) irradiated polarized light (electric vector), (b) transition dipole moment (AZ), and (c) strong absorption (experimental result). 4 Journal of Applied Solution Chemistry and Modeling, 2016, Volume 5, No. 1 Ito et al. 44 Figure 11: Expected supramolecular orientation of a complex with AZ from polarized IR spectra: (a) irradiated polarized light (electric vector). 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Theoretical analysis of low-lying vibrational modes of free canonical 2-deoxyribonucleosides. Chem Phys 2000; 260: 317-25. http://dx.doi.org/10.1016/S0301-0104(00)00251-2 [64] Hovorun DM, Mishchuk TR, Yurenko YP. Biopolymer Cell 2002; 18: 219. [65] Martel P, Hennion B, Durand D, Calmettes P. Low-frequency vibrations of a nucleoside analog. J Biomol Struct Dyn 1994; 12: 401-11. http://dx.doi.org/10.1080/07391102.1994.10508748 [66] unaga N, Furuya S, Ito M, Kominato C, Akitsu T Computational Chemistry: Theory, Methods and Applications, Nova Science Publishers, Inc. 2014; 85. [67] Kominato C, Akitsu T. Lett Appl Nano BioSci 2015; 2: 264. [60] Gruger A, Le Calvé N, Dizabo P, Fillaux J. J Chim Phys 1972; 69: 291. [60] Gruger A, Le Calvé N, Dizabo P, Fillaux J. J Chim Phys 1972; 69: 291. canonical 2-deoxyribonucleosides. Chem Phys 2000; 260: 317-25. http://dx.doi.org/10.1016/S0301-0104(00)00251-2 [61] Varsanyi G, Assignments for vibrational Spectra of Seven hundred benzene derivatives, Vol 1, (Adam Hilger, London) 1974; 280. [64] Hovorun DM, Mishchuk TR, Yurenko YP. Biopolymer Cell 2002; 18: 219. [62] Agarwal P, Bee S, Gupta A, et al. Quantum chemical study on influence of intermolecular hydrogen bonding on the geometry, the atomic charges and the vibrational dynamics of 2,6-dichlorobenzonitrile. Spectrochim Acta A 2014; 121: 464-82. h //d d i /10 1016/j 2013 10 104 [65] Martel P, Hennion B, Durand D, Calmettes P. Low-frequency vibrations of a nucleoside analog. J Biomol Struct Dyn 1994; 12: 401-11. http://dx.doi.org/10.1080/07391102.1994.10508748 [66] unaga N, Furuya S, Ito M, Kominato C, Akitsu T Computational Chemistry: Theory, Methods and Applications, Nova Science Publishers, Inc. 2014; 85. Received on 01-09-2015 DOI: http://dx.doi.org/10.6000/1929-5030.2016.05.01.3 © 2016 Ito et al.; Licensee Lifescience Global. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.
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GBIS: the information system of the German Genebank
Database
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Leibniz-Institut fu¨r Pflanzengenetik und Kulturpflanzenforschung (IPK) Gatersleben, OT Gatersleben, Corrensstraße 3, 06466 Stadt Seeland, Germany Leibniz-Institut fu¨r Pflanzengenetik und Kulturpflanzenforschung (IPK) Gatersleben, OT Gatersleben, Corrensstraße 3, 06466 Stadt Seeland, Germany *Corresponding author: Tel:þ49 39482 5-360; Fax:þ49 39482 5-155; Email: oppermann@ipk-gatersleben.de Citation details: Oppermann,M.,Weise,S.,Dittmann,C., et al. GBIS: the information system of the German Genebank. Database (2015) Vol. 2015: article ID bav021; doi:10.1093/database/bav021 *Corresponding author: Tel:þ49 39482 5-360; Fax:þ49 39482 5-155; Email: oppermann@ipk-gatersleben.de *Corresponding author: Tel:þ49 39482 5-360; Fax:þ49 39482 5-155; Email: oppermann@ipk-gatersleben.de Citation details: Oppermann,M.,Weise,S.,Dittmann,C., et al. GBIS: the information system of the German Genebank. Database (2015) Vol. 2015: article ID bav021; doi:10.1093/database/bav021 Citation details: Oppermann,M.,Weise,S.,Dittmann,C., et al. GBIS: the information system of the German Gene Database (2015) Vol. 2015: article ID bav021; doi:10.1093/database/bav021 Received 17 December 2014; Revised 17 February 2015; Accepted 19 February 2015 Received 17 December 2014; Revised 17 February 2015; Accepted 19 February 2015 Abstract The German Federal ex situ Genebank of Agricultural and Horticultural Crop Species is the largest collection of its kind in the countries of the European Union and amongst the 10 largest collections worldwide. Beside its enormous scientific value as a safeguard of plant biodiversity, the plant genetic resources maintained are also of high importance for breeders to provide new impulses. The complex processes of managing such a collec- tion are supported by the Genebank Information System (GBIS). GBIS is an important source of information for researchers and plant breeders, e.g. for identifying appropri- ate germplasm for breeding purposes. In addition, the access to genebank material as a sovereign task is also of high interest to the general public. Moreover, GBIS acts as a data source for global information systems, such as the Global Biodiversity Information Facility (GBIF) or the European Search Catalogue for Plant Genetic Resources (EURISCO). Database URL: http://gbis.ipk-gatersleben.de/ V C The Author(s) 2015. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction indispensable to preserve plant genetic resources for future generations. Currently, there are about 250 000 higher plant species in the world [conservative estimate, according to Ungricht (1)]. These include 7000 crop plant species [35 000 if or- namentals and forest plants are included (2)]. Crop plants are a major source for human and animal nutrition. Plants are cultivated by man also for fibre, oil, starch and other industrial purposes. Moreover, they play an important role as medicinal plants, for chemical and pharmaceutical in- dustry and as renewable resources [e.g. (3, 4)]. Hence, it is The collection, maintenance and characterization of crop plants and their wild relatives are important contribu- tions towards the preservation of biological diversity. In particular, genebanks play an important role for the long- term conservation of plant genetic resources for food and agriculture (PGRFA). Around the world, there are about 1750 genebank collections (5). Therefore, about 625 col- lections are maintained in Europe comprising more than 2 Database, 2015, 1–8 doi: 10.1093/database/bav021 Original article V C The Author(s) 2015. Published by Oxford University Press. (page number not for citation purposes) Data domains and technical aspects Taking into account the different purposes of the Genebank Information System, the system structure can be categorized into data domains (Figure 1). The central ob- ject of the system is the accession in the sense of an anchor for the information belonging to the collection items. It is part of the passport data domain. In correspondence to the data domains, the implementation of the applications took place in different subprojects starting with a core system covering the basic functionalities. The genebank collection comprises 3220 plant species belonging to 768 genera (10). There are 31 genera with more than 1000 accessions each, but also 501 genera with less than 9 accessions each, including 199 genera repre- sented by a single accession each. The genebank has mater- ial from 150 countries from all continents except Antarctica. To give an impression about the scope of GBIS, the most important functional domains of Figure 1 are briefly described below. Page 1 of 8 (page number not for citation purposes) Database, Vol. 2015, Article ID bav021 Page 2 of 8 million accessions (6). Their focus is not only on the aspect of pure conservation. The large genetic diversity stored in genebanks is also used to provide new impulses to plant breeding, e.g. by adding new alleles to existing breeding stocks (7). (10). On request of users, i.e. plant breeders, researchers, but also private persons, the genebank is distributing research material amounting to over 30 000 samples in response to more than 1500 individual requests per year (10). The German Federal ex situ Genebank of Agricultural and Horticultural Crop Species maintained by IPK Gatersleben is amongst the 10 largest collections world- wide and the largest one in the countries of the European Union, totalling over 151 000 accessions. It has been con- tinuously developed over the past 70 years [e.g. (8, 9)]. Beside the headquarters located in Gatersleben, there exist two further branch stations. Since 2002, the material from the Braunschweig Genetic Resources Centre (BGRC), the former West German genebank, was transferred to IPK to form the Federal Collection. The highly complex work of the genebank maintenance is supported by the management component of the Genebank Information System (GBIS/M). Moreover, via the online search system GBIS/I (see below), information on genebank accessions is provided to external users around the world. The genebank information system of IPK The present Genebank Information System (GBIS) has been developed since 2002 in the frame of the fusion of the collection of the former BGRC and the IPK genebank (11). It subsequently replaced earlier versions of the IPK genetic resources documentation system (12–15), as well as docu- mentation systems of the BGRC collection (16) and the IPK branches (17, 18). Seed and line management The smallest unit kept separately in a genebank is called an accession, which is represented by physical objects such as seed samples, grow outs (plants from an accession grown together in a field plot) or lines (material preserved in vitro or in cryo). All these materializations of an accession share their origin and common information. Genebank accessions may enter the genebank collection in either of two ways: dir- ectly collected by a collector or an expedition, or provided by another collection or institution called ‘donor’. Information necessary for the management of the gene- bank objects, such as storage location, availability of seed samples or lines of each accession, germinability of seeds or results of storage disease tests, generated and managed mainly in genebank workflows. Details of the workflow support are described in ‘Internal GBIS management appli- cations GBIS/M and GBIS/B’. Passport data d Passport data includes basic information about the where, when and what of an accession, e.g. information about the collecting mission or the donor institute, collection site, names, numbers; complemented by data about the avail- ability and other status information, different crop classifi- cations, existence of specimens and images. A genebank information system at IPK has to meet nu- merous and complex requirements. Collecting expeditions and institutions The plant material preserved in the genebank originates partly from collecting expeditions in many countries (largely conducted by, or in collaboration with IPK) or from donor institutions (e.g. research institutes, plant breeders, botanical gardens or other genebanks). This data Tests Support for the just-in-time documentation of the results of health tests (e.g. for in vitro cultures) and germinability (of seed samples) on sample level. Except for the geo references, the remaining domains belong to common functionalities such as reporting for querying and statistics, and provisioning of internal and external user accounts. Observations and experiments The accessions are being preserved (i) as seed samples in cold storage facilities (ca. 96% of the collection), with the need for regular rejuvenation of the seed stock by growing in the field, (ii) vegetatively under field or greenhouse con- ditions, (iii) in vitro storage of plant tissues and (iv) cryo- preservation in liquid nitrogen. Presently, about 8000 accessions are being rejuvenated per year; thus, the average storage duration of a seed accession is around 20 years An indispensable part of the work of genebanks is the phenotypic characterization of accessions (19). Phenotypic data is also called characterization and evaluation (C&E) data. Characterization refers to recording highly heritable traits, usually controlled by a single or very few genes, while evaluation refers to traits depending on several to many genes, whose expression is usually environment-dependent. Database, Vol. 2015, Article ID bav021 Page 3 of 8 Figure 1. Data domains in GBIS. Figure 1. Data domains in GBIS. From the point of view of database management, however, we do not distinguish between characterization and evalu- ation data. Phenotypic data is primarily obtained by obser- vations during each regeneration cycle (20). Seed regeneration is usually driven by factors such as the avail- ability of seeds or their germination capacity, rather than by statistical considerations. Thus, the available phenotypic data forms a non-orthogonal data set (21). Nevertheless, the analysis of this data allows meaningful results, such as the identification of promising new alleles (22) to support the selection of material for breeding and research programmes. The data model is designed to handle observations on het- erogeneous accessions, i.e. populations consisting of differ- ent genotypes. There is a distinction between the definition of the traits with their different scales and the observations themselves that can be summarized in experiments. Traits can be organized in descriptors using a predefined crop- specific vocabulary derived from descriptor lists (23). However, this can only be guaranteed for data observed directly at IPK (characterization and primary evaluation data). Non-compliant data (secondary evaluation data) will be stored as accession-referenced files. allows for tracking the origin of an accession and provides access to further information about an accession. Ordering system To support the key task of genebanks, i.e. to provide plant (most often seed) samples to breeders, researchers and the public domain, comprehensive information needs to be managed. This includes, amongst others, the name and address of the person/institution requesting the material, the order (and the items ordered), the date of purchase and the status of the material to be delivered. Moreover, data which needs to be provided in accordance with the International Treaty (International Treaty on Plant Genetic Resources for Food and Agriculture, http://www.planttreaty.org/, as of 2014-11-28) are stored here. Document management To assist the user in managing all kinds of documents that refer to an accession. These can be herbarium specimens, as well as photos, scanned file cards or fieldbook pages and other digital documents. List management For many workflows, specific sets of accessions, samples or grow outs have to be defined, based on various criteria. Examples are: lists of accessions that need to be sown for re- generation, or lists of seed samples to be fetched out from the cold storage for distribution. Therefore, a very flexible query builder was implemented for list management and re- porting. As a special feature, lists of objects can be fixed, irrespectively of changes in the underlying data. Taxonomy Information about scientific names (present, but also his- torical determinations) of genebank accessions is funda- mental. The taxonomic hierarchy, e.g. family, genus, species and infraspecific taxa, is reflected in the system. GBIS applications GBIS consists of three subsystems: (i) a desktop application for supporting the internal collection management (GBIS/ Management), (ii) an application for mobile data Database, Vol. 2015, Article ID bav021 Page 4 of 8 two-faced tool that (i) has been designed to support a wide range of workflows within the genebank (Figure 5) and also (ii) to provide internal users with access to the GBIS database for curating genebank data outside a workflow context. All data domains described in ‘Data domains and technical aspects’ are reflected by the modules of this appli- cation (Figure 1). collection in the field (GBIS/Bonitur, German for scoring or (field) observations on plants) and (iii) an Internet search and order portal for external genebank users (GBIS/ Information, internet). The three applications share a com- mon database (Figure 2). Internal GBIS management applications GBIS/M and GBIS/B Accession management. Accessions are materialized, e.g. as seed samples, lines or grow-outs (plants grown together in the field). The accession management part of GBIS/M provides an accession-oriented access with the possibility of viewing and editing data. For every accession, it is pos- sible to trace histories of numbers and other identifiers, names, botanical determinations and origins as well as phenotypic data. Furthermore, preservation management data is accessible here. The first subsystem, GBIS/M (Figure 3), forms the internal management component of GBIS. It can be described as a Figure 2. Overview of the GBIS architecture. Work flow support. GBIS/M is highly integrated in the daily work routine of the genebank staff. For every sup- ported workflow, the data are preprocessed to give an optimized view of information to match the needs of the task. In Figure 4, the core objects of the system and the Figure 2. Overview of the GBIS architecture. Figure 3. Form with main data of an accession. It is part of the management module and enables access to all information about an accession allows the curators to manage all this information at a central location. Details of the domain objects related to the accession can be edited on the d ferent tabs. Figure 3. Form with main data of an accession. It is part of the management module and enables access to all information about an accession. It allows the curators to manage all this information at a central location. Details of the domain objects related to the accession can be edited on the dif- ferent tabs. Database, Vol. 2015, Article ID bav021 Page 5 of 8 Figure 4. Important domain objects and data management-related genebank workflows. Figure 5. Typical management workflows of the genebank, which are supported by GBIS. (a) shows the ‘seed cycle’ for seed-propagated material, whereas (b) shows the life cycle of plant material stored in vitro. atabase, o 0 5, t c e ba 0 age 5 o 8 Figure 4. Important domain objects and data management-related genebank workflows. Figure 4. Important domain objects and data management-related genebank workflows. ure 4. Important domain objects and data management-related genebank workflows. Figure 5. Typical management workflows of the genebank, which are supported by GBIS. (a) shows the ‘seed cycle’ for seed-propagated material, whereas (b) shows the life cycle of plant material stored in vitro. Figure 5. Implementation information about genebank material. It allows for search- ing and filtering accessions by various well-defined criteria and their combinations in a simple stacked way or by defining complex Boolean concatenation. For certain char- acteristics, a free-text search is implemented. If a crop cat- egory is chosen, also phenotypic data become searchable. The search results are first shown in an overview table where selected accessions can be put in a wish list for ordering or data export. By clicking on an accession num- ber, the user can view all available details of this accession. Moreover, GBIS/I offers a shopping-cart function enabling registered users to request material of interest online. It also fulfils the requirements of the International Treaty on Plant Genetic Resources for Food and Agriculture by implementing a click-wrap-mechanism for signing Standard Material Transfer Agreements (SMTA). The sys- tem is featured with an order tracking function. All data shown is refreshed at least daily, based on the real-time data in the common database. A compilation of screen- shots displaying accession detail information is shown in Figure 6. information about genebank material. It allows for search- ing and filtering accessions by various well-defined criteria and their combinations in a simple stacked way or by defining complex Boolean concatenation. For certain char- acteristics, a free-text search is implemented. If a crop cat- egory is chosen, also phenotypic data become searchable. The search results are first shown in an overview table where selected accessions can be put in a wish list for ordering or data export. By clicking on an accession num- ber, the user can view all available details of this accession. Moreover, GBIS/I offers a shopping-cart function enabling registered users to request material of interest online. It also fulfils the requirements of the International Treaty on Plant Genetic Resources for Food and Agriculture by implementing a click-wrap-mechanism for signing Standard Material Transfer Agreements (SMTA). The sys- tem is featured with an order tracking function. All data shown is refreshed at least daily, based on the real-time data in the common database. A compilation of screen- shots displaying accession detail information is shown in Figure 6. The database schema is realized on an Oracle 11g rela- tional database management system (Oracle Corporation, http://www.oracle.com/, as of 2014-10-13) in a top-down- design based on the object model developed during the re- quirement analysis phase (2002–2006). Internal GBIS management applications GBIS/M and GBIS/B Typical management workflows of the genebank, which are supported by GBIS. (a) shows the ‘seed cycle’ for seed-propagated material, whereas (b) shows the life cycle of plant material stored in vitro. information flow during some workflows between these objects are shown. field trials. For this, the relevant data (crop, traits to be observed, and observations from previous grow-outs of the same accession) is transferred from GBIS to a mobile device. After registering the observations in the field, the data are synchronized with the GBIS database. The whole surround- ing workflow is fully integrated in the GBIS/M subsystem. This strong integration of GBIS/M in the working proc- esses of the genebank leads, in case of seed and line man- agement, to a closed system (Figure 5). Each step is based on the previous one and can benefit from the information generated there. The second subsystem, GBIS/B, is a Java application that runs on mobile devices equipped with a barcode scanner (24). It supports the user in recording phenotypic data of External GBIS: Internet search and order portal GBIS/I For external users, especially plant breeders and re- searchers, the web portal GBIS/I is the main source of Database, Vol. 2015, Article ID bav021 Page 6 of 8 Implementation The comprehen- siveness results in a highly normalized database schema comprising 111 domain-specific tables as well as 78 look- up tables for decoding. To speed-up data retrieval, materialized views are used. As a sustainability-by-design feature, we keep the schema readable and understandable by non-informaticians or external IT specialists. The implementation is characterized by a strong custom- ization for the needs of the German genebank and IPK’s IT infrastructure, which is dominated by Oracle technologies. Both the desktop application GBIS/M and the web applica- tion GBIS/I make use of the Oracle Application Development Framework (http://www.oracle.com/technet- work/developer-tools/adf/, as of 2014-10-14) (25). GBIS/I runs on a Oracle WebLogic Server (http://www.oracle.com/ us/products/middleware/cloud-app-foundation/weblogic/, Figure 6. Compilation of screenshots of the accession details view in the GBIS/I application. It shows a kaleidoscope of data from different data d mains supporting scientists, breeders and other interested users to select appropriate genebank accessions. It represents the view for external use onto the data managed by the genebank curators as shown in Figure 3. Figure 6. Compilation of screenshots of the accession details view in the GBIS/I application. It shows a kaleidoscope of data from different data do- mains supporting scientists, breeders and other interested users to select appropriate genebank accessions. It represents the view for external users onto the data managed by the genebank curators as shown in Figure 3. Figure 6. Compilation of screenshots of the accession details view in the GBIS/I application. It shows a kaleidoscope of data from different data do- mains supporting scientists, breeders and other interested users to select appropriate genebank accessions. It represents the view for external users onto the data managed by the genebank curators as shown in Figure 3. Database, Vol. 2015, Article ID bav021 Page 7 of 8 Table 1. Origin of accessions in IPK’s genebank by continents. Continent Accessions Countries Most frequent countries Africa 12 139 35 Ethiopia (7462), Morocco (1731), Libya (1086) Americas 13 883 25 USA (3887), Peru (3130), Mexico (1402) Asia 23 734 40 Turkey (5020), Iran (3521), China (2384) Europe 75 136 49a Germany (20634), Italy (7264), former Soviet Union (4209) Oceania 507 2 Australia (413), New Zealand (94) Unknown 25 470 aIncluding countries not existing any longer. Table 1. Origin of accessions in IPK’s genebank by continents. aIncluding countries not existing any longer. as of 2014-10-14). Outlook In its present state, GBIS is already an important source of information for various external systems, such as the European search catalogue for plant genetic resources, EURISCO (European Search Catalogue for Plant Genetic Resources of the European Cooperative Programme for Plant Genetic Resources, http://eurisco.ecpgr.org/, as of 2014-10-14) (26), the Global Biodiversity Information Facility, GBIF (http://www.gbif.org/, as of 2014-10-14) (27), Mansfeld’s World Database of Agricultural and Horticultural Crops (http://mansfeld.ipk-gatersleben.de/, as of 2014-10-14) (28), the Garlic and Shallot Core Collection (GSCC) (29), or the Life Science search engine LAILAPS (30). Access to GBIS data is provided in several ways, e.g. using the Biocase PyWrapper (31), as file export or as direct database link. GBIS supports different data exchange standards, such as the Multi-Crop Passport Descriptors (FAO/Bioversity: Multi-Crop Passport Descriptors V.2 (MCPD V.2) (2012), http://www.bioversityinternational. org/e-library/publications/detail/faobioversity-multi-crop- passport-descriptors-v2-mcpd-v2/, as of 2015-02-02) and Access to Biological Collections Data (ABCD 2003: Content definition: the ABCD Schema. CODATA/TDWG Task Group on Access to Biological Collection Data. http:// www.bgbm.org/TDWG/CODATA/Schema/, as of 2015-02- 02). However, in order to increase the attractiveness for re- search and breeding, genebank information systems such as GBIS need to be more closely connected with data from be- yond the classical plant genetic resources domain. This in- cludes information from the—omics areas, such as genotyping, metabolomics and proteomics. In the future, such information will help improving the collection man- agement in terms of identifying duplicates, ensuring genetic purity or establishing core collections. Moreover, breeders will benefit from a better identification of appropriate germ- plasm for breeding purposes. This applies in particular to the enhancement of breeding lines with regard to With the experience from the current subsystems, a next generation of the system, GBIS2, is under development. It transforms the rich-client architecture into a state-of-the-art web-based system, to benefit from the use of the same tech- nology for all subsystems. This gives a chance to keep it open for further enhancements by a leaner development pro- cess. Restrictions of the current technology will be overcome and the subsystems can be built in a more scalable way. Implementation Due to the use of commercial software, the GBIS source code is not deposited in an open source re- pository but can be made available on request. phenotypic traits, such as drought tolerance. The added value that could be created by systematically using gene- bank material, cannot be estimated highly enough. As a proof of concept, IPK is currently preparing the genotyping of the whole barley collection (20 000 acces- sions). It is planned to connect the information gained from this initiative with GBIS. Acknowledgements The project ‘Establishment of the Federal ex situ Genebank for Agricultural and Horticultural Crops: Fusion of the Genebanks of IPK and BAZ Braunschweig’, workpackage 1 ‘Development of a new Genebank Information System’ (2002–2006) was jointly funded by the German Federal Ministries of Education and Research (BMBF) and of Food and Agriculture (BMEL). Since 2006, the further development of GBIS was financed with IPK core funds. We thank the following colleagues who participated in the develop- ment of specifications and the design and implementation of GBIS since 2002 (in alphabetical order): Steffen Flemming, Julia´n Daniel Jime´nez Krause, Werner Scho¨lch, Andreas Stephanik and Jo¨rn Vorwald. Staff of the companies MB Data Consulting and Opitz Consulting participated in the design and implementation of GBIS/ M and GBIS/I. We thank our colleagues Dag Terje Filip Endresen (Norwegian participant node of the Global Biodiversity Information Facility—GBIF) and Theo van Hintum (Centre for Genetic Resources, The Netherlands—CGN, Wageningen) for critical read- ing of the manuscript and for valuable comments. We like to thank the research group Bioinformatics and Information Technology for providing and maintaining the institute’s database environment. References 1. Ungricht,S. (2004) How many plant species are there? And how many are threatened with extinction? Endemic species in global biodiversity and conservation assessments. Taxon, 53, 481–484. 19. Vorwald,J. (2002) Verfu¨gbarmachung von Evaluierungsdaten im Genbankinformationssystem (GBIS) des IPK Gatersleben. Bericht u¨ber die 53. Tagung 2002 der Vereinigung der Pflanzenzu¨chter und Saatgutkaufleute O¨sterreichs, 26.–28. November 2002, BAL Gumpenstein, pp. 41–46. 2. Khoshbakht,K. and Hammer,K. (2008) How many plant species are cultivated? Genet. Resour. Crop. Evol., 55, 925–928. 20. Lehmann,C. and Mansfeld,R. (1957) Zur Technik der Sortimentserhaltung. Die Kulturpflanze, 5, 108–138. 3. Metzger,J.O. and Bornscheuer,U. (2006) Lipids as renewable resources: current state of chemical and biotechnological conver- sion and diversification. Appl. Microbiol. Biotechnol., 71, 13–22. 21. Knu¨pffer,H. (2001) Handling of characterization and evaluation data in crop databases. In: Maggioni L, Spellman O (eds). Report of a Network Coordinating Group on Cereals. Ad hoc meeting, 7–8 July 2000, Radziko´w, Poland. International Plant Genetic Resources Institute, Rome, Italy, pp. 58–65. 4. Tilman,D., Hill,J. and Lehman,C. (2006) Carbon-negative bio- fuels from low-input high-diversity grassland biomass. Science, 314, 1598–1600. 5. FAO (2010) The Second Report on the State of the World’s Plant Genetic Resources for Food and Agriculture. FAO, Rome. 22. Keilwagen,J., Kilian,B., O¨ zkan,H. et al. (2014) Separating the wheat from the chaff–a strategy to utilize plant genetic resources from ex situ genebanks. Sci. Rep., 4, 5231. 6. Engels,J.M.M. and Maggioni,L. (2012) AEGIS: a regionally based approach to PGR conservation. In: Maxted N, Dulloo M, Ford-Lloyd BV (eds). Agrobiodiversity Conservation: Securing the Diversity of Crop Wild Relatives and Landraces. CABI, Wallingford, pp. 321–326. 23. Gotor,E., Alercia,A., Rao,V.R. et al. (2008) The scientific infor- mation activity of Bioversity International: the descriptor lists. Genet. Resour. Crop. Evol., 55, 757–772. 24. Bienert,J. (2005) Konzeption und Entwicklung eines Erfassungstools fu¨r Feldbeobachtungen an pflanzengenetischen Ressourcen auf mobilen Endgera¨ten mit Anbindung an das Genbankinformationssystem GBIS am IPK Gatersleben, Hochschule Harz, Wernigerode, Germany. 7. Hoisington,D., Khairallah,M., Reeves,T. et al. (1999) Plant gen- etic resources: What can they contribute toward increased crop productivity? Proc. Natl Acad. Sci., 96, 5937–5943. 8. Hammer,K., Ga¨de,H. and Knu¨pffer,H. (1994) 50 Jahre Genbank Gatersleben–eine U¨ bersicht. Vortra¨ge fu¨r Pflanzenzu¨chtung, 27, 333–383. 25. Oppermann,M., Koller,D., Winterberg,T. (2006) Praxiserfahrungen mit dem Oracle Application Development Framework (ADF). Java Spektrum, 01/2006, 26–30. 9. Mu¨ntz,K. and Wobus,U. (2013) Das Institut Gatersleben und seine Geschichte: Genetik und Kulturpflanzenforschung in drei politischen Systemen. Springer, Berlin-Heidelberg. 26. Dias,S., Dulloo,M., Arnaud,E. Funding The development of the GBIS was initially funded by the German Federal Ministry of Education and Research and the German Federal Ministry of Food and Agriculture. Since 2007, the Database, Vol. 2015, Article ID bav021 Page 8 of 8 Begemann F, Hammer K, Schmidt F (eds). Pflanzengenetische Ressourcen — Situationsanalyse und Dokumentationssituation, Bonn, pp. 124–125. Begemann F, Hammer K, Schmidt F (eds). Pflanzengenetische Ressourcen — Situationsanalyse und Dokumentationssituation, Bonn, pp. 124–125. development is self-funded by IPK. Funding open access charge: self- funded by IPK. Conflict of interest. None declared. 18. Schu¨ler,K. (1993) Stand der Dokumentation genetischer Ressourcen in der Genbank-Außenstelle Groß Lu¨sewitz. Pflanzengenetische Ressourcen–Situationsanalyse und Dokumentationssysteme, pp. 126–128. References (2012) The role of EURISCO in promoting use of agricultural biodiversity. In: Maxted N, Dulloo M, Ford-Lloyd BV et al (eds). Agrobiodiversity Conservation: Securing the Diversity of Crop Wild Relatives and Landraces, pp. 270–277. 10. IPK (2014) Scientific Report 2012/2013. Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, pp. 225. 11. Oppermann,M. and Knu¨pffer,H. (2006) GBIS–das neue Genbank-Informationssystem am IPK. Vortra¨ge fu¨r Pflanzenzu¨chtung, 70, 47–49. 27. Endresen,D.T.F. and Knu¨pffer,H. (2012) The Darwin Core ex- tension for genebanks opens up new opportunities for sharing genebank datasets. Biodivers. Inform., 8, 12–29. 12. Knu¨pffer,H. and Freytag,U. (1993) Dokumentation pflanzengene- tischer Ressourcen in der Genbank des Instituts fu¨r Pflanzengenetik und Kulturpflanzenforschung Gatersleben. Pflanzengenetische Ressourcen–Situationsanalyse und Dokumentationssysteme. Landwirtschaftsverlag, Mu¨nster, pp. 113–123. 28. Knu¨pffer,H., Ochsmann,J. and Biermann,N. (2003) The "Mansfeld Database" in its national and international context. Schriften zu Genetischen Ressourcen, 22, 32–34. 29. Colmsee,C., Joachim Keller,E.R., Zanke,C. et al. (2012) The Garlic and Shallot core collection image database of IPK present- ing two vegetatively maintained crops in the Federal ex situ gene- bank for agricultural and horticultural crops at Gatersleben, Germany. Genet. Resour. Crop. Evol., 59, 1407–1415. 13. Knu¨pffer,H., Harrer,S., Jime´nez Krause,J.D. et al. (1998) Informationsangebote der Genbank im Internet. Schriften zu Genetischen Ressourcen, 8, 239–243. 14. Knu¨pffer,H. (1999) Dokumentation pflanzengenetischer Ressourcen in der Genbank des IPK Gatersleben. Schriften zu Genetischen Ressourcen, 12, 7–12. 30. Lange,M., Spies,K., Bargsten,J. et al. (2010) The LAILAPS search engine: relevance ranking in life science databases. J. Integr. Bioinform., 7, 110. 15. Knu¨pffer,H. (1983) Computer in Genbanken–eine U¨ bersicht. Die Kulturpflanze, 31, 77–143. 31. Gu¨ntsch,A., Do¨ring,M., Berendsohn,W. (2006) Mobilisierung von prima¨ren Biodiversita¨tsdaten: Das BioCASe Protokoll und seine Anwendung in internationalen Netzwerken. Workshop des Arbeitskreises ,,Umweltdatenbanken“ der Fachgruppe ,,Informatik im Umweltschutz“, 6.–7. June 2005, Hanover, Germany. Umweltbundesamt, pp. 129–138. 16. Germeier,C.U., Frese,L. and Bu¨cken,S. (2003) Concepts and data models for treatment of duplicate groups and sharing of responsibilities in genetic resources information systems. Genet. Resour. Crop. Evol., 50, 693–705. 17. Willner,E. (1993) Stand der Dokumentation genetischer Ressourcen in der Genbank-Außenstelle Malchow/Poel. In:
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Supplementary Materials and Methods from Erlotinib Resistance in Lung Cancer Cells Mediated by Integrin β1/Src/Akt-Driven Bypass Signaling
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Reagents Gefitinib was provided from AstraZeneca Inc. and erlotinib was provided from F. Hoffmann-La Roche Ltd. SU11274 and Picropodophylin were purchased from Calbiochem and dasatinib was purchased from Bio Vision. Anti-HER2 and anti-phospho-HER2 were from Upstate Biotechnology. Anti-α-tubulin antibody was from Sigma-aldrich Co. Anti-EGFR, anti-phospho-HER3, anti-Erk1/2, anti-phospho-Erk1/2, anti-Akt, anti-phospho-Akt (S473), anti-phospho-EGFR (Y1068), anti-Src, and anti-phospho-Src (Y416) antibodies were purchased from Cell Signaling Technology. Anti-integrinβ1for immunoblotting or FACS analysis, anti-integrinα5, and anti-integrinα2 antibodies were purchased from Becton, Dickinson and Company. Anti-Met, and anti-mouse IgG, anti-HER3 antibodies and neutralizing antibody against integrinβ1 (P5D2) were purchased from Santa Cruz Biotechnology. cDNA for integrinβ1 was purchased from Origene. The small interfering RNA (siRNA) corresponding to integrinβ1 (5’- UACACUUACAGACACACCACACUCGCA-3’), integrinα2 (5’-AUGAGAUGUCUUCUGG ACAAUGGUC-3’), integrinα5 (5’-UUGAUGAGCUCAUA GACAUGGUGGA -3’), and Src (5’- Gefitinib was provided from AstraZeneca Inc. and erlotinib was provided from F. Hoffmann-La Roche Ltd. SU11274 and Picropodophylin were purchased from Calbiochem and dasatinib was purchased from Bio Vision. Anti-HER2 and anti-phospho-HER2 were from Upstate Biotechnology. Anti-α-tubulin antibody was from Sigma-aldrich Co. Anti-EGFR, anti-phospho-HER3, anti-Erk1/2, anti-phospho-Erk1/2, anti-Akt, anti-phospho-Akt (S473), anti-phospho-EGFR (Y1068), anti-Src, and anti-phospho-Src (Y416) antibodies were purchased from Cell Signaling Technology. Anti-integrinβ1for immunoblotting or FACS analysis, anti-integrinα5, and anti-integrinα2 antibodies were purchased from Becton, Dickinson and Company. Anti-Met, and anti-mouse IgG, anti-HER3 antibodies and neutralizing antibody against integrinβ1 (P5D2) were purchased from Santa Cruz Biotechnology. cDNA for integrinβ1 was purchased from Origene. The small interfering RNA (siRNA) corresponding to integrinβ1 (5’- UACACUUACAGACACACCACACUCGCA-3’), integrinα2 (5’-AUGAGAUGUCUUCUGG ACAAUGGUC-3’), integrinα5 (5’-UUGAUGAGCUCAUA GACAUGGUGGA -3’), and Src (5’- Immunoprecipitation analysis Total cell lysates using lysis buffer (500 mmol/L Tris-HCl, 150 mmol/L NaCl, 1% Triton X-100, 0.5% deoxycholic acid, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 1 mmol/L sodium orthovanadate, 10μmol/mL MG132 and 1mmol/L N-ethylmaleimide) was incubated for 2hr with anti- integrinβ1 antibody and Protein A/G plusagarose (Santa Cruz Biotechnology) and gently shaken. The precipitates were washed twice with ice-cold lysis buffer and resolved by SDS-PAGE followed by Western blot analysis. Anti-integrinβ1 antibody for immunoprecipitation was purchased from Millipore Corporation. rchased from Millipore Corporation.
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ERROR: type should be string, got "https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. Roles of the Inner Eyewall Structure in the Secondary Eyewall Formation of Simulated \n1 \nTropical Cyclones \n2 \nNannan Qin1,2, Liguang Wu1,4, Qingyuan Liu3 \n3 \n1Department of Atmospheric and Oceanic Sciences and Institute of Atmospheric Sciences, Fudan \n4 \nUniversity, Shanghai, 200438, China \n5 \n2State Key Laboratory of Severe Weather, Chinese Academy of Meteorological Sciences, \n6 \nBeijing, 100081, China \n7 \n3Nanjing Joint Institute for Atmospheric Sciences, Nanjing, China \n8 \n4Innovation Center of Ocean and Atmosphere System, Zhuhai Fudan Innovation Research \n9 \nInstitute,Zhuhai, 518057, China \n10 \n \n13 \n \n14 \n \n15 \n \n16 \n \n17 \nCorrespondence to: Dr. Liguang Wu (liguangwu@fudan.edu.cn) \n18 \n \n19 \n \n20 \n11 \n \n12 Roles of the Inner Eyewall Structure in the Secondary Eyewall Formation of Simulated \n1 \nTropical Cyclones \n2 \nNannan Qin1,2, Liguang Wu1,4, Qingyuan Liu3 \n3 \n1Department of Atmospheric and Oceanic Sciences and Institute of Atmospheric Sciences, Fudan \n4 \nUniversity, Shanghai, 200438, China \n5 \n2State Key Laboratory of Severe Weather, Chinese Academy of Meteorological Sciences, \n6 \nBeijing, 100081, China \n7 \n3Nanjing Joint Institute for Atmospheric Sciences, Nanjing, China \n8 \n4Innovation Center of Ocean and Atmosphere System, Zhuhai Fudan Innovation Research \n9 \nInstitute,Zhuhai, 518057, China \n10 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. Abstract. It has been suggested that the inner eyewall structure may play an important role in the \n21 \nsecondary eyewall formation (SEF) of tropical cyclones (TCs). This study is to further examine \n22 \nthe role of the inner eyewall structure by comparing two numerical experiments, which were \n23 \nconducted with the same large-scale environment and initial and boundary conditions but different \n24 \ngrid sizes. The SEF was simulated in the experiment with the finer grid spacing, but not in the \n25 \nother. 26 Abstract. It has been suggested that the inner eyewall structure may play an important role in the \n21 \nsecondary eyewall formation (SEF) of tropical cyclones (TCs). This study is to further examine \n22 \nthe role of the inner eyewall structure by comparing two numerical experiments, which were \n23 \nconducted with the same large-scale environment and initial and boundary conditions but different \n24 \ngrid sizes. The SEF was simulated in the experiment with the finer grid spacing, but not in the \n25 \nother. 1 Introduction \n39 Many intense tropical cyclones (TCs) usually undergo the secondary eyewall formation (SEF) \n40 \n(Fortner, 1958; Willoughby, 1982). Observational study shows that about 80% of intense TCs \n41 \n(maximum surface wind > 62 m s-1) in the western North Pacific, 70% in the Atlantic, and 50% in \n42 \nthe eastern Pacific possessed concentric eyewalls at least once (Hawkins and Helveston, 2008). 43 \nThe eyewall replacement circle is one of the most important issues remaining in understanding \n44 \nand predicting the change of TC intensity due to the resulting dramatic intensity fluctuations \n45 \n(Samsury and Zipser, 1995; Terwey and Montgomery, 2008; Bell et al., 2012). Although much \n46 \neffort has been made to understand the mechanisms of the SEF, a consensus has not been reached \n47 \nso far. 48 Many intense tropical cyclones (TCs) usually undergo the secondary eyewall formation (SEF) \n40 \n(Fortner, 1958; Willoughby, 1982). Observational study shows that about 80% of intense TCs \n41 \n(maximum surface wind > 62 m s-1) in the western North Pacific, 70% in the Atlantic, and 50% in \n42 \nthe eastern Pacific possessed concentric eyewalls at least once (Hawkins and Helveston, 2008). 43 \nThe eyewall replacement circle is one of the most important issues remaining in understanding \n44 \nand predicting the change of TC intensity due to the resulting dramatic intensity fluctuations \n45 \n(Samsury and Zipser, 1995; Terwey and Montgomery, 2008; Bell et al., 2012). Although much \n46 \neffort has been made to understand the mechanisms of the SEF, a consensus has not been reached \n47 \nso far. 48 Previous studies have pointed out the dynamic importance of the vortex circulation in the \n49 \nSEF (Montgomery and Kallenbach, 1997; Chen and Yau, 2001; Qiu et al., 2010). Given a negative \n50 \nradial gradient of vorticity outside the primary eyewall, vortex Rossby waves (VRWs) propagate \n51 \noutward and stop at a stagnation radius, where the mean flow strengthens through the interaction \n52 \nof eddies with the azimuthal-mean vortex (Montgomery and Kallenbach, 1997; Qiu et al., 2010; \n53 \nChen and Yau, 2001; Hogsett and Zhang, 2009; Dai et al., 2021). With the strengthening mean \n54 \nflow, the outer convection occurs and evolves into an outer eyewall through the wind-induced \n55 \nsurface heat exchange (Emanuel, 1986). Terwey and Montgomery (2008) proposed that cumulus \n56 \nconvection forms and maintains in a far-field region with a weak negative radial gradient of \n57 \nvorticity (the β –skirt) and moderate stretching time. 26 Comparing the eyewall structure in the simulated TCs with and without the SEF indicates \n27 \nthat the eyewall structure can play an important role in the SEF. For the simulated TC with the \n28 \nSEF, the eyewall is more upright with stronger updrafts, accompanied by a wide eyewall anvil at \n29 \na higher altitude. Compared to the simulated TC without the SEF, diagnostic analysis reveals that \n30 \nthe cooling outside the inner eyewall is induced by the sublimation, melting and evaporation of \n31 \nhydrometeors falling from the eyewall anvil. The cooling also induces upper-level dry, cool inflow \n32 \nbelow the anvil, prompting the subsidence and moat formation between the inner eyewall and the \n33 \nspiral rainband. In the simulated TC without the SEF, the cooling induced by the falling \n34 \nhydrometeors is significantly reduced and offset by the diabatic warming. There is no upper-level \n35 \ndry inflow below the anvil and no moat formation between the inner eyewall and the spiral \n36 \nrainband. This study suggests that a realistic simulation of the intense eyewall convection is \n37 \nimportant to the prediction of the SEF in the numerical forecasting model. 38 Comparing the eyewall structure in the simulated TCs with and without the SEF indicates \n27 \nthat the eyewall structure can play an important role in the SEF. For the simulated TC with the \n28 \nSEF, the eyewall is more upright with stronger updrafts, accompanied by a wide eyewall anvil at \n29 \na higher altitude. Compared to the simulated TC without the SEF, diagnostic analysis reveals that \n30 \nthe cooling outside the inner eyewall is induced by the sublimation, melting and evaporation of \n31 \nhydrometeors falling from the eyewall anvil. The cooling also induces upper-level dry, cool inflow \n32 \nbelow the anvil, prompting the subsidence and moat formation between the inner eyewall and the \n33 \nspiral rainband. In the simulated TC without the SEF, the cooling induced by the falling \n34 \nhydrometeors is significantly reduced and offset by the diabatic warming. There is no upper-level \n35 \ndry inflow below the anvil and no moat formation between the inner eyewall and the spiral \n36 \nrainband. This study suggests that a realistic simulation of the intense eyewall convection is \n37 \nimportant to the prediction of the SEF in the numerical forecasting model. 38 2 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. 1 Introduction \n39 \nMany intense tropical cyclones (TCs) usually undergo the secondary eyewall formation (SEF) \n40 \n(Fortner, 1958; Willoughby, 1982). Observational study shows that about 80% of intense TCs \n41 \n(maximum surface wind > 62 m s-1) in the western North Pacific, 70% in the Atlantic, and 50% in \n42 \nthe eastern Pacific possessed concentric eyewalls at least once (Hawkins and Helveston, 2008). 43 \nThe eyewall replacement circle is one of the most important issues remaining in understanding \n44 \nand predicting the change of TC intensity due to the resulting dramatic intensity fluctuations \n45 \n(Samsury and Zipser, 1995; Terwey and Montgomery, 2008; Bell et al., 2012). Although much \n46 \neffort has been made to understand the mechanisms of the SEF, a consensus has not been reached \n47 \nso far. 48 \nPrevious studies have pointed out the dynamic importance of the vortex circulation in the \n49 \nSEF (Montgomery and Kallenbach, 1997; Chen and Yau, 2001; Qiu et al., 2010). Given a negative \n50 \nradial gradient of vorticity outside the primary eyewall, vortex Rossby waves (VRWs) propagate \n51 \noutward and stop at a stagnation radius, where the mean flow strengthens through the interaction \n52 \nof eddies with the azimuthal-mean vortex (Montgomery and Kallenbach, 1997; Qiu et al., 2010; \n53 \nChen and Yau, 2001; Hogsett and Zhang, 2009; Dai et al., 2021). With the strengthening mean \n54 \nflow, the outer convection occurs and evolves into an outer eyewall through the wind-induced \n55 \nsurface heat exchange (Emanuel, 1986). Terwey and Montgomery (2008) proposed that cumulus \n56 \nconvection forms and maintains in a far-field region with a weak negative radial gradient of \n57 \nvorticity (the β –skirt) and moderate stretching time. A secondary eyewall forms through the \n58 \nupscale cascade and axisymmetrization of eddy vorticities in the sustained convection. It has been \n59 \nfound that the secondary eyewall can be simulated in a barotropic model when the preexisting \n60 \nouter convection is stretched into a closed vorticity band by the rotation of the inner vortex (Kuo \n61 1 Introduction \n39 A secondary eyewall forms through the \n58 \nupscale cascade and axisymmetrization of eddy vorticities in the sustained convection. It has been \n59 \nfound that the secondary eyewall can be simulated in a barotropic model when the preexisting \n60 \nouter convection is stretched into a closed vorticity band by the rotation of the inner vortex (Kuo \n61 3 3 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. et al., 2004, 2008). Based on a nonlinear boundary layer model, Kepert (2013) proposed that the \n62 \nsecondary eyewall can form through a positive feedback among the local enhancement of the radial \n63 \nvorticity gradient, the frictional updraft. Those studies highlight the positive feedback of the eddy \n64 \nkinetic energy to the storm-scale flow through the dynamics of the VRW, vorticity interaction, and \n65 \nthe Ekman pumping. However, the convective activity related to the generation of the eddy kinetic \n66 \nenergy is not fully addressed. 67 Some studies focused on spiral rainbands because the SEF generally starts from convective \n68 \nrainbands (Houze, 2007; Zhao et al., 2008, 2016; Kossin and Sitkowski, 2009). When the rainband \n69 \noutside the eyewall is enhanced by adding a large diabatic heating rate to the rainband in a \n70 \nnumerical simulation, the TC can experience the SEF (Wang, 2009). Zhu and Zhu (2014) \n71 \nemphasized that a critical strength of the rainbands is needed for the formation of a secondary wind \n72 \nmaximum through diabatic heating. Idealized numerical simulations indicated that the sustained \n73 \nconvection in the SEF region was enhanced by the interaction between the unbalanced boundary \n74 \nlayer process and the asymmetric inflows induced by the outside rainbands that propagated inward \n75 \n(Qiu and Tan, 2013; Wang and Tan, 2020). Recent studies revealed that, for SEF cases, the \n76 \ndescending inflow in the downwind portion of the spiral rainbands transfers high angular \n77 \nmomentum inward, leading to the outward expansion of the wind field (Didlake et al., 2018; \n78 \nWunsch and Didlake, 2018; Wang et al., 2019; Yu et al., 2020). A radial expansion of storm wind \n79 \nprecedes the SEF through the boundary layer processes and coupled convective dynamics (Rozoff \n80 \net al., 2012; Huang et al., 2012; Abarca and Montgomery, 2013; Sun et al., 2013). However, not \n81 \nall the spiral rainbands outside of the TC eyewall can evolve into a closed outer eyewall. 1 Introduction \n39 82 Many numerical studies have also shown the importance of various microphysical processes \n83 \nin the SEF since the inner-core structure and intensity of TCs are sensitive to the microphysical \n84 Many numerical studies have also shown the importance of various microphysical processes \n83 \nin the SEF since the inner-core structure and intensity of TCs are sensitive to the microphysical \n84 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. processes (Wang, 2002; Zhu and Zhang, 2006). Zhu and Zhang (2006) proposed that varying cloud \n85 \nmicrophysics processes affect the timing of the spinup of the secondary eyewall since the \n86 \ndifferences in the inner eyewall convection and the rainband structure of the simulated TCs. 87 \nNumerical simulations showed that changing the terminal velocity of snow led to changes in the \n88 \nmagnitude and distribution of the diabatic heating of inner-core convection at outer radii, which is \n89 \nimportant for the SEF (Zhu and Zhu, 2015). Influenced by the evaporative cooling from the fallout \n90 \nof hydrometeors, the penetrative downdrafts can promote the local convection outside the primary \n91 \neyewall, where the SEF occurs (Tyner et al., 2018). Moreover, microphysical processes are also \n92 \nimportant to the occurrence of the moat, by which the spiral rainband is separated from the inner \n93 \neyewall with a chance to become a secondary eyewall. Willoughby et al. (1982) considered the \n94 \nmoat generated with subsidence as the evaporative cooling of precipitation falling from the \n95 \ncumulus anvil. In our previous study based on a numerical modeling simulation (Qin et al., 2021), \n96 \nwe demonstrated that the moat subsidence is mainly caused by the negative buoyancy resulting \n97 \nfrom the cooling from sublimation, melting and evaporation processes of hydrometeors from the \n98 \ncumulus eyewall and the related well-developed anvil, and the moat subsidence is further enhanced \n99 \nby the compensating upper-level dry-air inflows. 100 processes (Wang, 2002; Zhu and Zhang, 2006). Zhu and Zhang (2006) proposed that varying cloud \n85 \nmicrophysics processes affect the timing of the spinup of the secondary eyewall since the \n86 \ndifferences in the inner eyewall convection and the rainband structure of the simulated TCs. 87 \nNumerical simulations showed that changing the terminal velocity of snow led to changes in the \n88 \nmagnitude and distribution of the diabatic heating of inner-core convection at outer radii, which is \n89 \nimportant for the SEF (Zhu and Zhu, 2015). 1 Introduction \n39 Influenced by the evaporative cooling from the fallout \n90 \nof hydrometeors, the penetrative downdrafts can promote the local convection outside the primary \n91 \neyewall, where the SEF occurs (Tyner et al., 2018). Moreover, microphysical processes are also \n92 \nimportant to the occurrence of the moat, by which the spiral rainband is separated from the inner \n93 \neyewall with a chance to become a secondary eyewall. Willoughby et al. (1982) considered the \n94 \nmoat generated with subsidence as the evaporative cooling of precipitation falling from the \n95 \ncumulus anvil. In our previous study based on a numerical modeling simulation (Qin et al., 2021), \n96 \nwe demonstrated that the moat subsidence is mainly caused by the negative buoyancy resulting \n97 \nfrom the cooling from sublimation, melting and evaporation processes of hydrometeors from the \n98 \ncumulus eyewall and the related well-developed anvil, and the moat subsidence is further enhanced \n99 \nby the compensating upper-level dry-air inflows. 100 The objective of this study is to further examine the roles of the inner eyewall structure and \n101 \nthe associated cooling in the formation of the moat between the inner eyewall and the spiral \n102 \nrainband that later becomes the outer eyewall. Our examination was based on two numerical \n103 \nexperiments with the same large-scale environment and initial and boundary conditions, but \n104 \ndifferent grid sizes since the strength and distribution of the inner-core convection of TCs are \n105 \nsensitive to the horizontal resolutions (Zhang et al., 2015; Qin and Zhang, 2018; Wu et al., 2018, \n106 \n2019). This paper is organized as follows. Section 2 briefly summarizes the experimental design. 107 5 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. Section 3 describes the simulated TCs with different horizontal spacings. In Section 4, different \n108 \neyewall structures are identified, followed by Section 5, in which how the differences in the \n109 \neyewall structure affect the SEF is investigated. A summary and concluding remarks are given in \n110 \nthe final section. 111 Section 3 describes the simulated TCs with different horizontal spacings. In Section 4, different \n108 \neyewall structures are identified, followed by Section 5, in which how the differences in the \n109 \neyewall structure affect the SEF is investigated. A summary and concluding remarks are given in \n110 \nthe final section. 111 Section 3 describes the simulated TCs with different horizontal spacings. 1 Introduction \n39 In Section 4, different \n108 \neyewall structures are identified, followed by Section 5, in which how the differences in the \n109 \neyewall structure affect the SEF is investigated. A summary and concluding remarks are given in \n110 \nthe final section. 111 2 Experimental design \n112 In our previous study, a 72-h simulation (CTL) was conducted using the Weather Research \n113 \nand Forecasting model (WRF, version 3.2.1) with quintuply nested domains (27/9/3/1/0.3 km), in \n114 \nwhich the simulated TC experienced the SEF (Qin et al., 2021). In order to understand the \n115 \ninfluence of structural changes of the eyewall on the SEF, a sensitivity experiment (NSEF) was \n116 \nfurther designed by removing the innermost domain used in CTL. The two experiments were \n117 \nconducted over the open ocean for 72 hours. The sea surface temperature is fixed at 29 ℃. The \n118 \noutermost domain is centered at 30 °N, 132.5 °E. The domains with the grid spacing of 3 km and \n119 \nless moved with the TC center. There are 75 vertical levels with the model top at 50 hPa. The \n120 \nNational Centers for Environmental Prediction (NCEP) Final Operational Global Analysis data (1°\n121 \n×1°) is utilized for the initial and lateral boundary conditions. The simulations begin with a TC-\n122 \nlike vortex. 123 Model physics options are the same as those used in Chen and Wu (2016) and Qin et al. 124 \n(2021). The major model physics options include the single-moment 3-class microphysics scheme \n125 \nfor the outermost domain, the single-moment 6-class microphysics scheme for the rest of the \n126 \ndomains, the Yonsei University planetary boundary layer (PBL) scheme (Noh et al., 2003), the \n127 \nlongwave radiation scheme of the Rapid Radiative Transfer Model (RRTM, Mlawer et al., 1997), \n128 \nthe shortwave radiation scheme of the Dudhia (Dudhia, 1989). The Kain-Fritsch cumulus \n129 \nparameterization scheme (Kain and Fritsch, 1993) is applied only in the outermost domain. 130 Model physics options are the same as those used in Chen and Wu (2016) and Qin et al. 124 \n(2021). The major model physics options include the single-moment 3-class microphysics scheme \n125 \nfor the outermost domain, the single-moment 6-class microphysics scheme for the rest of the \n126 \ndomains, the Yonsei University planetary boundary layer (PBL) scheme (Noh et al., 2003), the \n127 \nlongwave radiation scheme of the Rapid Radiative Transfer Model (RRTM, Mlawer et al., 1997), \n128 \nthe shortwave radiation scheme of the Dudhia (Dudhia, 1989). The Kain-Fritsch cumulus \n129 \nparameterization scheme (Kain and Fritsch, 1993) is applied only in the outermost domain. 130 6 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. 3 Evolution of the simulated TCs \n131 There is little difference in the track of the simulated storms largely because the large-scale \n132 \nenvironmental conditions are the same in the two experiments (figure not shown). Despite the \n133 \nsame large-scale environment and initial and boundary conditions, the intensity evolution is \n134 \ndifferent in the two simulations (Fig. 1). In NSEF, after an 18-h spin-up, the near-surface maximum \n135 \nwind speed (VMAX) experiences a persistent increase and reaches its peak intensity of 62.5 m s-1 at \n136 \n57 h, then the VMAX decreases. In CTL, the storm experiences a weakening stage from 32 h to 48 \n137 \nh and a reintensification from 48 h to 63 h due to the SEF. There are pronounced fluctuations in \n138 \nthe VMAX in CTL, suggesting the influence of the small-scale structures simulated with the finer \n139 \ngrid size in the innermost domain. 140 The different eyewall structures between the two experiments can be seen from the evolution \n141 \nof the azimuthal-mean tangential wind. Figure 2 compares the time-radius cross-sections of the \n142 \nazimuthal-mean tangential wind and the vertical motion at 0.5 km between the two simulations. In \n143 \nNSEF, a single maximum wind core maintains and the tangential wind expands radially outward \n144 \nduring the intensification. It is indicated that no SEF occurs in this experiment. In CTL, the \n145 \nsimulated TC experiences the SEF, as discussed in Qin et al. (2021). The formation of the \n146 \nsecondary eyewall begins around 32 h with a secondary maximum tangential wind of over 35 m \n147 \ns-1 at the radius of 85 km. After the SEF, the outer eyewall contracts and intensifies with its strength \n148 \ncatching up with the primary eyewall at t = 40 h (Fig. 2b). A few hours later, the primary eyewall \n149 \nweakens and is replaced by the new eyewall around 46 h. 150 The different eyewall structures between the two experiments can be seen from the evolution \n141 \nof the azimuthal-mean tangential wind. Figure 2 compares the time-radius cross-sections of the \n142 \nazimuthal-mean tangential wind and the vertical motion at 0.5 km between the two simulations. In \n143 \nNSEF, a single maximum wind core maintains and the tangential wind expands radially outward \n144 \nduring the intensification. It is indicated that no SEF occurs in this experiment. In CTL, the \n145 \nsimulated TC experiences the SEF, as discussed in Qin et al. (2021). 3 Evolution of the simulated TCs \n131 Figure 3 \n159 \nshows the horizontal distributions of the 5-km radar reflectivity at the selected times. In NSEF, the \n160 \nbroad and active rainband is evident in the downshear quadrant of the storm, while the rainband in \n161 \nthe upshear quadrant is weak with sporadic convection located around the radii of 100-150 km at \n162 \n28 h. Afterward, the rainbands contract and merge with the inner eyewall, leading to an expansion \n163 \nof the wind field and a broad single eyewall without the formation of a moat. In CTL, the rainbands \n164 \nshow a pattern similar to that in NSEF by 28 h (Fig. 3d), but the rainbands are elongated \n165 \nazimuthally and became a closed ring outside the primary eyewall by 32 h. A clear moat region \n166 \nforms at 50-60 km radii between the primary and the outer eyewalls. After a weakening stage, the \n167 \nprimary eyewall almost dissipates by 44 h (Fig. 3f), followed by an inward contraction of the outer \n168 \neyewall. 169 Moreover, the simulated inner rainbands evolve differently in the two experiments. Figure 3 \n159 \nshows the horizontal distributions of the 5-km radar reflectivity at the selected times. In NSEF, the \n160 \nbroad and active rainband is evident in the downshear quadrant of the storm, while the rainband in \n161 \nthe upshear quadrant is weak with sporadic convection located around the radii of 100-150 km at \n162 \n28 h. Afterward, the rainbands contract and merge with the inner eyewall, leading to an expansion \n163 \nof the wind field and a broad single eyewall without the formation of a moat. In CTL, the rainbands \n164 \nshow a pattern similar to that in NSEF by 28 h (Fig. 3d), but the rainbands are elongated \n165 \nazimuthally and became a closed ring outside the primary eyewall by 32 h. A clear moat region \n166 \nforms at 50-60 km radii between the primary and the outer eyewalls. After a weakening stage, the \n167 \nprimary eyewall almost dissipates by 44 h (Fig. 3f), followed by an inward contraction of the outer \n168 \neyewall. 169 3 Evolution of the simulated TCs \n131 The formation of the \n146 \nsecondary eyewall begins around 32 h with a secondary maximum tangential wind of over 35 m \n147 \ns-1 at the radius of 85 km. After the SEF, the outer eyewall contracts and intensifies with its strength \n148 \ncatching up with the primary eyewall at t = 40 h (Fig. 2b). A few hours later, the primary eyewall \n149 \nweakens and is replaced by the new eyewall around 46 h. 150 The different inner-core structures can also be seen in the evolution of the azimuthal-mean \n151 \nvertical motion (Figs. 2c and 2d). In NSEF, the strong upward motion in the single eyewall is \n152 \nmaintained during the 72-h simulation. The vertical motion in the eyewall with a speed larger than \n153 The different inner-core structures can also be seen in the evolution of the azimuthal-mean \n151 \nvertical motion (Figs. 2c and 2d). In NSEF, the strong upward motion in the single eyewall is \n152 \nmaintained during the 72-h simulation. The vertical motion in the eyewall with a speed larger than \n153 7 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. 0.2 m s-1 extends about 20-30 km at 0.5-km height. In CTL, prior to the SEF, the primary eyewall \n154 \ncontracts inward and the width of the eyewall with the upward motion over 0.2 m s-1 is reduced. 155 \nAround 34 h, a secondary maximum upward motion occurs at a radius of around 60 km. After the \n156 \nSEF, the weakening and dissipation of the primary eyewall can also be seen from the upward \n157 \nmotion shown in Fig. 2d. It is seen that the double eyewall structure exists for 14 h. 158 0.2 m s-1 extends about 20-30 km at 0.5-km height. In CTL, prior to the SEF, the primary eyewall \n154 \ncontracts inward and the width of the eyewall with the upward motion over 0.2 m s-1 is reduced. 155 \nAround 34 h, a secondary maximum upward motion occurs at a radius of around 60 km. After the \n156 \nSEF, the weakening and dissipation of the primary eyewall can also be seen from the upward \n157 \nmotion shown in Fig. 2d. It is seen that the double eyewall structure exists for 14 h. 158 Moreover, the simulated inner rainbands evolve differently in the two experiments. 4 Differences in the vertical structures of the eyewall \n170 One of the major differences in the eyewall between NSEF and CTL is the magnitude and the \n171 \nvertical distribution of the vertical motion, which can be examined with the azimuthally averaged \n172 \nvertical motion within the radius of 100 km (Fig. 4) and the contoured frequency by altitude \n173 \ndiagram (CFAD) of the vertical motion (Fig. 5a). The CFAD illustrates the frequency distribution \n174 \nof the vertical motion of the indicated values at each altitude in the region of the 10-km radially \n175 \ninside and outside of the radius of the maximum tangential wind (RMW) for two simulations. The \n176 8 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. azimuthal-mean upward motion in the eyewall is stronger in CTL than that in NSEF (Fig. 4). 177 \nSpecifically, the upward motion in the eyewall in NSEF is maximized at 10- and 12-km height \n178 \nwith peaks of 11 and 13 m s-1 for the 0.1 % and 0.05% percentiles (Fig. 5a), respectively. The weak \n179 \nupward motion between 45- to 75-km radii is associated with the broad rainbands (Figs. 4a and \n180 \n4b). In contrast, the maximum upward motion in CTL is 12 and 14 m s-1 for the 0.1 % and 0.05% \n181 \npercentiles (Fig. 5a). The stronger upward motion in CTL indicates that a higher resolution in the \n182 \nmodel simulation can resolve more intense eyewall updrafts (Yau et al., 2004). Moreover, the \n183 \neyewall with strong updrafts in CTL is more upright in the vertical direction. 184 azimuthal-mean upward motion in the eyewall is stronger in CTL than that in NSEF (Fig. 4). 177 \nSpecifically, the upward motion in the eyewall in NSEF is maximized at 10- and 12-km height \n178 \nwith peaks of 11 and 13 m s-1 for the 0.1 % and 0.05% percentiles (Fig. 5a), respectively. The weak \n179 \nupward motion between 45- to 75-km radii is associated with the broad rainbands (Figs. 4a and \n180 \n4b). In contrast, the maximum upward motion in CTL is 12 and 14 m s-1 for the 0.1 % and 0.05% \n181 \npercentiles (Fig. 5a). The stronger upward motion in CTL indicates that a higher resolution in the \n182 \nmodel simulation can resolve more intense eyewall updrafts (Yau et al., 2004). Moreover, the \n183 \neyewall with strong updrafts in CTL is more upright in the vertical direction. 4 Differences in the vertical structures of the eyewall \n170 184 Another important difference in the eyewall between NSEF and CTL is the feature of the \n185 \nupper-level outflow layer. Figure 5b compares the upper-level outflow in the two simulations by \n186 \nshowing the 0.1% and 0.05% contoured frequency of the radial wind in the region of a radial \n187 \ndistance of 60 km starting from the radius of 10-km outside the eyewall. In NSEF, the upper-level \n188 \noutflow peaks around the 11-km height with maxima of 28 and 26 m s-1 for the 0.05% and 0.1% \n189 \npercentages, respectively. The outflow layer is deep with a magnitude of over 15 m s-1 extending \n190 \ndownward to 8 km. In CTL, the maximum outflow with values of over 29 m s-1 is located around \n191 \nthe 14-km height. The outflow layer at the higher altitude in CTL is associated with the strong \n192 \nupward motion in the eyewall that can lift the hydrometeors much higher. 193 Another important difference in the eyewall between NSEF and CTL is the feature of the \n185 \nupper-level outflow layer. Figure 5b compares the upper-level outflow in the two simulations by \n186 \nshowing the 0.1% and 0.05% contoured frequency of the radial wind in the region of a radial \n187 \ndistance of 60 km starting from the radius of 10-km outside the eyewall. In NSEF, the upper-level \n188 \noutflow peaks around the 11-km height with maxima of 28 and 26 m s-1 for the 0.05% and 0.1% \n189 \npercentages, respectively. The outflow layer is deep with a magnitude of over 15 m s-1 extending \n190 \ndownward to 8 km. In CTL, the maximum outflow with values of over 29 m s-1 is located around \n191 \nthe 14-km height. The outflow layer at the higher altitude in CTL is associated with the strong \n192 \nupward motion in the eyewall that can lift the hydrometeors much higher. 193 The different eyewall structures can also be seen in the horizontal distribution of the cloud-\n194 \ntop temperature (Fig. 6). In NSEF, the eyewall is wider and possesses relatively weaker convection \n195 \nas indicated by the cloud-top temperature of above -750C (Fig. 6a). In CTL, the cloud associated \n196 \nwith the eyewall is deeper since the coldest cloud-top temperature is below -75 0C (Fig. 6b). The \n197 \ncoldest cloud-top temperature is located at the downshear- and upshear-right region due to the \n198 \ninfluence of the southeastward VWS. 4 Differences in the vertical structures of the eyewall \n170 The strong eyewall convection is accompanied by the strong \n199 The different eyewall structures can also be seen in the horizontal distribution of the cloud-\n194 \ntop temperature (Fig. 6). In NSEF, the eyewall is wider and possesses relatively weaker convection \n195 \nas indicated by the cloud-top temperature of above -750C (Fig. 6a). In CTL, the cloud associated \n196 \nwith the eyewall is deeper since the coldest cloud-top temperature is below -75 0C (Fig. 6b). The \n197 \ncoldest cloud-top temperature is located at the downshear- and upshear-right region due to the \n198 \ninfluence of the southeastward VWS. The strong eyewall convection is accompanied by the strong \n199 9 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. and high-altitude outflow compared to that in NSEF. 200 \n5 Influence of the eyewall structure on the moat formation \n201 \n5.1 Buoyancy effects \n202 \nAs discussed above, the rainbands can encircle into a closed outer eyewall instead of merging \n203 \nwith the inner eyewall in CTL due to the formation of the moat. First, we investigate the \n204 \nrelationship between the distribution of buoyancy and the occurrence of the moat subsidence. 205 \nMany studies indicated that buoyance, which is determined by temperature perturbation, affects \n206 \nthe vertical motion tendency (e.g., Zhang et al., 2000; Braun, 2002; Miller et al., 2015). It is \n207 \nintended to examine how the buoyancy changes when the SEF fails in NSEF with a different \n208 \neyewall structure. Following the method used by Braun (2002), the perturbation associated with \n209 \nthe buoyance calculation is defined as 𝐴𝐴′(𝜆𝜆, 𝑟𝑟, 𝑧𝑧) = 𝐴𝐴(𝜆𝜆, 𝑟𝑟, 𝑧𝑧) −𝐴𝐴0(𝑧𝑧) −𝐴𝐴0,1(𝜆𝜆, 𝑟𝑟, 𝑧𝑧) , where A \n210 \nrepresents any variable in a cylindrical coordinate (𝜆𝜆, 𝑟𝑟, 𝑧𝑧), 𝜆𝜆, r, z are the azimuthal angle, the \n211 \nradius from the TC center, and the vertical height axis, respectively, 𝐴𝐴0 is averaged over the whole \n212 \narea of the 1-km domain, 𝐴𝐴0,1 are the wavenumber-0 and -1 components of the perturbation field \n213 \nfrom 𝐴𝐴0. 𝐴𝐴0 + 𝐴𝐴0,1 denotes the reference state for the buoyance analysis. Following Houze (1993) \n214 \nand Braun (2002), buoyancy (B) is defined as: \n215 \n𝐵𝐵\n𝑔𝑔൤\n𝜃𝜃𝑣𝑣′𝜃𝑣𝜃𝑣\n+ (𝜅𝜅\n1)\n𝑝𝑝′𝑝𝑝\n𝑞𝑞′൨\n(1)\n216 As discussed above, the rainbands can encircle into a closed outer eyewall instead of merging \n203 \nwith the inner eyewall in CTL due to the formation of the moat. 4 Differences in the vertical structures of the eyewall \n170 First, we investigate the \n204 \nrelationship between the distribution of buoyancy and the occurrence of the moat subsidence. 205 \nMany studies indicated that buoyance, which is determined by temperature perturbation, affects \n206 \nthe vertical motion tendency (e.g., Zhang et al., 2000; Braun, 2002; Miller et al., 2015). It is \n207 \nintended to examine how the buoyancy changes when the SEF fails in NSEF with a different \n208 \neyewall structure. Following the method used by Braun (2002), the perturbation associated with \n209 \nthe buoyance calculation is defined as 𝐴𝐴′(𝜆𝜆, 𝑟𝑟, 𝑧𝑧) = 𝐴𝐴(𝜆𝜆, 𝑟𝑟, 𝑧𝑧) −𝐴𝐴0(𝑧𝑧) −𝐴𝐴0,1(𝜆𝜆, 𝑟𝑟, 𝑧𝑧) , where A \n210 \nrepresents any variable in a cylindrical coordinate (𝜆𝜆, 𝑟𝑟, 𝑧𝑧), 𝜆𝜆, r, z are the azimuthal angle, the \n211 \nradius from the TC center, and the vertical height axis, respectively, 𝐴𝐴0 is averaged over the whole \n212 \narea of the 1-km domain, 𝐴𝐴0,1 are the wavenumber-0 and -1 components of the perturbation field \n213 \nfrom 𝐴𝐴0. 𝐴𝐴0 + 𝐴𝐴0,1 denotes the reference state for the buoyance analysis. Following Houze (1993) \n214 \nand Braun (2002), buoyancy (B) is defined as: \n215 𝐵𝑔𝜃𝑣𝜅𝑝𝑞 𝐵𝐵= 𝑔𝑔൤\n𝜃𝜃𝑣𝑣′\n𝜃𝜃𝑣𝑣0+𝜃𝜃𝑣𝑣\n0,1 + (𝜅𝜅−1)\n𝑝𝑝′\n𝑝𝑝0+𝑝𝑝0,1 −𝑞𝑞′൨, (1) \n216 𝜃𝑣𝜅 𝐵𝐵= 𝑔𝑔൤\n𝜃𝜃𝑣𝑣′\n𝜃𝜃𝑣𝑣0+𝜃𝜃𝑣𝑣\n0,1 + (𝜅𝜅−1)\n𝑝𝑝′\n𝑝𝑝0+𝑝𝑝0,1 −𝑞𝑞′൨, (1) \n216 𝜃𝑣𝜅 (1) 10𝐵𝑔𝜃𝑣𝜃𝑣𝜅𝑝𝑝𝑞\nwhere g is the gravitational acceleration, 𝜃𝜃𝑣𝑣 is the virtual potential temperature, 𝜅𝜅= 0.286, p is \n217 \nthe pressure, and q is the hydrometeor mixing ratio, including the mixing ratio of the cloud water \n218 \n(qc), rain water (qr), graupel (qg), snow (qs), and ice (qi). Terms on the right-hand side of Eq. (1) \n219 \nare the thermal buoyancy, the dynamic buoyancy, and the hydrometeor loading, respectively. 220 \nConsidering that the rainband distributes asymmetrically prior to the SEF, it is appropriate to use \n221 \nquarter-mean variables, i.e. the upshear-right quadrant, to analyze the moat and outer eyewall \n222 10 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. formation in our following discussions. 223 \nFor the convenience of the following buoyancy analysis, Fig. 7 shows the radius-height cross \n224 \nsections of the perturbation virtual potential temperature (𝜃𝜃𝑣𝑣\n′). Consistent with the broad eyewall \n225 \nconvection in NSEF, positive 𝜃𝜃𝑣𝑣\n′ appears under the eyewall anvil, which may force the air parcel \n226 \nupward since the local 𝜃𝜃𝑣𝑣 exceeds the ambient environmental value. 4 Differences in the vertical structures of the eyewall \n170 On the contrary, the 𝜃𝜃𝑣𝑣\n′ is \n227 \nnegative outside and underneath the inner eyewall in CTL (Fig. 7e), which will suppress the \n228 \nupward motion there. 229 𝜃𝑣 Given the different distribution of 𝜃𝜃𝑣𝑣\n′, we next examine the related thermal buoyancy, as well \n230 \nas the dynamic buoyancy and the hydrometeor loading (Fig. 8). In NSEF, we note that the positive \n231 \nbuoyancy coinciding with the positive 𝜃𝜃𝑣𝑣\n′ appears outside the inner eyewall since the buoyancy is \n232 \nlargely determined by the thermal buoyancy and the dynamical buoyancy and the water loading \n233 \neffects are relatively small (Figs. 8a-8d). The positive buoyancy forces upward motion, leading to \n234 \nthe widespread upward motion in NSEF (Fig. 8b). There is no moat formation without the \n235 \nconsiderable subsidence, and the spiral rainband merges with the primary eyewall and no SEF \n236 \noccurs. In CTL, the emergence of the moat subsidence is largely caused by the negative buoyancy, \n237 \nespecially the negative thermal buoyancy in response to the negative 𝜃𝜃𝑣𝑣\n′ outside the inner eyewall. 238 \nMoreover, the dynamic buoyancy and the water loading effect both contribute to the enhancement \n239 \nof the moat subsidence. Note that the negative buoyancy also occurs in the inner eyewall below 6-\n240 \nkm height (Fig. 8e), which is consistent with the weakening of the primary eyewall when the outer \n241 \neyewall intensifies. These results indicate that the negative buoyancy beneath the high-altitude \n242 \neyewall anvil from the inner eyewall is crucial for the subsidence generation and the moat \n243 \nemergence. The moat plays an important role in the SEF by separating the preexisting spiral \n244 \nrainbands from the inner eyewall. 245 Given the different distribution of 𝜃𝜃𝑣𝑣\n′, we next examine the related thermal buoyancy, as well \n230 \nas the dynamic buoyancy and the hydrometeor loading (Fig. 8). In NSEF, we note that the positive \n231 \nbuoyancy coinciding with the positive 𝜃𝜃𝑣𝑣\n′ appears outside the inner eyewall since the buoyancy is \n232 \nlargely determined by the thermal buoyancy and the dynamical buoyancy and the water loading \n233 \neffects are relatively small (Figs. 8a-8d). The positive buoyancy forces upward motion, leading to \n234 \nthe widespread upward motion in NSEF (Fig. 8b). There is no moat formation without the \n235 \nconsiderable subsidence, and the spiral rainband merges with the primary eyewall and no SEF \n236 \noccurs. 4 Differences in the vertical structures of the eyewall \n170 In CTL, the emergence of the moat subsidence is largely caused by the negative buoyancy, \n237 \nespecially the negative thermal buoyancy in response to the negative 𝜃𝜃𝑣𝑣\n′ outside the inner eyewall. 238 \nMoreover, the dynamic buoyancy and the water loading effect both contribute to the enhancement \n239 \nof the moat subsidence. Note that the negative buoyancy also occurs in the inner eyewall below 6-\n240 \nkm height (Fig. 8e), which is consistent with the weakening of the primary eyewall when the outer \n241 \neyewall intensifies. These results indicate that the negative buoyancy beneath the high-altitude \n242 \neyewall anvil from the inner eyewall is crucial for the subsidence generation and the moat \n243 \nemergence. The moat plays an important role in the SEF by separating the preexisting spiral \n244 \nrainbands from the inner eyewall. 245 11 11 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. To further demonstrate the tendency of the vertical motion, the perturbation vertical pressure \n246 \ngradient force and the net force among the buoyancy and the perturbation vertical pressure gradient \n247 \nforce are shown in Fig. 9. Even though the perturbation vertical pressure gradient force exhibits \n248 \noppositely to the buoyancy in the two simulated TCs, the net force contributes to the upward \n249 \nacceleration of updrafts in NSEF (Fig. 9c) but the downward acceleration of the subsidence outside \n250 \nthe primary eyewall in CTL (Fig. 9f). As a result, the subsidence outside the inner eyewall in CTL \n251 \ncontributes to the formation of the moat, followed by the SEF, while no SEF occurs in NSEF \n252 \nwithout the emergence of the moat. 253 To further demonstrate the tendency of the vertical motion, the perturbation vertical pressure \n246 \ngradient force and the net force among the buoyancy and the perturbation vertical pressure gradient \n247 \nforce are shown in Fig. 9. Even though the perturbation vertical pressure gradient force exhibits \n248 \noppositely to the buoyancy in the two simulated TCs, the net force contributes to the upward \n249 \nacceleration of updrafts in NSEF (Fig. 9c) but the downward acceleration of the subsidence outside \n250 \nthe primary eyewall in CTL (Fig. 9f). As a result, the subsidence outside the inner eyewall in CTL \n251 \ncontributes to the formation of the moat, followed by the SEF, while no SEF occurs in NSEF \n252 \nwithout the emergence of the moat. 253 5.2 Diabatic heating \n254 279 The evolution of subsidence in the moat area is largely controlled by the distribution of the \n269 \ndiabatic cooling. The azimuthal extension of the diabatic cooling and the related moat subsidence \n270 \nand their differences in CTL and NSEF at upper levels are further examined in Fig. 11. Before 24 \n271 \nh, both the diabatic cooling and the related subsidence in NSEF and CTL are characterized by a \n272 \nhighly asymmetric structure with intense subsidence/cooling located in the upshear-left quadrant \n273 \n(see Figs. 11a, 11b, and Fig. 3). After 24 h, the evolution of the diabatic cooling and subsidence \n274 \ndiffers in the two simulations. The asymmetric structure of the intense diabatic cooling and \n275 \nsubsidence maintains in NSEF, while the diabatic cooling and subsidence extend cyclonically from \n276 \nthe upshear-left quadrant to the downshear-right quadrant, ending with a quasi-symmetric structure \n277 \nfrom 24 h to 32 h (Fig. 11b), which is also confirmed by showing the differences in the diabatic \n278 \ncooling and moat subsidence between NSEF and CTL in Fig. 11c. 279 5.3 Subsidence in response to the diabatic cooing \n280 5.2 Diabatic heating \n254 Since the negative buoyancy is associated with the negative temperature disturbance, the \n255 \ndiabatic heating is examined. Figure 10 shows the radius-height distributions of the diabatic \n256 \nheating and diabatic cooling induced by the sublimation, melting, and evaporation of hydrometeors. 257 \nIn NSEF, the diabatic warming dominates the region within the radius of 100 km, except for the \n258 \neye and a local area around 40-km radius from 1- to 6-km heights (Fig. 10a). This broad warming \n259 \nis caused by the wider eyewall convection as shown in Fig. 3 and Fig 4a. Although the diabatic \n260 \ncooling related to the sublimation, melting and evaporation processes always exist in the storm \n261 \n(Figs. 10b-10d), the low- to middle-level convection outside the inner eyewall produces much \n262 \ndiabatic warming than cooling, leading to net diabatic warming appearing outside the primary \n263 \neyewall in NSEF. In contrast, instead of diabatic warming, the net diabatic cooling appears with \n264 \nthe absence of convection outside of the inner eyewall in CTL (Fig. 10e). This cooling is \n265 \nmaximized at 6- to 10-km height, which is largely induced by the sublimation of hydrometeors \n266 \nbeneath the eyewall anvil (Figs. 10f-10h), while the cooling located below 6-km height is caused \n267 \nby the melting and evaporative processes. 268 12 12 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. The evolution of subsidence in the moat area is largely controlled by the distribution of the \n269 \ndiabatic cooling. The azimuthal extension of the diabatic cooling and the related moat subsidence \n270 \nand their differences in CTL and NSEF at upper levels are further examined in Fig. 11. Before 24 \n271 \nh, both the diabatic cooling and the related subsidence in NSEF and CTL are characterized by a \n272 \nhighly asymmetric structure with intense subsidence/cooling located in the upshear-left quadrant \n273 \n(see Figs. 11a, 11b, and Fig. 3). After 24 h, the evolution of the diabatic cooling and subsidence \n274 \ndiffers in the two simulations. The asymmetric structure of the intense diabatic cooling and \n275 \nsubsidence maintains in NSEF, while the diabatic cooling and subsidence extend cyclonically from \n276 \nthe upshear-left quadrant to the downshear-right quadrant, ending with a quasi-symmetric structure \n277 \nfrom 24 h to 32 h (Fig. 11b), which is also confirmed by showing the differences in the diabatic \n278 \ncooling and moat subsidence between NSEF and CTL in Fig. 11c. 5.3 Subsidence in response to the diabatic cooing \n280 13\nThe Sawyer-Eliassen equation (SEE) is used to better understand how the diabatic heating \n281 \nwith the different eyewall structures affects the evolution of the moat and outer eyewall without \n282 \nconsidering the momentum forcing. The SEE is a useful analytical tool for diagnosing the response \n283 \nof the transverse circulation to diabatic heating (Smith et al., 2005; Bui et al., 2009; Zhu and Zhu, \n284 \n2014; Qin et al., 2021). According to Qin et al. (2021), the SEE used in this study is \n285 \n𝜕𝜕 \n𝜕𝜕𝜕𝜕ቂ\n𝜒𝜒\n𝜌𝜌𝜌𝜌\n𝜕𝜕𝜕𝜕\n𝜕𝜕𝜕𝜕\n𝜕𝜕𝜕𝜕\n𝜕𝜕𝜕𝜕−\n𝜒𝜒\n𝜌𝜌𝜌𝜌\n𝜕𝜕𝜕𝜕\n𝜕𝜕𝜕𝜕\n𝜕𝜕𝜕𝜕\n𝜕𝜕𝜕𝜕 ቃ+\n𝜕𝜕 \n𝜕𝜕𝜕𝜕ቂቀ𝜒𝜒𝜒𝜒𝜁𝜁𝑎𝑎−\n𝐶𝐶𝜒𝜒\n𝑔𝑔\n𝜕𝜕𝜕𝜕\n𝜕𝜕𝜕𝜕ቁ\n1\n𝜌𝜌𝜌𝜌\n𝜕𝜕𝜕𝜕\n𝜕𝜕𝜕𝜕−\n𝜒𝜒\n𝜌𝜌𝜌𝜌\n𝜕𝜕𝜕𝜕 \n𝜕𝜕𝜕𝜕\n𝜕𝜕𝜕𝜕\n𝜕𝜕𝜕𝜕ቃ= 𝑔𝑔\n𝜕𝜕𝜒𝜒2𝑄𝑄 \n𝜕𝜕𝜕𝜕\n+\n𝜕𝜕𝐶𝐶𝐶𝐶2𝑄𝑄 \n𝜕𝜕𝜕𝜕\n , (2) \n286 \nwhere 𝜒𝜒= 1/𝜃𝜃 and 𝜃𝜃 is the potential temperature, 𝜌𝜌 is the density, b is the buoyancy term, 𝜓𝜓 is a \n287 \nstream function, 𝜉𝜉= 2𝑣𝑣/𝑟𝑟+ 𝑓𝑓 is the local Coriolis parameter and f is the Coriolis parameter, 𝜁𝜁𝑎𝑎 \n288 \nis the vertical component of the absolute vorticity, 𝐶𝐶= 𝑣𝑣2/𝑟𝑟+ 𝑓𝑓𝑓𝑓 is the sum of the centrifugal \n289 \nforce and Coriolis force where v is the tangential wind, g is the gravitational acceleration, r and z \n290 \nare the radial and vertical coordinate, and 𝑄𝑄 is the diabatic heating rate (heating forcing). Note that, \n291 The Sawyer-Eliassen equation (SEE) is used to better understand how the diabatic heating \n281 \nwith the different eyewall structures affects the evolution of the moat and outer eyewall without \n282 \nconsidering the momentum forcing. The SEE is a useful analytical tool for diagnosing the response \n283 \nof the transverse circulation to diabatic heating (Smith et al., 2005; Bui et al., 2009; Zhu and Zhu, \n284 \n2014; Qin et al., 2021). According to Qin et al. 5.3 Subsidence in response to the diabatic cooing \n280 These results suggest that the formation of the moat is \n303 \nsensitive to the diabatic heating, especially the diabatic cooling beneath the eyewall anvil. 304 Figure 12 shows the radius-height cross sections of the SEE-diagnosed vertical and radial \n296 \nwind forced by the diabatic heating, diabatic cooling, and cooling induced by the sublimation, \n297 \nmelting and evaporation of hydrometeors, respectively. Significantly, in NSEF, the diabatic heating \n298 \nreleased by the wider eyewall convection induces the upward motion and deep-layer outflows \n299 \noutside the inner eyewall, and a compensated downdraft in the eye (Fig. 12a). In CTL, intense \n300 \ndiabatic cooling forces subsidence of over -0.3 m s-1 outside the inner eyewall, which contributes \n301 \nto the formation of the moat. Meanwhile, the diabatic cooling also induces the upper-level inflow \n302 \nbelow the strong outflow layer (Fig. 12e). These results suggest that the formation of the moat is \n303 \nsensitive to the diabatic heating, especially the diabatic cooling beneath the eyewall anvil. 304 Although the diabatic cooling caused by phase changes usually occurs in TCs, the magnitude \n305 \nof the diabatic cooling, especially the cooling due to the sublimation of ice particles, matters much \n306 \nin the formation of the moat (Figs. 12c and 12g). The cooling due to the sublimation process is \n307 \nmuch less outside the primary eyewall in NSEF compared to that in CTL (cf. Figs. 12c and 12g). 308 \nIn NSEF, the diabatic warming released by the low- to middle-level convection outside the primary \n309 \neyewall exceeds the cooling, resulting in net diabatic warming (Fig. 12a). Thus, a wider eyewall \n310 \nwith warming-forced upward motion prevails due to the positive feedback among the diabatic \n311 \nheating and the convection (Fig. 12a). In addition, the warming-forced upward motion is \n312 \naccompanied by the deep-layer outflow (Fig. 13a), under which the low-level inflow appears \n313 \nbelow the 8-km height (Fig. 13a), which brings moist air enhancing the convection in NSEF. In \n314 Although the diabatic cooling caused by phase changes usually occurs in TCs, the magnitude \n305 \nof the diabatic cooling, especially the cooling due to the sublimation of ice particles, matters much \n306 \nin the formation of the moat (Figs. 12c and 12g). The cooling due to the sublimation process is \n307 \nmuch less outside the primary eyewall in NSEF compared to that in CTL (cf. Figs. 12c and 12g). 5.3 Subsidence in response to the diabatic cooing \n280 (2021), the SEE used in this study is \n285 𝜕𝜒𝜕𝜕𝜕𝜕𝜒𝜕𝜕𝜕𝜕𝜕𝜒𝜒𝜁𝐶𝜒𝜕𝜕𝜕𝜕𝜒𝜕𝜕𝜕𝜕𝑔𝜕𝜒𝑄𝜕𝐶𝐶𝑄 𝜕𝜕 \n𝜕𝜕𝜕𝜕ቂ\n𝜒𝜒\n𝜌𝜌𝜌𝜌\n𝜕𝜕𝜕𝜕\n𝜕𝜕𝜕𝜕\n𝜕𝜕𝜕𝜕\n𝜕𝜕𝜕𝜕−\n𝜒𝜒\n𝜌𝜌𝜌𝜌\n𝜕𝜕𝜕𝜕\n𝜕𝜕𝜕𝜕\n𝜕𝜕𝜕𝜕\n𝜕𝜕𝜕𝜕 ቃ+\n𝜕𝜕 \n𝜕𝜕𝜕𝜕ቂቀ𝜒𝜒𝜒𝜒𝜁𝜁𝑎𝑎−\n𝐶𝐶𝜒𝜒\n𝑔𝑔\n𝜕𝜕𝜕𝜕\n𝜕𝜕𝜕𝜕ቁ\n1\n𝜌𝜌𝜌𝜌\n𝜕𝜕𝜕𝜕\n𝜕𝜕𝜕𝜕−\n𝜒𝜒\n𝜌𝜌𝜌𝜌\n𝜕𝜕𝜕𝜕 \n𝜕𝜕𝜕𝜕\n𝜕𝜕𝜕𝜕\n𝜕𝜕𝜕𝜕ቃ= 𝑔𝑔\n𝜕𝜕𝜒𝜒2𝑄𝑄 \n𝜕𝜕𝜕𝜕\n+\n𝜕𝜕𝐶𝐶𝐶𝐶2𝑄𝑄 \n𝜕𝜕𝜕𝜕\n , (2) \n286 𝜒𝜃𝜃𝜌𝜓 (2) 𝜓 where 𝜒𝜒= 1/𝜃𝜃 and 𝜃𝜃 is the potential temperature, 𝜌𝜌 is the density, b is the buoyancy term, 𝜓𝜓 is a \n287 \nstream function, 𝜉𝜉= 2𝑣𝑣/𝑟𝑟+ 𝑓𝑓 is the local Coriolis parameter and f is the Coriolis parameter, 𝜁𝜁𝑎𝑎 \n288 \nis the vertical component of the absolute vorticity, 𝐶𝐶= 𝑣𝑣2/𝑟𝑟+ 𝑓𝑓𝑓𝑓 is the sum of the centrifugal \n289 \nforce and Coriolis force where v is the tangential wind, g is the gravitational acceleration, r and z \n290 \nare the radial and vertical coordinate, and 𝑄𝑄 is the diabatic heating rate (heating forcing). Note that, \n291 13 𝑄 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. in the absence of the momentum forcing, the diagnosed radial inflow within the boundary layer is \n292 \nlargely underestimated, while the diagnosed secondary circulation above the boundary is \n293 \ncomparable to the simulated results, which is also found in other studies (Bui et al., 2009; Zhu and \n294 \nZhu, 2014; Qin et al., 2021). 295 in the absence of the momentum forcing, the diagnosed radial inflow within the boundary layer is \n292 \nlargely underestimated, while the diagnosed secondary circulation above the boundary is \n293 \ncomparable to the simulated results, which is also found in other studies (Bui et al., 2009; Zhu and \n294 \nZhu, 2014; Qin et al., 2021). 295 Figure 12 shows the radius-height cross sections of the SEE-diagnosed vertical and radial \n296 \nwind forced by the diabatic heating, diabatic cooling, and cooling induced by the sublimation, \n297 \nmelting and evaporation of hydrometeors, respectively. Significantly, in NSEF, the diabatic heating \n298 \nreleased by the wider eyewall convection induces the upward motion and deep-layer outflows \n299 \noutside the inner eyewall, and a compensated downdraft in the eye (Fig. 12a). In CTL, intense \n300 \ndiabatic cooling forces subsidence of over -0.3 m s-1 outside the inner eyewall, which contributes \n301 \nto the formation of the moat. Meanwhile, the diabatic cooling also induces the upper-level inflow \n302 \nbelow the strong outflow layer (Fig. 12e). 5.3 Subsidence in response to the diabatic cooing \n280 308 \nIn NSEF, the diabatic warming released by the low- to middle-level convection outside the primary \n309 \neyewall exceeds the cooling, resulting in net diabatic warming (Fig. 12a). Thus, a wider eyewall \n310 \nwith warming-forced upward motion prevails due to the positive feedback among the diabatic \n311 \nheating and the convection (Fig. 12a). In addition, the warming-forced upward motion is \n312 \naccompanied by the deep-layer outflow (Fig. 13a), under which the low-level inflow appears \n313 \nbelow the 8-km height (Fig. 13a), which brings moist air enhancing the convection in NSEF. In \n314 14 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. CTL, with the considerable cooling forced by the sublimation outside of the inner eyewall (Fig. 315 \n12g), the subsidence is induced outside the primary eyewall. Of importance is that the cooling-\n316 \nforced inflow is located at a higher level, which send dry air inward (Fig. 13b). The penetration of \n317 \ndry air through the upper-level inflow plays an important role in increasing the diabatic cooling \n318 \nthat promotes the subsidence. The cooling-induced subsidence contributes significantly to the \n319 \nformation of the moat. Subsequently, the spiral rainband evolves into the separated outer eyewall \n320 \nin CTL with the formation of the moat. 321 CTL, with the considerable cooling forced by the sublimation outside of the inner eyewall (Fig. 315 \n12g), the subsidence is induced outside the primary eyewall. Of importance is that the cooling-\n316 \nforced inflow is located at a higher level, which send dry air inward (Fig. 13b). The penetration of \n317 \ndry air through the upper-level inflow plays an important role in increasing the diabatic cooling \n318 \nthat promotes the subsidence. The cooling-induced subsidence contributes significantly to the \n319 \nformation of the moat. Subsequently, the spiral rainband evolves into the separated outer eyewall \n320 \nin CTL with the formation of the moat. 321 The differences in the azimuthal distributions of the subsidence and the related upper-level \n322 \nradial inflow between NSEF and CTL are shown in Fig. 14. In NSEF, a highly asymmetric \n323 \nstructure with strong subsidence located in the upshear-left quadrant persists during 18-32 hours \n324 \n(Fig. 14a). Although there is upper-level inflow appearing in the upshear-right region (Fig. 14a), \n325 \nit is largely offset by the strong upper-level outflow, leading to net outflow at upper levels as seen \n326 \nin Fig. 13a. 5.3 Subsidence in response to the diabatic cooing \n280 In contrast, the subsidence in CTL extends azimuthally, ending with a quasi-symmetric \n327 \nstructure from 18 h to 32 h (Fig. 14b). The azimuthal extension of the subsidence follows the \n328 \nappearance of the upper-level inflow, which advects dry air inward to enhance the cooling \n329 \nprocesses. Therefore, without the penetration of dry air by the upper-level descending inflow, the \n330 \ndiabatic cooling and the cooling-forced subsidence beneath the eyewall anvil are limited, which \n331 \nare unfavorable for the moat formation outside the inner eyewall. 332 In this study, two numerical experiments are conducted with the same large-scale \n334 \nenvironment and initial and boundary conditions. The simulated inner eyewall structures are \n335 \ndifferent due to different grid spacings used in the two experiments. The SEF occurs in CTL with \n336 \na fine resolution of 333 m, while no SEF occurs in the other with a coarse resolution of 1 km. 337 15 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. There are two major differences in the eyewall structure by comparing the simulated TCs \n338 \nwith and without the SEF. For the simulated TC with the SEF, the eyewall updrafts are stronger \n339 \nand the eyewall is more upright. The eyewall anvil is located at a higher altitude together with the \n340 \ndeep eyewall convection. As illustrated in Fig. 15, the upper-level dry-air inflow beneath the high-\n341 \naltitude anvil from the inner eyewall is important to the formation of the moat. Diagnostic analysis \n342 \nindicates that the dry inflow below the eyewall anvil is induced by the diabatic cooling released \n343 \nby the sublimation, melting and evaporation of hydrometeors falling from the eyewall anvil. With \n344 \nthe penetration of dry air by the upper-level inflow, subsidence occurs, promoting the formation \n345 \nof the moat between the inner eyewall and the spiral rainband. Afterward, the SEF occurs by \n346 \nshowing the double eyewall structure and the moat with subsidence. In the TC without the SEF, \n347 \nthe eyewall updrafts are weak and tilt much outward, showing an eyewall anvil at the lower altitude. 348 \nThe cooling induced by the falling hydrometeors beneath the low-altitude eyewall anvil is \n349 \nsignificantly reduced and offset by the diabatic warming. As a result, no upper-level dry-air inflow \n350 \noccurs below the eyewall anvil, and no moat forms between the inner eyewall and the spiral \n351 \nrainband. 5.3 Subsidence in response to the diabatic cooing \n280 In addition to the mesoscale descending inflow at \n362 \nmiddle levels, the upper-level dry inflow also contributes to the SEF by forcing the moat formation. 363 \nIn this sense, both the inner-eyewall and the rainband structures are important to the SEF. In \n364 \naddition, our study suggests that a realistic simulation of the eyewall convection is important to \n365 \nthe prediction of the SEF in the numerical forecasting model. 366 trigger new convective updrafts that are important to the subsequent SEF (Didlake et al., 2018; \n361 \nWunsch and Didlake, 2018; Yu et al., 2020). In addition to the mesoscale descending inflow at \n362 \nmiddle levels, the upper-level dry inflow also contributes to the SEF by forcing the moat formation. 363 \nIn this sense, both the inner-eyewall and the rainband structures are important to the SEF. In \n364 \naddition, our study suggests that a realistic simulation of the eyewall convection is important to \n365 \nthe prediction of the SEF in the numerical forecasting model. 366 Data availability. The simulation data is archived at the High-Performance Computing Center of \n368 \nNanjing University of Information Science and Technology and is available upon request. 369 Data availability. The simulation data is archived at the High-Performance Computing Center of \n368 \nNanjing University of Information Science and Technology and is available upon request. 369 Data availability. The simulation data is archived at the High-Performance Computing Center of \n368 \nNanjing University of Information Science and Technology and is available upon request. 369 Author contributions. LW designed research; NQ conceptualized the analysis and wrote the \n371 \nmanuscript; LW provided scientific suggestions for the manuscript. QL carried out the simulations \n372 \nand modified the model code. All authors were involved in helpful discussions and contributions \n373 \nto the manuscript. 374 Author contributions. LW designed research; NQ conceptualized the analysis and wrote the \n371 \nmanuscript; LW provided scientific suggestions for the manuscript. QL carried out the simulations \n372 \nand modified the model code. All authors were involved in helpful discussions and contributions \n373 \nto the manuscript. 374 Author contributions. LW designed research; NQ conceptualized the analysis and wrote the \n371 \nmanuscript; LW provided scientific suggestions for the manuscript. QL carried out the simulations \n372 \nand modified the model code. All authors were involved in helpful discussions and contributions \n373 \nto the manuscript. 374 Competing interests. The authors declare that they have no conflict of interest. 375 Acknowledgments. 5.3 Subsidence in response to the diabatic cooing \n280 352 There are two major differences in the eyewall structure by comparing the simulated TCs \n338 \nwith and without the SEF. For the simulated TC with the SEF, the eyewall updrafts are stronger \n339 \nand the eyewall is more upright. The eyewall anvil is located at a higher altitude together with the \n340 \ndeep eyewall convection. As illustrated in Fig. 15, the upper-level dry-air inflow beneath the high-\n341 \naltitude anvil from the inner eyewall is important to the formation of the moat. Diagnostic analysis \n342 \nindicates that the dry inflow below the eyewall anvil is induced by the diabatic cooling released \n343 \nby the sublimation, melting and evaporation of hydrometeors falling from the eyewall anvil. With \n344 \nthe penetration of dry air by the upper-level inflow, subsidence occurs, promoting the formation \n345 \nof the moat between the inner eyewall and the spiral rainband. Afterward, the SEF occurs by \n346 \nshowing the double eyewall structure and the moat with subsidence. In the TC without the SEF, \n347 \nthe eyewall updrafts are weak and tilt much outward, showing an eyewall anvil at the lower altitude. 348 \nThe cooling induced by the falling hydrometeors beneath the low-altitude eyewall anvil is \n349 \nsignificantly reduced and offset by the diabatic warming. As a result, no upper-level dry-air inflow \n350 \noccurs below the eyewall anvil, and no moat forms between the inner eyewall and the spiral \n351 \nrainband. 352 Our study highlights the importance of the inner eyewall structure in the moat formation \n353 \nthrough the cooling-induced upper-level inflow and subsidence beneath the eyewall anvil. While \n354 \nthe subsidence induced by the evaporative cooling from the precipitation at the outer radii from \n355 \nthe anvil is revealed by Tyner et al. (2018), who emphasized the role of the penetrative downdraft \n356 \nin promoting the local rainband convection and the subsequent outer eyewall formation, the \n357 \ncooling-induced upper-level dry-air inflow and its important role in the moat formation are not \n358 \nmentioned in their study. Recent studies indicated that a mesoscale descending inflow driven by \n359 \nthe middle-level melting and evaporative cooling in the downwind portion of the rainband can \n360 16 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. trigger new convective updrafts that are important to the subsequent SEF (Didlake et al., 2018; \n361 \nWunsch and Didlake, 2018; Yu et al., 2020). 5.3 Subsidence in response to the diabatic cooing \n280 This study was jointly supported by the National Natural Science Foundation \n376 \nof China (41730961, 41675009, 42075072, 41905001), the Postdoctoral Science Foundation of \n377 \nChina (2019M661342), the Natural Science Foundation of Jiangsu Province (BK20201505), and \n378 \nthe Open Research Program of the State Key Laboratory of Severe Weather (2019LASW-A02). 379 \nWe would like to acknowledge the use of computational resources for conducting the simulations \n380 \nat the High-Performance Computing Center of Nanjing University of Information Science and \n381 \nTechnology. 382 17 17 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. References 383 Abarca, S. F. and Montgomery, M. 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Atmos., \n531 \n120, 590-622, doi: 10.1002/2014JD022056, 2015. 532 24 534 \nFigure 1. Time series of intensity changes for the maximum azimuthal-mean near-surface wind \n535 \n(VMAX, m s-1) during the 72-h sensitivity (NSEF, black) and control run (CTL, red). The gray \n536 \nshading denotes the period of the eyewall replacement circle. 537 \nhttps://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. 534 \nFigure 1. Time series of intensity changes for the maximum azimuthal-mean near-surface wind\n535\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. 534 \nFigure 1. Time series of intensity changes for the maximum azimuthal-mean near-surface wind \n535 \n(VMAX, m s-1) during the 72-h sensitivity (NSEF, black) and control run (CTL, red). The gray \n536 \nshading denotes the period of the eyewall replacement circle. 537 534 Figure 1. Time series of intensity changes for the maximum azimuthal-mean near-surface wind \n535 \n(VMAX, m s-1) during the 72-h sensitivity (NSEF, black) and control run (CTL, red). The gray \n536 \nshading denotes the period of the eyewall replacement circle. 537 25 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. 538 \nFigure 2. Time-radius cross-sections of the azimuthal-mean (a, b) tangential wind (m s-1) and (c\n539 \nd) vertical motion (m s-1) at 0.5-km height for (a, c) NSEF and (b, d) CTL. The solid lines\n540 \nindicate the radius of the maximum tangential wind (RMW). doi: The black dashed lines indicate\n541 \nthe SEF, while the blue dashed lines denote the time when the secondary maximum wind is\n542 \nequal to the primary maximum wind. 543 538 538 \nFigure 2. Time-radius cross-sections of the azimuthal-mean (a, b) tangential wind (m s-1) and (c, \n539 \nd) vertical motion (m s-1) at 0.5-km height for (a, c) NSEF and (b, d) CTL. The solid lines \n540 \nindicate the radius of the maximum tangential wind (RMW). The black dashed lines indicate \n541 \nthe SEF, while the blue dashed lines denote the time when the secondary maximum wind is \n542 \nequal to the primary maximum wind. 543 26 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. 544 \nFigure 3. Horizontal distributions of the radar reflectivity at 5-km height at (a, d) 28, (b, e) 32, and \n545 \n(c, f) 44 h for (a-c) NSEF and (d-f) CTL. Vectors are the large-scale vertical wind shear (VWS, \n546 \nVspeed (200 hPa) -Vspeed (850 hPa)). 547 Figure 3. Horizontal distributions of the radar reflectivity at 5-km height at (a, d) 28, (b, e) 32, and \n545 \n(c, f) 44 h for (a-c) NSEF and (d-f) CTL. Vectors are the large-scale vertical wind shear (VWS, \n546 \nVspeed (200 hPa) -Vspeed (850 hPa)). 547 27 548 \nFigure 4. Radius-height cross-sections of the azimuthal-mean vertical motion (shaded, m s-1), in-\n549 \nplain flow (vector, m s-1) and radial inflows of -1 and -3 m s-1 (white contours) at (a, c) 30 h \n550 \nand (b, d) 32 h for (a, b) NSEF and (c, d) CTL. The white dashed arrows denote the eyewall. 551 \n \n \n552 \nhttps://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. 548 \nFigure 4 Radius-height cross-sections of the azimuthal-mean vertical motion (shaded m s-1) in-\n549 548 \nFigure 4. Radius-height cross-sections of the azimuthal-mean vertical motion (shaded, m s-1), in-\n549 \nplain flow (vector, m s-1) and radial inflows of -1 and -3 m s-1 (white contours) at (a, c) 30 h \n550 \nand (b, d) 32 h for (a, b) NSEF and (c, d) CTL. The white dashed arrows denote the eyewall. 551 \n \n \n552 Figure 4. doi: Radius-height cross-sections of the azimuthal-mean vertical motion (shaded, m s-1), in-\n549 \nplain flow (vector, m s-1) and radial inflows of -1 and -3 m s-1 (white contours) at (a, c) 30 h \n550 \nand (b, d) 32 h for (a, b) NSEF and (c, d) CTL. The white dashed arrows denote the eyewall. 551 \n552 28 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. 553 \n554 553 \n \n554 \nFigure 5. The contoured frequency by altitude diagram (CFAD, %) of (a) the vertical motion for \n555 \nthe 10-km radially inside and outside of the RMW and (b) the radial wind within a radial \n556 \ndistance of 60 km starting from the radius of 10-km outside the eyewall (RMW+10 km to \n557 \nRMW+70 km) for CTL (dashed lines) and NSEF (solid lines) at 30 h. The red and blue lines \n558 \nare the 0.1, and 0.05 percentile. 559 553 \n554 Figure 5. The contoured frequency by altitude diagram (CFAD, %) of (a) the vertical motion for \n555 \nthe 10-km radially inside and outside of the RMW and (b) the radial wind within a radial \n556 \ndistance of 60 km starting from the radius of 10-km outside the eyewall (RMW+10 km to \n557 \nRMW+70 km) for CTL (dashed lines) and NSEF (solid lines) at 30 h. The red and blue lines \n558 \nare the 0.1, and 0.05 percentile. 559 29 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. 560 560 \nFigure 6. Horizontal distribution of the cloud-top temperature (CTT, shaded, Co) superimposed \n561 \nwith the 15-km horizontal wind field (vector, m s-1) and RMW (white circle) at 30 h for (a) \n562 \nNSEF and (b) CTL. The black circle indicates the radius of 100 km relative to the TC center. 563 560 560 Figure 6. Horizontal distribution of the cloud-top temperature (CTT, shaded, Co) superimposed \n561 \nwith the 15-km horizontal wind field (vector, m s-1) and RMW (white circle) at 30 h for (a) \n562 \nNSEF and (b) CTL. The black circle indicates the radius of 100 km relative to the TC center. 563 30 564 \nFigure 7. doi: Radius-height cross-sections of the upshear-right quadrant-mean perturbation virtual \n565 \npotential temperature (shaded, K) at 30 h for (a) NSEF and (b) CTL. Contours are the vertical \n566 \n1\nhttps://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. 564 \nFigure 7. Radius-height cross-sections of the upshear-right quadrant-mean perturbation virtual \n565 \npotential temperature (shaded, K) at 30 h for (a) NSEF and (b) CTL. Contours are the vertical \n566 \nmotion (updraft, black solid contours: 0.5 m s-1; downdrafts, black dashed contours: 0.05 m \n567 \ns-1). The black dashed arrows denote the eyewall. 568 564 \nFigure 7. Radius-height cross-sections of the upshear-right quadrant-mean perturbation virtual \n565 \npotential temperature (shaded, K) at 30 h for (a) NSEF and (b) CTL. Contours are the vertical \n566 \nmotion (updraft, black solid contours: 0.5 m s-1; downdrafts, black dashed contours: 0.05 m \n567 \ns-1). The black dashed arrows denote the eyewall. 568 Figure 7. Radius-height cross-sections of the upshear-right quadrant-mean perturbation virtual \n565 \npotential temperature (shaded, K) at 30 h for (a) NSEF and (b) CTL. Contours are the vertical \n566 \nmotion (updraft, black solid contours: 0.5 m s-1; downdrafts, black dashed contours: 0.05 m \n567 \ns-1). The black dashed arrows denote the eyewall. 568 31 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. 569 \nFigure 8. Radius-height cross-sections of the upshear-right quadrant-mean (a, e) buoyance force \n570 \n(shaded, 10-3 m s-2), (b, f) the thermal buoyancy (shaded, 10-3 m s-2), (c, g) the dynamic \n571 \nbuoyancy (shaded, 10-3 m s-2), and (d, h) the hydrometeor loading (shaded, 10-3 m s-2) \n572 \nsuperimposed with the vertical motion (updraft, black solid contours: 0.5 m s-1; downdrafts, \n573 \nblack dashed contours: 0.05 m s-1) at 30 h for (a-d) NSEF and (e-h) CTL. The black dashed \n574 \narrows denote the eyewall. 575 Figure 8. Radius-height cross-sections of the upshear-right quadrant-mean (a, e) buoyance force \n570 \n(shaded, 10-3 m s-2), (b, f) the thermal buoyancy (shaded, 10-3 m s-2), (c, g) the dynamic \n571 \nbuoyancy (shaded, 10-3 m s-2), and (d, h) the hydrometeor loading (shaded, 10-3 m s-2) \n572 \nsuperimposed with the vertical motion (updraft, black solid contours: 0.5 m s-1; downdrafts, \n573 \nblack dashed contours: 0.05 m s-1) at 30 h for (a-d) NSEF and (e-h) CTL. The black dashed \n574 \narrows denote the eyewall. doi: 575 Figure 8. Radius-height cross-sections of the upshear-right quadrant-mean (a, e) buoyance force \n570 \n(shaded, 10-3 m s-2), (b, f) the thermal buoyancy (shaded, 10-3 m s-2), (c, g) the dynamic \n571 \nbuoyancy (shaded, 10-3 m s-2), and (d, h) the hydrometeor loading (shaded, 10-3 m s-2) \n572 \nsuperimposed with the vertical motion (updraft, black solid contours: 0.5 m s-1; downdrafts, \n573 \nblack dashed contours: 0.05 m s-1) at 30 h for (a-d) NSEF and (e-h) CTL. The black dashed \n574 \narrows denote the eyewall. 575 32 576 \nFigure 9. Radius-height cross-sections of the upshear-right quadrant-mean (a, b) vertical pressure \n577 \ngradient force (shaded, 10-3 m s-2) and (c, d) the sum of buoyancy and vertical pressure \n578 \ngradient force (shaded, 10-3 m s-2), superimposed with the vertical motion (updraft, black solid \n579 \ncontours: 0.5 m s-1; downdrafts, black dashed contours: 0.05 m s-1) at 30 h for (a, c) NSEF \n580 \nand (b, d) CTL. The black dashed arrows denote the eyewall. 581 \nhttps://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. 576 \nFigure 9. Radius-height cross-sections of the upshear-right quadrant-mean (a, b) vertical pressure \n577 \ngradient force (shaded, 10-3 m s-2) and (c, d) the sum of buoyancy and vertical pressure \n578 \ngradient force (shaded, 10-3 m s-2), superimposed with the vertical motion (updraft, black solid \n579 \ncontours: 0.5 m s-1; downdrafts, black dashed contours: 0.05 m s-1) at 30 h for (a, c) NSEF \n580 \nand (b, d) CTL. The black dashed arrows denote the eyewall. 581 576 \nFi\n9 R di\nh i ht\nti\nf th\nh\ni ht\nd\nt\n(\nb)\nti\nl\n577 Figure 9. Radius-height cross-sections of the upshear-right quadrant-mean (a, b) vertical pressure \n577 \ngradient force (shaded, 10-3 m s-2) and (c, d) the sum of buoyancy and vertical pressure \n578 \ngradient force (shaded, 10-3 m s-2), superimposed with the vertical motion (updraft, black solid \n579 \ncontours: 0.5 m s-1; downdrafts, black dashed contours: 0.05 m s-1) at 30 h for (a, c) NSEF \n580 \nand (b, d) CTL. The black dashed arrows denote the eyewall. 581 33 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. 582 \nFigure 10. doi: Radius-height cross-sections of the upshear-right quadrant-mean (a, e) diabatic heating\n583 \nrate (shaded, 10-3 K s-1), (b, f) cooling rate including evaporation, melting and sublimation\n584 \nprocesses (shaded, 10-3 K s-1), (c, g) sublimation cooling rate, and (d, h) melting and\n585 \nevaporation cooling rates superimposed with the vertical motion (updraft, solid lines of 0.5\n586 \nm s-1; downdrafts, dashed lines of -0.05 and -0.3 m s-1) at 30 h for (a-d) NSEF and (e-h) CTL\n587 \nThe black dashed arrows denote the eyewall. 588 582 \nFigure 10. Radius-height cross-sections of the upshear-right quadrant-mean (a, e) diabatic heating \n583 \nrate (shaded, 10-3 K s-1), (b, f) cooling rate including evaporation, melting and sublimation \n584 \nprocesses (shaded, 10-3 K s-1), (c, g) sublimation cooling rate, and (d, h) melting and \n585 \nevaporation cooling rates superimposed with the vertical motion (updraft, solid lines of 0.5 \n586 \nm s-1; downdrafts, dashed lines of -0.05 and -0.3 m s-1) at 30 h for (a-d) NSEF and (e-h) CTL. 587 \nThe black dashed arrows denote the eyewall. 588 582 \nFigure 10. Radius-height cross-sections of the upshear-right quadrant-mean (a, e) diabatic heating \n583 \nrate (shaded, 10-3 K s-1), (b, f) cooling rate including evaporation, melting and sublimation \n584 \nprocesses (shaded, 10-3 K s-1), (c, g) sublimation cooling rate, and (d, h) melting and \n585 \nevaporation cooling rates superimposed with the vertical motion (updraft, solid lines of 0.5 \n586 \nm s-1; downdrafts, dashed lines of -0.05 and -0.3 m s-1) at 30 h for (a-d) NSEF and (e-h) CTL. 587 \nThe black dashed arrows denote the eyewall. 588 34 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. Figure 11. Azimuthal-time cross-sections of the layer-mean (11.5-12.5 km) diabatic cooling \n(shaded, 10-3 K s-1) and subsidence (contour, m s-1) averaged within a radial distance of 25 \nkm starting from the radius of 5-km outside the eyewall (RMW+5 km to RMW+30 km) of \n(a) NSEF, (b) CTL, and (c) differences between CTL and NSEF. The black contours are -0.8 \nm s-1 in (a, b) and -0.4 m s-1 in (c). Figure 11. doi: Azimuthal-time cross-sections of the layer-mean (11.5-12.5 km) diabatic cooling \n590 \n(shaded, 10-3 K s-1) and subsidence (contour, m s-1) averaged within a radial distance of 25 \n591 \nkm starting from the radius of 5-km outside the eyewall (RMW+5 km to RMW+30 km) of \n592 \n(a) NSEF, (b) CTL, and (c) differences between CTL and NSEF. The black contours are -0.8 \n593 \nm s-1 in (a, b) and -0.4 m s-1 in (c). 594 35 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. 595 \nFigure 12. Radius-height cross-sections of the upshear-right quadrant-mean vertical (shaded\n596 \n1) and radial motion (contours, m s-1) forced by the (a, e) diabatic heating, (b, f) hydrome\n597 \ncooling, (c, g) sublimation cooling, (d, h) melting and evaporation cooling at 30 h for \n598 \nNSEF and (c-h) CTL. Note that the radial wind is at 2 m s-1 intervals in (a) and (e), and\n599 \ns-1 intervals in others. The white dashed lines with 0.5 m s-1 vertical motion indicat\n600 \neyewall convection region. The black dashed arrows denote the eyewall. 601 Figure 12. Radius-height cross-sections of the upshear-right quadrant-mean vertical (shaded, m s-\n596 \n1) and radial motion (contours, m s-1) forced by the (a, e) diabatic heating, (b, f) hydrometeors \n597 \ncooling, (c, g) sublimation cooling, (d, h) melting and evaporation cooling at 30 h for (a-d) \n598 \nNSEF and (c-h) CTL. Note that the radial wind is at 2 m s-1 intervals in (a) and (e), and 1 m \n599 \ns-1 intervals in others. The white dashed lines with 0.5 m s-1 vertical motion indicate the \n600 \neyewall convection region. The black dashed arrows denote the eyewall. 601 36 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. 602 \nFigure 13. Radius-height cross-sections of the the upshear-right quadrant-mean relative humidity \n603 602 \nFigure 13. Radius-height cross-sections of the the upshear-right quadrant-mean relative humidity \n603 \n(shaded, %) and in-plain flows (vector, m s-1) at t = 30 h for (a) NSEF and (b) CTL. The black \n604 \nand purple lines are radial inflows of 0 01 and 1 m s-1 respectively The red dashed arrow\n605 Figure 13. doi: Radius-height cross-sections of the the upshear-right quadrant-mean relative humidity \n603 \n(shaded, %) and in-plain flows (vector, m s-1) at t = 30 h for (a) NSEF and (b) CTL. The black \n604 \nand purple lines are radial inflows of -0.01 and -1 m s-1, respectively. The red dashed arrow \n605 \nindicates the upper-level dry inflows. 606 37 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. 607 \nFigure 14. Same as in Fig. 11 but for subsidence (shaded, m s-1) and radial inflows (contour, m s-\n608 \n1) of (a) NSEF, (b) CTL, and (c) differences between CTL and NSEF. 609 \n \n \n610 607 \nFigure 14. Same as in Fig. 11 but for subsidence (shaded, m s-1) and radial inflows (contour, m s-\n608 \n1) of (a) NSEF, (b) CTL, and (c) differences between CTL and NSEF. 609 \n610 607 \nFigure 14. Same as in Fig. 11 but for subsidence (shaded, m s-1) and radial inflows (contour, m s-\n608 \n1) of (a) NSEF, (b) CTL, and (c) differences between CTL and NSEF. 609 \n610 38 38 https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. https://doi.org/10.5194/acp-2021-147\nPreprint. Discussion started: 7 April 2021\nc⃝Author(s) 2021. CC BY 4.0 License. 611 611 \n \n612 \nFigure 15. The inner-core structures of a hurricane undergoing the eyewall replacement circle, \n613 \nincluding the eye, the primary eyewall, the moat, and the rainband of evolving into the \n614 \nsecondary eyewall. The black solid arrows denote the air motion relative to the TC. The black \n615 \ndashed arrows show the upper-level descending inflows beneath the cumulus anvil from the \n616 \ninner eyewall. The light blue shading indicates the cooling induced by the sublimation, \n617 \nmelting, and evaporation of hydrometeors (ice, snow, graupel, and raindrops) associated with \n618 \nthe moat subsidence. The gray dashed lines indicate the precipitation below the clouds. The \n619 \nred solid line denotes the 0 oC temperature of the melting level. 620 611 \n612 Figure 15. The inner-core structures of a hurricane undergoing the eyewall replacement circle, \n613 \nincluding the eye, the primary eyewall, the moat, and the rainband of evolving into the \n614 \nsecondary eyewall. The black solid arrows denote the air motion relative to the TC. doi: The black \n615 \ndashed arrows show the upper-level descending inflows beneath the cumulus anvil from the \n616 \ninner eyewall. The light blue shading indicates the cooling induced by the sublimation, \n617 \nmelting, and evaporation of hydrometeors (ice, snow, graupel, and raindrops) associated with \n618 \nthe moat subsidence. The gray dashed lines indicate the precipitation below the clouds. The \n619 \nred solid line denotes the 0 oC temperature of the melting level. 620 39"
https://openalex.org/W2984361962
https://europepmc.org/articles/pmc6862283?pdf=render
English
null
VXC-72R/ZrO2/GCE-Based Electrochemical Sensor for the High-Sensitivity Detection of Methyl Parathion
Materials
2,019
cc-by
8,486
Received: 6 October 2019; Accepted: 1 November 2019; Published: 5 November 2019 Received: 6 October 2019; Accepted: 1 November 2019; Published: 5 November 2019 Abstract: In this work, a carbon black (VXC-72R)/zirconia (ZrO2) nanocomposite-modified glassy carbon electrode (GCE) was designed, and a VXC-72R/ZrO2/GCE-based electrochemical sensor was successfully fabricated for the high-sensitivity detection of methyl parathion (MP). Electrochemical measurements showed that the VXC-72R/ZrO2/GCE-based electrochemical sensor could make full use of the respective advantages of the VXC-72R and ZrO2 nanoparticles to enhance the MP determination performance. The VXC-72R nanoparticles had high electrical conductivity and a large surface area, and the ZrO2 nanoparticles possessed a strong affinity to phosphorus groups, which could achieve good organophosphorus adsorption. On the basis of the synergistic effect generated from the interaction between the VXC-72R and ZrO2 nanoparticles, the VXC-72R/ZrO2/GCE-based electrochemical sensor could show excellent trace analysis determination performance. The low detection limit could reach up to 0.053 µM, and there was a linear concentration range of 1 µM to 100 µM. Such a high performance indicates that the VXC-72R/ZrO2/GCE-based electrochemical sensor has potential in numerous foreground applications. Keywords: VXC-72R/ZrO2/GCE nanocomposite electrode; synergetic effect; electrochemical sensor; methyl parathion materials materials materials Materials 2019, 12, 3637; doi:10.3390/ma12213637 VXC-72R/ZrO2/GCE-Based Electrochemical Sensor for the High-Sensitivity Detection of Methyl Parathion Runqiang Liu 1,2,3,*, Yashuang Wang 3, Bo Li 3, Binbin Liu 3, Huina Ma 3, Dongdong Li 3, Li Dong 3, Fang Li 3, Xiling Chen 3,* and Xinming Yin 2,* 1 Postdoctoral Research Base, Henan Institute of Science and Technology, Xinxiang 453003, China 1 Postdoctoral Research Base, Henan Institute of Science and Technology, Xinxiang 453003, China 2 College of Plant Protections, Henan Agricultural University, Zhengzhou 450002, China 3 School of Resources and Environment, Henan Institute of Science and Technology, Xinxiang 453003, China; yashuangwang1102@126.com (Y.W.); boli9277@163.com (B.L.); LiuBinbin4118@163.com (B.L.); mhn18438020581@163.com (H.M.); Lidongdong1994@126.com (D.L.); ledong181255@163.com (L.D.); lifangday@163.com (F.L.) 1 Postdoctoral Research Base, Henan Institute of Science and Technology, Xinxiang 453003, China 2 College of Plant Protections, Henan Agricultural University, Zhengzhou 450002, China gy g 2 College of Plant Protections, Henan Agricultural University, Zhengzhou 450002, China g g y g 3 School of Resources and Environment, Henan Institute of Science and Technology, Xinxiang 453003, China; yashuangwang1102@126.com (Y.W.); boli9277@163.com (B.L.); LiuBinbin4118@163.com (B.L.); mhn18438020581@163.com (H.M.); Lidongdong1994@126.com (D.L.); ledong181255@163.com (L.D.); lifangday@163.com (F.L.) * Correspondence: liurunqiang1983@126.com (R.L.); chenxiling@hist.edu.cn (X.C.); xmyin11@163.com (X 1. Introduction As a classic pesticide, methyl parathion (MP) has made important contributions to the field of crop protection and pest control [1]. However, the problem of MP residues has a serious impact on human health and the environment [2–4]. Therefore, research and development for a convenient and efficient detecting method for MP at trace levels have attracted more and more research interests from scientific researchers. Although traditional analysis methods have played a certain role in detecting MP, their complex working procedures and high compliance costs make it difficult to meet the demand for fast speeds and high, efficient detection [5–7]. Thus, it is of great significance to design a simple, low-cost, and sensitive analytical technique for the detection of MP. At present, electrochemical sensors have been shown to enhance detection efficiency and reduce operation costs, which accelerates the development of high-performance pesticide detection technology [8–13]. It should also be noted that the preparation of high-performance electrochemical sensors is largely dependent on chemically modified electrodes. Among the many modification materials, carbon materials (graphene [4,14–17], carbon nanotubes [18–20], mesoporous carbon [21,22], Materials 2019, 12, 3637; doi:10.3390/ma12213637 www.mdpi.com/journal/materials www.mdpi.com/journal/materials 2 of 11 Materials 2019, 12, 3637 etc.) play a significant role in improving electrochemical sensor performance. Moreover, zirconia (ZrO2) has been extensively used in the field of electrochemical sensors [14,23–25]. This material has a strong affinity to phosphorus groups, which makes ZrO2-based electrochemical sensors possess selective recognition and adsorption functions for MP [14,26,27]. In particular, nanostructured ZrO2 particles show large specific surface areas, which can further enhance MP detection performance. Moreover, ZrO2 possesses high chemical stability and a lack of toxicity, which contributes to the extensive use of this material. According to the available literature, the collaborative use of carbon materials and ZrO2 nanoparticles can result in better MP detection performance [28,29]. Dai et al. have fabricated a highly sensitive electrochemical sensor based on the nanocomposites of carbon nanofibers and ultrafine zirconia nanoparticles (ZrO2–CNFs) [26]. The research results showed that the ZrO2–CNF-based electrochemical sensor could present with high sensitivity and a good linear relationship between the peak current and MP concentration due to its strong affinity and adsorption properties in terms of methyl parathion. Furthermore, Gong et al. have successfully synthesized zirconia nanoparticle-decorated graphene nanosheets (ZrO2 NPs–GNs) through a facile electrochemical approach [14]. 1. Introduction The corresponding electrochemical sensor can give full play to the advantages of ZrO2 NPs (high recognition and enrichment capability for phosphoric moieties) and GNs (large surface area and high electrical conductivity) to significantly enhance MP detection performance. The above-mentioned analysis indicated that the collaborative use of carbon materials and ZrO2 nanoparticles possesses a synergistic effect. However, it should be noted that these experimental strategies have some weaknesses in spite of their excellent determination performance. The corresponding preparation methods, including electrochemical deposition and an electrospinning technique, involve more complex experimental operations, and some experimental equipment is very expensive. These problems have restricted the practical applications for electrochemical sensors based on nanocomposites of carbon materials and ZrO2 nanoparticles. Therefore, it is quite necessary to develop a simple, rapid, and low-cost technique to prepare electrochemical sensors with excellent determination performance. In this work, we fabricated a high-sensitive electrochemical sensor based on a carbon black (VXC-72R)/ZrO2 nanocomposite electrode. The VXC-72R/ZrO2/glassy carbon electrode (GCE)-based electrochemical sensor can make full use of the respective advantages of VXC-72R and ZrO2 nanoparticles to enhance MP determination performance. More than anything, the present experimental strategy is simple, rapid, and low-cost, which can help facilitate the practical application of electrochemical sensors based on nanocomposites of carbon materials and ZrO2 nanoparticles. To the best of our knowledge, there has been no report about VXC-72R/ZrO2/GCE-based electrochemical sensors. Moreover, the obtained electrochemical sensor has excellent trace analysis determination performance. 3. Results and Discussion 3. Results and Discussion Figure 1a,b shows the XRD patterns of the VXC-72R and VXC-72R/ZrO2 nanocomposites. It can be seen that the VXC-72R nanoparticles presented obvious characteristic diffraction peaks. For the VXC-72R/ZrO2 nanocomposite, the XRD pattern showed some well-defined diffraction peaks that were in complete agreement with the standard diffraction peak of VXC-72R and ZrO2 (JGCEDS No. 17-0923) [8,24]. Figure 1c shows the XPS spectra of the VXC-72R/ZrO2 nanocomposite. We can clearly see that the characteristic peaks of the elements C1s, Zr3d, and O1s appeared in the XPS spectra. It needs to be noted that the Zr element contained two splitting peaks, which corresponded with Zr 3d5/2 (182.3 eV) and Zr 3d3/2 (184.5 eV) [26]. Figure 1a,b shows the XRD patterns of the VXC-72R and VXC-72R/ZrO2 nanocomposites. It can be seen that the VXC-72R nanoparticles presented obvious characteristic diffraction peaks. For the VXC-72R/ZrO2 nanocomposite, the XRD pattern showed some well-defined diffraction peaks that were in complete agreement with the standard diffraction peak of VXC-72R and ZrO2 (JGCEDS No. 17-0923) [8,24]. Figure 1c shows the XPS spectra of the VXC-72R/ZrO2 nanocomposite. We can clearly see that the characteristic peaks of the elements C1s, Zr3d, and O1s appeared in the XPS spectra. It needs to be noted that the Zr element contained two splitting peaks, which corresponded with Zr 3d5/2 (182.3 eV) and Zr 3d3/2 (184.5 eV) [26]. ( ) ( ) [ ] Figure 1. X-ray diffraction (XRD) patterns of (a) carbon black (VXC-72R) and (b) VXC-72R/ZrO2 nanocomposites. X-ray photoelectron spectroscopy (XPS) spectra of (c) VXC-72R/ZrO2 nanocomposites and the (d) Zr element. Figure 1. X-ray diffraction (XRD) patterns of (a) carbon black (VXC-72R) and (b) VXC-72R/ZrO2 nanocomposites. X-ray photoelectron spectroscopy (XPS) spectra of (c) VXC-72R/ZrO2 nanocomposites and the (d) Zr element. Figure 1. X-ray diffraction (XRD) patterns of (a) carbon black (VXC-72R) and (b) VXC-72R/ZrO2 nanocomposites. X-ray photoelectron spectroscopy (XPS) spectra of (c) VXC-72R/ZrO2 nanocomposites and the (d) Zr element Figure 1. X-ray diffraction (XRD) patterns of (a) carbon black (VXC-72R) and (b) VXC-72R/ZrO2 nanocomposites. X-ray photoelectron spectroscopy (XPS) spectra of (c) VXC-72R/ZrO2 nanocomposites and the (d) Zr element. It is generally known that the microscopic morphology and particle size distribution of the modification material always have a great impact on the detection performance of an electrochemical sensor. Figure 2 shows SEM images of the VXC-72R and VXC-72R/ZrO2 nanocomposite. 2. Materials and Methods The VXC-72R/ZrO2/GCE-based electrochemical sensor was successfully fabricated through drop-coating technology. First, a certain amount of VXC-72R (Cabot Corporation, Boston, MA, USA) was homogeneously dispersed in dimethylformamide (DMF) solvent to obtain a VXC-72R suspension (40 mL, 0.5 mg mL−1) with the help of ultrasonic dispersion. Then, 120 mg of ZrO2 nanoparticles (99.99%, ≤100 nm, Shanghai Aladdin Bio-Chem Technology Co., LTD, Shanghai, China) was added into the VXC-72R suspension through vigorous stirring (30 min). Subsequently, the mixed VXC-72R/ZrO2 suspension (5 µL, 0.5 mg mL−1) was coated on the surface of a GCE. After heat treatment (15 min) with the help of an infrared lamp (Hwato 150 W, Chengdu, China), the VXC-72R/ZrO2/GCE-based electrochemical sensor was successfully fabricated. A VXC-72R/GCE-based electrochemical sensor was obtained using a similar technique. The structure and morphology were studied through X-ray diffraction (XRD, Bruker DX-1000, Karlsruhe, Germany), X-ray photoelectron spectroscopy (XPS, Thermo Fisher Scientific, Waltham, MA, USA), and scanning electron microscopy (SEM, JEOL JSM-6360LV, Tokyo, Japan). The electrochemical 3 of 11 Materials 2019, 12, 3637 measurements were carried out using a CHI660E electrochemical workstation (CH Instruments, Shanghai, China). Modified GCE composite electrodes were used as a working electrode, with platinum wire and a saturated calomel electrode (SCE) as a counterelectrode and reference electrode, respectively. A certain amount of the mixed solution of NaH2PO4 and Na2HPO4 was prepared to be used as phosphate-buffered solution (PBS, 0.1 M, pH 7.0). Materials 2019, 12, x FOR PEER REVIEW 3 of 11 electrode, with platinum wire and a saturated calomel electrode (SCE) as a counterelectrode and reference electrode, respectively. A certain amount of the mixed solution of NaH2PO4 and Na2HPO4 was prepared to be used as phosphate-buffered solution (PBS, 0.1 M, pH 7.0). 3. Results and Discussion 3. Results and Discussion The corresponding CV results are shown in Figure 3. It was found that the CV curve of the undecorated GCE-based electrochemical sensor had a pair of reversible redox peaks, which agreed with the research results [7]. For the VXC-72R/GCE-based electrochemical sensor, the current response of the reversible redox peaks significantly increased due to the high electric conductivity and large surface area of the VXC-72R nanoparticles [26,29]. Furthermore, the VXC-72R/ZrO2/GCE-based electrochemical sensor also showed obvious redox peaks with relatively satisfactory peak currents. When the good organophosphorus adsorption and large surface area of the VXC-72R/ZrO2 nanocomposites are taken into account, the VXC-72R/ZrO2/GCE-based electrochemical sensor may present excellent MP determination performance A cyclic voltammetry (CV) test was applied to explore the performance of the electrochemical sensors based on the undecorated GCEs, VXC-72R/GCEs, and VXC-72R/ZrO2/GCEs. The corresponding CV results are shown in Figure 3. It was found that the CV curve of the undecorated GCE-based electrochemical sensor had a pair of reversible redox peaks, which agreed with the research results [7]. For the VXC-72R/GCE-based electrochemical sensor, the current response of the reversible redox peaks significantly increased due to the high electric conductivity and large surface area of the VXC-72R nanoparticles [26,29]. Furthermore, the VXC-72R/ZrO2/GCE-based electrochemical sensor also showed obvious redox peaks with relatively satisfactory peak currents. When the good organophosphorus adsorption and large surface area of the VXC-72R/ZrO2 nanocomposites are taken into account, the VXC-72R/ZrO2/GCE-based electrochemical sensor may present excellent MP determination performance. present excellent MP determination performance. In order to investigate the MP determination performance, electrochemical sensors based on the undecorated GCEs, VXC-72R/GCEs, and VXC-72R/ZrO2/GCEs were tested in PBS solution (0.1 M, pH 7.0) of 0.1 mM MP. The corresponding CV results are shown in Figure 4. It can be obviously seen that there was an irreversible reduction peak in the CV curve of the GCE-based electrochemical sensor because of the irreversible reduction of the nitro group into the hydroxylamine group [30]. Meanwhile, the CV curve also contained a pair of redox peaks that corresponded to the reversible redox reaction of the hydroxylamine group [7,31,32]. For the VXC-72R/GCE-based electrochemical sensor, the peak current response significantly increased due to the high electrical conductivity and large surface area of the VXC-72R. In contrast, the VXC-72R/ZrO2/GCE-based electrochemical sensor presented a slightly lower peak current response, but the redox peaks were quite sharp, suggesting an optimum electrocatalytic performance [23]. 3. Results and Discussion 3. Results and Discussion It can be seen from Figure 2a that the VXC-72R nanoparticles presented a uniform particle size distribution and that the particle size was in the range of a nanometer. As shown here, the scale was 500 nm, and the average particle size was obviously less than 100 nm, which completely aligned with the manufacturer's data (≈30 nm). Figure 2b shows SEM images of the VXC-72R/ZrO2 nanocomposite. It is generally known that the microscopic morphology and particle size distribution of the modification material always have a great impact on the detection performance of an electrochemical sensor. Figure 2 shows SEM images of the VXC-72R and VXC-72R/ZrO2 nanocomposite. It can be seen from Figure 2a that the VXC-72R nanoparticles presented a uniform particle size distribution and that the particle size was in the range of a nanometer. As shown here, the scale was 500 nm, and the average particle size was obviously less than 100 nm, which completely aligned with the manufacturer’s data (≈30 nm). Figure 2b shows SEM images of the VXC-72R/ZrO2 nanocomposite. We found that the 4 of 11 Materials 2019, 12, 3637 introduction of ZrO2 nanoparticles had no major influence on the particle size distribution. In this work, the ZrO2 nanoparticles were purchased from Shanghai Aladdin Bio-Chem Technology Co., LTD. The purity in the office data was 99.99%, and the particle size was less than 100 nm. As shown in Figure 2b (with a 500-nm scale), both the micromorphology and particle size distribution were similar to the VXC-72R nanoparticles. Although these two pictures are not clear enough, the good dispersity and nanostructure morphology can still be observed: these could enhance the MP detection performance of the VXC-72R/ZrO2/GCE-based electrochemical sensor. Materials 2019, 12, x FOR PEER REVIEW 4 of 11 distribution were similar to the VXC-72R nanoparticles. Although these two pictures are not clear enough, the good dispersity and nanostructure morphology can still be observed: these could enhance the MP detection performance of the VXC 72R/ZrO2/GCE based electrochemical sensor Figure 2. SEM images of (a) VXC-72R and (b) VXC-72R/ZrO2 nanocomposites. Figure 2. SEM images of (a) VXC-72R and (b) VXC-72R/ZrO2 nanocomposites. Figure 2 SEM images of (a) VXC-72R and (b) VXC-72R/ZrO2 nanocomposites Figure 2. SEM images of (a) VXC-72R and (b) VXC-72R/ZrO2 nanocomposites. A cyclic voltammetry (CV) test was applied to explore the performance of the electrochemical sensors based on the undecorated GCEs, VXC-72R/GCEs, and VXC-72R/ZrO2/GCEs. 3. Results and Discussion 3. Results and Discussion [ ( ) ] [ ( ) ] g In order to investigate the MP determination performance, electrochemical sensors based on ndecorated GCEs, VXC-72R/GCEs, and VXC-72R/ZrO2/GCEs were tested in PBS solution (0.1 Figure 4. CV curves of 0.1 mM methyl parathion (MP) in phosphate-buffered saline (PBS) (0.1 M, pH 7.0) and the electrochemical sensors based on the undecorated GCEs, VXC-72R/GCEs, and VXC- 72R/ZrO2/GCEs. Figure 4. CV curves of 0.1 mM methyl parathion (MP) in phosphate-buffered saline (PBS) (0.1 M, pH 7.0) and the electrochemical sensors based on the undecorated GCEs, VXC-72R/GCEs, and VXC-72R/ZrO2/GCEs. [ ( ) ] [ ( ) ] g In order to investigate the MP determination performance, electrochemical sensors based on ndecorated GCEs, VXC-72R/GCEs, and VXC-72R/ZrO2/GCEs were tested in PBS solution (0. Figure 4. CV curves of 0.1 mM methyl parathion (MP) in phosphate-buffered saline (PBS) (0.1 M, pH 7.0) and the electrochemical sensors based on the undecorated GCEs, VXC-72R/GCEs, and VXC- 72R/ZrO2/GCEs. Figure 4. CV curves of 0.1 mM methyl parathion (MP) in phosphate-buffered saline (PBS) (0.1 M, pH 7.0) and the electrochemical sensors based on the undecorated GCEs, VXC-72R/GCEs, and VXC-72R/ZrO2/GCEs. Figure 4. CV curves of 0.1 mM methyl parathion (MP) in phosphate-buffered saline (PBS) (0.1 M, pH 7.0) and the electrochemical sensors based on the undecorated GCEs, VXC-72R/GCEs, and VXC- 72R/ZrO2/GCEs Figure 4. CV curves of 0.1 mM methyl parathion (MP) in phosphate-buffered saline (PBS) (0.1 M, pH 7.0) and the electrochemical sensors based on the undecorated GCEs, VXC-72R/GCEs, and VXC-72R/ZrO2/GCEs. In order to enhance MP detection performance, the effects of ZrO2 concentration and the pH value on the current response were studied through the CV and differential pulse voltammetry (DPV) methods, as shown in Figure 5. Figure 5a shows the effect of ZrO2 concentration on the oxidation current response with the CV method, where the VXC-72R concentration was 0.5 mg mL−1. When a small amount of ZrO2 nanoparticles was introduced, the peak current response increased gradually with the increase in ZrO2 concentration due to the strong affinity to phosphorus groups, which made the ZrO2-based electrochemical sensor possess a selective recognition and adsorption function for MP. However, it is a pity that the excessive concentration could produce serious negative effects because of the increased electron transfer resistance. Figure 5b shows the effects of the pH value on the current response, as studied through DPV methods. 3. Results and Discussion 3. Results and Discussion For the VXC-72R/GCE-based nsor, the peak current response significantly increased due to the high electrical co rge surface area of the VXC-72R. In contrast, the VXC-72R/ZrO2/GCE-based electro esented a slightly lower peak current response, but the redox peaks were quite sh n optimum electrocatalytic performance [23]. This can be explained by the synergi XC-72R and ZrO2 nanoparticles. The VXC-72R nanoparticles had high electrical con rge surface area, and the ZrO2 nanoparticles possessed a strong affinity to phos hich could achieve good organophosphorus adsorption [24] On the basis of the s an ir of th CV Figure 3. Cyclic voltammetry (CV) curves of the electrochemical sensors based on the undecorated glassy carbon electrodes (GCEs), VXC-72R/GCEs, and VXC-72R/ZrO2/GCEs in 5 mM of K3[Fe(CN)6]/K4[Fe(CN)6] solution containing 0.1 mM KCl. In order to investigate the MP determination performance, electrochemical sensors based on ndecorated GCEs, VXC-72R/GCEs, and VXC-72R/ZrO2/GCEs were tested in PBS solution (0. Figure 3. Cyclic voltammetry (CV) curves of the electrochemical sensors based on the undecorated glassy carbon electrodes (GCEs), VXC-72R/GCEs, and VXC-72R/ZrO2/GCEs in 5 mM of K3[Fe(CN)6]/K4[Fe(CN)6] solution containing 0.1 mM KCl. nsor, the peak current response significantly increased due to the high electrical conductivity rge surface area of the VXC-72R. In contrast, the VXC-72R/ZrO2/GCE-based electrochemical se esented a slightly lower peak current response, but the redox peaks were quite sharp, sugges n optimum electrocatalytic performance [23]. This can be explained by the synergistic effect o XC-72R and ZrO2 nanoparticles. The VXC-72R nanoparticles had high electrical conductivity a rge surface area, and the ZrO2 nanoparticles possessed a strong affinity to phosphorus gro hich could achieve good organophosphorus adsorption [24]. On the basis of the synergistic e nerated from the interaction between the VXC-72R and ZrO2 nanoparticles, the V R/ZrO2/GCE-based electrochemical sensor had an excellent trace analysis determina erformance. Figure 3. Cyclic voltammetry (CV) curves of the electrochemical sensors based on the undecorated glassy carbon electrodes (GCEs), VXC-72R/GCEs, and VXC-72R/ZrO2/GCEs in 5 mM of K3[Fe(CN)6]/K4[Fe(CN)6] solution containing 0 1 mM KCl Figure 3. Cyclic voltammetry (CV) curves of the electrochemical sensors based on the undecorated glassy carbon electrodes (GCEs), VXC-72R/GCEs, and VXC-72R/ZrO2/GCEs in 5 mM of K3[Fe(CN)6]/K4[Fe(CN)6] solution containing 0.1 mM KCl. g g p p p y g nerated from the interaction between the VXC-72R and ZrO2 nanoparticles, the V R/ZrO2/GCE-based electrochemical sensor had an excellent trace analysis determina rformance. 3. Results and Discussion 3. Results and Discussion This can be explained by the synergistic effect of the VXC-72R and ZrO2 nanoparticles. The VXC-72R nanoparticles had high electrical conductivity and a large surface area, and the ZrO2 nanoparticles possessed a strong affinity to phosphorus groups, which could achieve good organophosphorus adsorption [24]. On the basis of the synergistic effect generated from the interaction between the VXC-72R and ZrO2 nanoparticles, the VXC-72R/ZrO2/GCE-based electrochemical sensor had an excellent trace analysis determination performance. 5 of 11 nsor may Materials 2019, 12, 3637 nanocomposites a ll M resent excellent MP determination performance. Figure 3. Cyclic voltammetry (CV) curves of the electrochemical sensors based on the undecorated glassy carbon electrodes (GCEs), VXC-72R/GCEs, and VXC-72R/ZrO2/GCEs in 5 mM of K3[Fe(CN)6]/K4[Fe(CN)6] solution containing 0.1 mM KCl. In order to investigate the MP determination performance, electrochemical sensors based on ndecorated GCEs, VXC-72R/GCEs, and VXC-72R/ZrO2/GCEs were tested in PBS solution (0.1 Figure 3. Cyclic voltammetry (CV) curves of the electrochemical sensors based on the undecorated glassy carbon electrodes (GCEs), VXC-72R/GCEs, and VXC-72R/ZrO2/GCEs in 5 mM of K3[Fe(CN)6]/K4[Fe(CN)6] solution containing 0.1 mM KCl. aterials 2019, 12, x FOR PEER REVIEW 5 o H 7.0) of 0.1 mM MP. The corresponding CV results are shown in Figure 4. It can be obviously s hat there was an irreversible reduction peak in the CV curve of the GCE-based electrochem ensor because of the irreversible reduction of the nitro group into the hydroxylamine group [ Meanwhile, the CV curve also contained a pair of redox peaks that corresponded to the revers dox reaction of the hydroxylamine group [7,31,32]. For the VXC-72R/GCE-based electrochem ensor, the peak current response significantly increased due to the high electrical conductivity rge surface area of the VXC-72R. In contrast, the VXC-72R/ZrO2/GCE-based electrochemical sen resented a slightly lower peak current response, but the redox peaks were quite sharp, sugges n optimum electrocatalytic performance [23]. This can be explained by the synergistic effect of XC-72R and ZrO2 nanoparticles. The VXC-72R nanoparticles had high electrical conductivity an rge surface area, and the ZrO2 nanoparticles possessed a strong affinity to phosphorus grou hich could achieve good organophosphorus adsorption [24]. On the basis of the synergistic ef enerated from the interaction between the VXC-72R and ZrO2 nanoparticles, the V 2R/ZrO2/GCE-based electrochemical sensor had an excellent trace analysis determina erformance. Figure 4. 3. Results and Discussion 3. Results and Discussion CV curves of 0.1 mM methyl parathion (MP) in phosphate-buffered saline (PBS) (0.1 M, pH 7.0) and the electrochemical sensors based on the undecorated GCEs, VXC-72R/GCEs, and VXC- 72R/ZrO2/GCEs. Figure 4. CV curves of 0.1 mM methyl parathion (MP) in phosphate-buffered saline (PBS) (0.1 M, pH 7.0) and the electrochemical sensors based on the undecorated GCEs, VXC-72R/GCEs, and VXC-72R/ZrO2/GCEs. Figure 3. Cyclic voltammetry (CV) curves of the electrochemical sensors based on the undecorated glassy carbon electrodes (GCEs), VXC-72R/GCEs, and VXC-72R/ZrO2/GCEs in 5 mM of K3[Fe(CN)6]/K4[Fe(CN)6] solution containing 0 1 mM KCl Figure 3. Cyclic voltammetry (CV) curves of the electrochemical sensors based on the undecorated glassy carbon electrodes (GCEs), VXC-72R/GCEs, and VXC-72R/ZrO2/GCEs in 5 mM of K3[Fe(CN)6]/K4[Fe(CN)6] solution containing 0.1 mM KCl. Materials 2019, 12, x FOR PEER REVIEW 5 o pH 7.0) of 0.1 mM MP. The corresponding CV results are shown in Figure 4. It can be obviously s hat there was an irreversible reduction peak in the CV curve of the GCE-based electrochem ensor because of the irreversible reduction of the nitro group into the hydroxylamine group [3 Meanwhile, the CV curve also contained a pair of redox peaks that corresponded to the reversi edox reaction of the hydroxylamine group [7,31,32]. For the VXC-72R/GCE-based electrochem ensor, the peak current response significantly increased due to the high electrical conductivity a arge surface area of the VXC-72R. In contrast, the VXC-72R/ZrO2/GCE-based electrochemical sen presented a slightly lower peak current response, but the redox peaks were quite sharp, suggest n optimum electrocatalytic performance [23]. This can be explained by the synergistic effect of VXC-72R and ZrO2 nanoparticles. The VXC-72R nanoparticles had high electrical conductivity an arge surface area, and the ZrO2 nanoparticles possessed a strong affinity to phosphorus grou which could achieve good organophosphorus adsorption [24]. On the basis of the synergistic ef generated from the interaction between the VXC-72R and ZrO2 nanoparticles, the VX 72R/ZrO2/GCE-based electrochemical sensor had an excellent trace analysis determinat performance. aterials 2019, 12, x FOR PEER REVIEW H 7.0) of 0.1 mM MP. The corresponding CV results are shown in Figure 4. It can be at there was an irreversible reduction peak in the CV curve of the GCE-based nsor because of the irreversible reduction of the nitro group into the hydroxylam eanwhile, the CV curve also contained a pair of redox peaks that corresponded t dox reaction of the hydroxylamine group [7,31,32]. 3. Results and Discussion 3. Results and Discussion It was found that the peak current response first increased and then decreased with an increase in the pH value and the optimal value was 7 In order to enhance MP detection performance, the effects of ZrO2 concentration and the pH value on the current response were studied through the CV and differential pulse voltammetry (DPV) methods, as shown in Figure 5. Figure 5a shows the effect of ZrO2 concentration on the oxidation current response with the CV method, where the VXC-72R concentration was 0.5 mg mL−1. When a small amount of ZrO2 nanoparticles was introduced, the peak current response increased gradually with the increase in ZrO2 concentration due to the strong affinity to phosphorus groups, which made the ZrO2-based electrochemical sensor possess a selective recognition and adsorption function for MP. However, it is a pity that the excessive concentration could produce serious negative effects because of the increased electron transfer resistance. Figure 5b shows the effects of the pH value on the current response, as studied through DPV methods. It was found that the peak current response first increased and then decreased with an increase in the pH value, and the optimal value was 7, which could 6 of 11 Materials 2019, 12, 3637 be mainly attributed to the degradation effect of alkaline medium on MP and the close relationship between proton and redox reactions. Furthermore, the inset in Figure 5b shows the effects of pH value on the peak potential. It can be observed that the peak potential presented a tendency to decrease with an increase in the pH value, and the corresponding slope value was −60.8. Since the Nernst equation is E = E0 + (59.16 m/n) pH (m: proton number; n: electron number), this slope value means that the ratio value of m/n was equal to about 1, which suggests that the proton number and electron number are equal in the redox process. Materials 2019, 12, x FOR PEER REVIEW 6 of 11 value means that the ratio value of m/n was equal to about 1, which suggests that the proton number and electron number are equal in the redox process Materials 2019 12 x FOR PEER REVIEW 6 of 11 q p Figure 5. The effects of (a) ZrO2 concentration and (b) the pH value on the current response were studied through CV and differential pulse voltammetry (DPV) methods. 3. Results and Discussion 3. Results and Discussion The inset in Figure 5b shows the effects of pH value on the peak potential. Figure 5. The effects of (a) ZrO2 concentration and (b) the pH value on the current response were studied through CV and differential pulse voltammetry (DPV) methods. The inset in Figure 5b shows the effects of pH value on the peak potential. value means that the ratio value of m/n was equal to about 1, which suggests that the proton number and electron number are equal in the redox process. Figure 5. The effects of (a) ZrO2 concentration and (b) the pH value on the current response were studied through CV and differential pulse voltammetry (DPV) methods. The inset in Figure 5b shows value means that the ratio value of m/n was equa and electron number are equal in the redox proce to about 1, which suggests that the proton number ss. Figure 5. The effects of (a) ZrO2 concentration and (b) the pH value on the current response were studied through CV and differential pulse voltammetry (DPV) methods. The inset in Figure 5b shows the effects of pH value on the peak potential. Figure 5. The effects of (a) ZrO2 concentration and (b) the pH value on the current response were studied through CV and differential pulse voltammetry (DPV) methods. The inset in Figure 5b shows the effects of pH value on the peak potential. Figure 5. The effects of (a) ZrO2 concentration and (b) the pH value on the current response were studied through CV and differential pulse voltammetry (DPV) methods. The inset in Figure 5b shows Figure 6 shows the CV curves of the VXC-72R/ZrO2/GCE-based electrochemical sensor in PBS solution (0.1 M, pH 7.0) of 50 μM MP at different scan rates. The corresponding scan rate was 50, 100, 150, 200, 300, and 400 mV/s. As shown here, with an increasing scan rate, all of the peak current responses gradually increased, indicating the close correlation between the scan rate and MP determination performance. Moreover, it should be noted that the peak current values were almost linear with the scan rate, as shown in Figure 7. 3. Results and Discussion 3. Results and Discussion These results suggest that the reduction of MP was related to both a diffusion-controlled process and an adsorption-controlled process, which were mainly contributed by the good organophosphorus adsorption, high electrical conductivity, and large surface area of the VXC 72R/ZrO2 nanocomposites [7] Figure 6 shows the CV curves of the VXC-72R/ZrO2/GCE-based electrochemical sensor in PBS solution (0.1 M, pH 7.0) of 50 µM MP at different scan rates. The corresponding scan rate was 50, 100, 150, 200, 300, and 400 mV/s. As shown here, with an increasing scan rate, all of the peak current responses gradually increased, indicating the close correlation between the scan rate and MP determination performance. Moreover, it should be noted that the peak current values were almost linear with the scan rate, as shown in Figure 7. These results suggest that the reduction of MP was related to both a diffusion-controlled process and an adsorption-controlled process, which were mainly contributed by the good organophosphorus adsorption, high electrical conductivity, and large surface area of the VXC-72R/ZrO2 nanocomposites [7]. the effects of pH value on the peak potential. Figure 6 shows the CV curves of the VXC-72R/ZrO2/GCE-based electrochemical sensor in PBS solution (0.1 M, pH 7.0) of 50 μM MP at different scan rates. The corresponding scan rate was 50, 100, 150, 200, 300, and 400 mV/s. As shown here, with an increasing scan rate, all of the peak current responses gradually increased, indicating the close correlation between the scan rate and MP determination performance. Moreover, it should be noted that the peak current values were almost linear with the scan rate, as shown in Figure 7. These results suggest that the reduction of MP was related to both a diffusion-controlled process and an adsorption-controlled process, which were mainly contributed by the good organophosphorus adsorption, high electrical conductivity, and large surface area of the VXC-72R/ZrO2 nanocomposites [7]. Figure 6. CV curves of 0.1 mM MP in PBS (0.1 M, pH 7.0) and the electrochemical sensor based on the VXC-72R/ZrO2/GCEs at different scan rates. Figure 6. CV curves of 0.1 mM MP in PBS (0.1 M, pH 7.0) and the electrochemical sensor based on the VXC-72R/ZrO2/GCEs at different scan rates. Figure 6. CV curves of 0.1 mM MP in PBS (0.1 M, pH 7.0) and the electrochemical sensor based on the VXC-72R/ZrO2/GCEs at different scan rates. Figure 6. 3. Results and Discussion 3. Results and Discussion CV curves of 0.1 mM MP in PBS (0.1 M, pH 7.0) and the electrochemical sensor based on the VXC-72R/ZrO2/GCEs at different scan rates. 7 of 11 7 of 11 Materials 2019, 12, 3637 Materials 2019, 12, x FOR Figure 7. A plot of peak currents versus the scan rate based on CV curves at a scan rate of 20–300 Figure 7. A plot of peak currents versus the scan rate based on CV curves at a scan rate of 20–300 mV·s−1. Figure 7. A plot of peak currents versus the scan rate based on CV curves at a scan rate of 20–300 mV·s−1. Figure 7 A plot of peak currents versus the scan rate based on CV curves at a scan rate of 20–300 Figure 7. A plot of peak currents versus the scan rate based on CV curves at a scan rate of 20–300 mV·s−1. Figure 7. A plot of peak currents versus the scan rate based on CV curves at a scan rate of 20–300 mV·s−1. g p p mV·s−1. Figure 8 shows the differential pulse voltammetry (DPV) measurements of the VXC- 72R/ZrO2/GCE-based electrochemical sensor in the MP solution at different concentrations. It can be seen that the reduction current had much to do with the MP concentration. Moreover, the peak current values had a linear relation with the MP concentration at two ranges of 1–100 μM, as shown in Figure 9. The detection limit of the VXC-72R/ZrO2/GCE composite could reach up to 0.053 μM, and the linear relationship between the peak current and the MP concentration could be applied to a relatively wide MP concentration range. Table 1 lists the research results of the related electrochemical sensor based on different modification materials. It can be seen that the VXC- 72R/ZrO2/GCE-based electrochemical sensor presented excellent sensitive detection performance for MP. Although the performance of the obtained sensor in this work was slightly lower than that of other ZrO2-based sensors, the present work involved a simple, rapid, and low-cost technique. This suggests that the collaborative use of the VXC 72R and ZrO nanoparticles is of great significance in Figure 8 shows the differential pulse voltammetry (DPV) measurements of the VXC-72R/ZrO2/GCE-based electrochemical sensor in the MP solution at different concentrations. It can be seen that the reduction current had much to do with the MP concentration. 3. Results and Discussion 3. Results and Discussion Moreover, the peak current values had a linear relation with the MP concentration at two ranges of 1–100 µM, as shown in Figure 9. The detection limit of the VXC-72R/ZrO2/GCE composite could reach up to 0.053 µM, and the linear relationship between the peak current and the MP concentration could be applied to a relatively wide MP concentration range. Table 1 lists the research results of the related electrochemical sensor based on different modification materials. It can be seen that the VXC-72R/ZrO2/GCE-based electrochemical sensor presented excellent sensitive detection performance for MP. Although the performance of the obtained sensor in this work was slightly lower than that of other ZrO2-based sensors, the present work involved a simple, rapid, and low-cost technique. This suggests that the collaborative use of the VXC-72R and ZrO2 nanoparticles is of great significance in promoting the practical application of high-performance electrochemical sensors. Figure 8 shows the differential pulse voltammetry (DPV) measurements of the VXC- 72R/ZrO2/GCE-based electrochemical sensor in the MP solution at different concentrations. It can be seen that the reduction current had much to do with the MP concentration. Moreover, the peak current values had a linear relation with the MP concentration at two ranges of 1–100 μM, as shown in Figure 9. The detection limit of the VXC-72R/ZrO2/GCE composite could reach up to 0.053 μM, and the linear relationship between the peak current and the MP concentration could be applied to a relatively wide MP concentration range. Table 1 lists the research results of the related electrochemical sensor based on different modification materials. It can be seen that the VXC- 72R/ZrO2/GCE-based electrochemical sensor presented excellent sensitive detection performance for MP. Although the performance of the obtained sensor in this work was slightly lower than that of other ZrO2-based sensors, the present work involved a simple, rapid, and low-cost technique. This suggests that the collaborative use of the VXC-72R and ZrO2 nanoparticles is of great significance in promoting the practical application of high performance electrochemical sensors uggests that the collaborative use of the VXC 72R and ZrO2 nanoparticles is of great significance romoting the practical application of high-performance electrochemical sensors. Figure 8. DPV for the determination of MP in 0.1 M PBS (pH = 7.0) with VXC-72R/ZrO2/GCEs at MP promoting the practical application of high performance electrochemical sensors. Figure 8. 3. Results and Discussion 3. Results and Discussion Electrode Analytical Method Detection Limit (µM) Linear Range (µM) Reference CPME–AB DPAdSV 3.9 × 104 0.1–70 [21] AuNPs/Nafion/GCE SWV 0.1 0.5–120 [32] OMC/GCE LSV 7.6 × 103 0.09–61 [33] Pd/MWCNTs DPV 0.19 0.38–53.2 [31] BCL@MOF/nanofibers/chitosan/GCE DPV 0.067 0.1–38 [34] ZrO2 NPs–GNs SWV 2.28 × 10−3 0.002–0.9 [14] ZrO2–Au nanocomposite SWV 0.011 0.02–0.140 [23] ZrO2–CNFs DPV 1.29 × 10−3 1 × 10-3–2 × 10−2 [26] VXC-72R/ZrO2/GCE DPV 0.053 1–100 This work ZrO2–Au nanocomposite SWV 0.011 0.02–0.140 [23] ZrO2–CNFs DPV 1.29 × 10−3 1 × 10-3–2 × 10−2 [26] VXC-72R/ZrO2/GCE DPV 0.053 1–100 This work To demonstrate the applicability of the proposed sensor for MP determination in two real water samples containing tap water and river water, two real water samples were first filtered using a standard 0.22-μm filter and then spiked with MP standard solution at three concentrations. The recovery amount of each sample was the average of three measured concentrations using the To demonstrate the applicability of the proposed sensor for MP determination in two real water samples containing tap water and river water, two real water samples were first filtered using a standard 0.22-µm filter and then spiked with MP standard solution at three concentrations. The recovery amount of each sample was the average of three measured concentrations using the standard addition method. As listed in Table 2, the recoveries of the river water and tap water samples varied from 90% to 97.68% and from 97.74% to 100.3%, respectively. Therefore, this result showed that the proposed sensor possessed excellent practicability and accuracy for the determination of MP in real water samples. ndard addition method. As listed in Table 2, the recoveries of the river water and tap mples varied from 90% to 97.68% and from 97.74% to 100.3%, respectively. Therefore, this wed that the proposed sensor possessed excellent practicability and accuracy fo ermination of MP in real water samples. Table 2. Analytical results of MP in real samples using the proposed method (n = 3). Sample MP Added (μM) MP Found (μM) Recovery (%) RSD (%) River water 1 3.0 2.93 97.68 3.3 River water 2 10 9.00 90.00 0.6 Table 2. Analytical results of MP in real samples using the proposed method (n = 3). 3. Results and Discussion 3. Results and Discussion DPV for the determination of MP in 0.1 M PBS (pH = 7.0) with VXC-72R/ZrO2/GCEs at MP concentrations ranging from 1 to 100 μM (1, 3, 5, 7, 9, 10, 30, 50, and 100 μM). Figure 8. DPV for the determination of MP in 0.1 M PBS (pH = 7.0) with VXC-72R/ZrO2/GCEs at MP concentrations ranging from 1 to 100 µM (1, 3, 5, 7, 9, 10, 30, 50, and 100 µM). Figure 8. DPV for the determination of MP in 0.1 M PBS (pH = 7.0) with VXC-72R/ZrO2/GCEs at MP Figure 8. DPV for the determination of MP in 0.1 M PBS (pH = 7.0) with VXC-72R/ZrO2/GCEs at MP concentrations ranging from 1 to 100 μM (1, 3, 5, 7, 9, 10, 30, 50, and 100 μM). Figure 8. DPV for the determination of MP in 0.1 M PBS (pH = 7.0) with VXC-72R/ZrO2/GCEs at MP concentrations ranging from 1 to 100 µM (1, 3, 5, 7, 9, 10, 30, 50, and 100 µM). 8 of 11 8 of 11 Materials 2019, 12, 3637 Materials 2019, 12, x FO Figure 9. The linear relationship between the oxidation peak current and MP concentration (based on DPV). Figure 9. The linear relationship between the oxidation peak current and MP concentration (based on DPV). Figure 9. The linear relationship between the oxidation peak current and MP concentration (based on DPV) Figure 9. The linear relationship between the oxidation peak current and MP concentration (based on DPV). Figure 9. The linear relationship between the oxidation peak current and MP concentration (based on DPV) Figure 9. The linear relationship between the oxidation peak current and MP concentration (based on DPV). T bl 1 C i f th f b t i ti t d thi k Table 1. Comparison of the performance between existing reports and this work. Table 1. Comparison of the performance between existing reports and this work. Electrode Analytical Method Detection Limit (μM) Linear Range (μM) Reference CPME–AB DPAdSV 3.9 × 104 0.1–70 [21] AuNPs/Nafion/GCE SWV 0.1 0.5–120 [32] OMC/GCE LSV 7.6 × 103 0.09–61 [33] Pd/MWCNTs DPV 0.19 0.38–53.2 [31] BCL@MOF/nanofibers/chitosan/GCE DPV 0.067 0.1–38 [34] ZrO2 NPs–GNs SWV 2.28 × 10−3 0.002–0.9 [14] Table 1. Comparison of the performance between existing reports and this work. 3. Results and Discussion 3. Results and Discussion Sample MP Added (µM) MP Found (µM) Recovery (%) RSD (%) River water 1 3.0 2.93 97.68 3.3 River water 2 10 9.00 90.00 0.6 River water 3 100 95.63 95.63 3.9 Tap water 1 3.0 2.93 97.74 2.5 Tap water 2 10 10.03 100.3 7.3 Tap water 3 100 98.77 98.77 4.4 addition method. As listed in Table 2, the recoveries of the river water an varied from 90% to 97.68% and from 97.74% to 100.3%, respectively. Therefor Table 2. Analytical results of MP in real samples using the proposed method (n = 3). River Tap 4. Conclusions Tap water 1 3.0 2.93 97.74 2.5 Tap water 2 10 10.03 100.3 7.3 Tap water 3 100 98.77 98.77 4.4 To summarize, a simple and low-cost VXC-72R/ZrO2/GCE-based electrochemical sensor was successfully fabricated for the high-sensitivity detection of MP. Electrochemical measurements showed that the VXC-72R/ZrO2/GCE-based electrode had a relatively satisfactory peak current and quite 9 of 11 Materials 2019, 12, 3637 a small charge transfer resistance. The VXC-72R nanoparticles had high electrical conductivity and a large surface area, and the ZrO2 nanoparticles possessed good adsorption–recognition ability for MP. The synergistic effect from the VXC-72R/ZrO2 nanocomposites significantly optimized the MP determination performance. The low detection limit and wide MP concentration range of the VXC-72R/ZrO2/GCE-based electrochemical sensor can promote the research and development of simple, low-cost, and efficient electrochemical sensors. Author Contributions: R.L., X.C., and X.Y. conceived of and designed the experiments; R.L., Y.W., B.L. (Bo Li), B.L. (Binbin Liu), H.M., D.L., and L.D. performed the experiments; all authors analyzed the data; R.L. and Y.W. cowrote the paper; and all authors discussed the results and commented on the paper. Funding: This research was funded by the National Key R&D Program of China (No. 2017YFD0301104), the High-Level Talents Startup Project of the Henan Institute of Science and Technology (No. 103010615001), and the Henan Postdoctoral Fund Project (No. 159831). Funding: This research was funded by the National Key R&D Program of China (No. 2017YFD0301104), the High-Level Talents Startup Project of the Henan Institute of Science and Technology (No. 103010615001), and the Henan Postdoctoral Fund Project (No. 159831). Conflicts of Interest: The authors declare no conflicts of interest. Conflicts of Interest: The authors declare no conflicts of interest. References . Akhtar, M.; Iqbal, S.; Bhanger, M.I.; Zia-Ul-Haq, M.; Moazzam, M. Sorption of organophosphorous pestic onto chickpea husk from aqueous solutions. Coll. Surf. B Biointerfaces 2009, 69, 63–70. [CrossRef] [PubM 1. Akhtar, M.; Iqbal, S.; Bhanger, M.I.; Zia-Ul-Haq, M.; Moazzam, M. 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Direct, selective and ultrasensitive electrochemical biosensing of methyl parathion in vegetables using Burkholderia cepacia lipase@MOF nanofibers-based biosensor. Talanta 2019, 197, 356–362. [CrossRef] [PubMed] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Developing a Prognostic Information System for Personalized Care in Real Time
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Developing a Prognostic Information System for Personalized Care in Real Time Tracy A. Lieu1,2, Lisa J. Herrinton1,2, Dimitri E. Buzkov1,2, Liyan Romain Neugebauer1,2, Tami Needham1,3, Daniel Ng1,2, Stephani Stewart1,3, Stephen K. Van Den Eeden1,2 and David M. Baer1,4 1 The Permanente Medical Group, Kaiser Permanente Northern California, US 2 Division of Research, Kaiser Permanente Northern California, US 3 TPMG Technology Group, Kaiser Permanente Northern California, US 4 Department of Oncology, Kaiser Permanente Oakland Medical Center, US Corresponding author: Tracy A. Lieu, MD, MPH (tracy.lieu@kp.org) Tracy A. Lieu1,2, Lisa J. Herrinton1,2, Dimitri E. Buzkov1,2, Liyan Liu1,2, Deborah Lyons1,3, Romain Neugebauer1,2, Tami Needham1,3, Daniel Ng1,2, Stephanie Prausnitz1,2, Kam Stewart1,3, Stephen K. Van Den Eeden1,2 and David M. Baer1,4 1 The Permanente Medical Group, Kaiser Permanente Northern California, US 2 Division of Research, Kaiser Permanente Northern California, US 3 TPMG Technology Group, Kaiser Permanente Northern California, US 4 Department of Oncology, Kaiser Permanente Oakland Medical Center, US Corresponding author: Tracy A. Lieu, MD, MPH (tracy.lieu@kp.org) Context: Electronic medical records hold promise to transform clinical practice. However, technological and other barriers may preclude using them to guide care in real time. We used the Virtual Data Warehouse (VDW) to develop a tool that enables physicians to generate real-time, personalized prognostic information about survival after cancer. Case description: Patients with cancer often ask their oncologists, “Have you ever seen a patient like me?” To help oncologists answer this question, we developed a prototype Prognostic Information System (PRISM), a web-based tool that gathers data about the index patient from Kaiser Permanente’s clinical information systems, selects a historical cohort of similar patients, and displays the survival curve of the similar patients relative to key points in their treatment course. p y p Findings and major themes: The prototype was developed by a multidisciplinary team with expertise in oncology, research, and technology. We have completed two rounds of user testing and refinement. Successful development rested on: (1) executive support and a clinical champion; (2) collaboration among experts from multiple disciplines; (3) starting with simple cases rather than ambitious ones; (4) extensive research experience with the Virtual Data Warehouse, related databases, and an existing query tool; and (5) following agile software development principles, especially iterative user testing. g g p p p p y g Conclusion: Clinical data stored in health care systems’ electronic medical records can be used to personalize clinical care in real time. Lieu, TR, et al. 2019 Developing a Prognostic Information System for Personalized Care in Real Time. eGEMs (Generating Evidence & Methods to improve patient outcomes), 7(1): 2, pp. 1–9. DOI: https://doi.org/10.5334/egems.266 Lieu, TR, et al. 2019 Developing a Prognostic Information System for Personalized Care in Real Time. eGEMs (Generating Evidence & Methods to improve patient outcomes), 7(1): 2, pp. 1–9. DOI: https://doi.org/10.5334/egems.266 Developing a Prognostic Information System for Personalized Care in Real Time Development of prognostic information systems can be accelerated by collaborations among researchers, technology specialists, and clinicians and by use of existing technology like the Virtual Data Warehouse. Keywords: Electronic health records; delivery of health care; organizational innovation; patient-centered care Context Physicians and health systems are eager to reap the benefits of using the evidence that can be generated from electronic medical records (EMRs), yet are frustrated that more useful tools are not yet available [1]. More than 9 of 10 physician practices have EMRs, creating the potential to use data to inform clinical decisions and support learning health systems [2]. However, a gap remains between the promise of data science to transform health care and the limitations of imple­ menting reliable user-tested tools in actual practice. g p Oncologists are often asked by patients newly diagnosed with cancer, “Have you ever seen a patient like me?” This question may convey the patient’s need to be treated as an individual as well as concern about the oncologist’s experi­ ence with their particular cancer, especially in the current era of targeted therapy. In oncology and other specialties, physicians could benefit from a tool that rapidly identifies and analyzes a group of patients similar to the patient they are counseling. Providing data on the actual outcomes of similar patients would enable conversations that are more personalized and credible than those based solely on published literature [3]. The need for personalized information has led to the development of “patients like me” websites and tools that connect groups of patients based on key char­ acteristics [4, 5]. In parallel, physicians would value tools that can rapidly analyze and display outcomes in the course of their conversations with patients. p Attempts to create “patients like me” tools by organizations such as the MD Anderson Cancer Center and the American Society of Clinical Oncology have not yet succeeded despite large financial investments [6, 7]. A major barrier has been Art. 2, page 2 of 9 Lieu et al: Developing a Prognostic Information System for Personalized Care in Real Time the lack of comprehensive patient-level data over multi-year periods that cover the course of the patient’s disease tra­ jectory and long-term outcomes including survival. A second major barrier is the technologic complexity of using elec­ tronic medical record data to create predictions rapidly enough to provide information to individual patients during clinical encounters. This case study describes how we developed a prototype that overcomes these barriers to provide physicians with real-time prognostic information tailored to individual patients for use during clinical encounters. p Setting and Conception This project was conducted by a multidisciplinary team within The Permanente Medical Group, which partners with Kaiser Foundation Health Plan to serve more than 4 million patients in Northern California. Our integrated health care system, Kaiser Permanente Northern California (KPNC), includes 21 medical centers and more than 100 oncologists. More than 17,000 patients in this population are newly diagnosed with cancer each year. Like all Kaiser Permanente regions, KPNC uses EPIC as the core of its EMR. In 2015, a KPNC oncology leader (D.M.B.) requested that our organization’s Division of Research develop a tool to gen­ erate personalized information from our own health care databases for use in counseling cancer patients. In response, we created a prototype Prognostic Information System (PRISM) to enable physicians to generate personalized informa­ tion for individual patients during real-time clinical encounters. PRISM generates personalized prognostic information by gathering key characteristics about the index patient, identifying a cohort of similar patients that are matched to the index patient on these characteristics, and displaying the survival curves of the similar patients. Data infrastructure PRISM builds on many years of development and research use of the Virtual Data Warehouse (VDW), a data model devel­ oped by the Health Care Systems Research Network to facilitate public domain health and health services research [8]. The VDW was initiated during the 1990s with support from the Cancer Research Network and increasingly expanded and standardized over the years with support from many other federally-sponsored research networks. It provides an effi­ cient, reliable, and convenient store of the data that are essential to our research activities. In KPNC, the VDW includes data on all diagnoses, treatments, and clinical outcomes for more than 9.6 million patients seen since 1996. Among the data elements relevant to oncology are cancer diagnoses, surgery, radiation, and intravenous and oral chemotherapy, and mortality data drawn from the KPNC Cancer Registry and multiple sources including pathology reports, diagnosis and treatment data, and multiple vital sources, including state death certificate data [8]. The VDW also incorporates a comorbidity score, the COPS2 score, We chose to use this comorbidity score because it better predicts outcomes than individual comorbidities and is simpler to display. It has been validated as an excellent predictor of mortality and has been updated every month for every KPNC member since 2010 [9]. The initial PRISM prototype relied on data architecture from the Division of Research Data-on-Demand (D3) tool (Figure 1), a web-based query tool implemented to increase the accessibility of data stored in the VDW [10, 11]. The D3 tool was developed in 2013 by one of our computer scientists (D.E.B.). It enables authorized staff to access data and perform simple analyses through a graphical user interface, instead of writing complex scripting or programming code. The D3 tool is a Java application with an HTML user interface supporting web browsers Internet Explorer and Chrome. The application and web services run on Apache Tomcat on a Windows VM server. The front-end application queries The initial PRISM prototype relied on data architecture from the Division of Research Data-on-Demand (D3) tool (Figure 1), a web-based query tool implemented to increase the accessibility of data stored in the VDW [10, 11]. The D3 tool was developed in 2013 by one of our computer scientists (D.E.B.). It enables authorized staff to access data and perform simple analyses through a graphical user interface, instead of writing complex scripting or programming code. Second Prototype: Pancreatic Cancer Based on the encouraging appraisal of the first prototype, we gained executive sponsorship and funding to develop a second. We chose pancreatic cancer because it has a low survival rate and the treatment has not changed dramatically in recent years We expanded the team to include an epidemiologist with expertise in pancreatic cancer (S.K.V.), as well as experts in technology (D.L., T.N., K.S., and others), a biostatistician (R.N.), and a data scientist (L.L.). We developed the second prototype using lessons learned from the first. We aimed for the second prototype to be simple, comprehensible, valid, and rapid enough for an oncologist to use on a computer screen during a clinical inter­ action with a patient. We developed new approaches to (1) enabling PRISM to be used at different points in a patient’s clinical course, not only at diagnosis; (2) identifying variables for use in selecting similar patients; (3) developing the user interface for ease of use and comprehension; and (4) training users to not use PRISM to select treatments. Enabling use at different points in a patient’s clinical course. The team’s oncologist asked for PRISM to predict survival at initial diagnosis and at other specific points during treatment. We diagrammed the possible treatment pathways for pancreatic cancer, including no treatment; surgery with or without adjuvant or neoadjuvant chemotherapy; radiation; and first-line, second-line, and third-line or higher-order palliative chemotherapy. We then designed PRISM to elicit input from the oncologist on which treatments the patient had completed, started, or planned. This input needed to be elicited from the oncologist because treatments that were planned would not yet be recorded in computerized data. We designed PRISM to select similar patients who had the same treatment history as the index patient, and to calculate survival from the start of that phase in treatment. Survival calculations are made at one of four time points: at diagnosis, at surgery, at the start of first-line palliative chemotherapy, or at the start of second-line palliative chemotherapy. For example, given an index patient who has had surgery and is now planning first-line palliative chemotherapy, PRISM selects similar patients who have had surgery and who had first-line palliative chemotherapy, and calculates survival from the start of that chemotherapy. Selecting similar-patient groups. To characterize similar patient groups, we first identified 4,431 patients with pancre­ atic cancer diagnosed within KPNC between 2005 and 2014. Data infrastructure perform simple analyses through a graphical user interface, instead of writing complex scripting or programming code. The D3 tool is a Java application with an HTML user interface supporting web browsers Internet Explorer and Chrome. The application and web services run on Apache Tomcat on a Windows VM server. The front-end application queries Figure 1: The Virtual Data Warehouse (VDW) of Kaiser Permanente Northern California, Division of Research Data on Demand (D3) Query Tool, and Prognostic Information System (PRISM). Figure 1: The Virtual Data Warehouse (VDW) of Kaiser Permanente Northern California, Division of Research Data on Demand (D3) Query Tool, and Prognostic Information System (PRISM). Lieu et al: Developing a Prognostic Information System for Personalized Care in Real Time Art. 2, page 3 of 9 a back-end database via ANSI SQL. The database is a customized version of the VDW, transformed into an OLAP star schema in Teradata. Data are refreshed via monthly extract, transform, and load (ETL) running in a SAS container on a Sun Solaris server, pulling from various source data including the VDW, Epic Clarity, and other local data. The most cur­ rent PRISM prototype is built in a .NET MVC framework, using Java to access the back-end database, which is an Oracle Exadata datamart composed of specific subsets of prospective and historical cancer patients. First Prototype: Diffuse B-Cell Lymphoma yp y p We selected diffuse B-cell lymphoma for PRISM’s first prototype because its treatment is simple in involving only chemotherapy and not surgery or radiation. We devoted 12 months to creating a user interface and query structure for PRISM that focused on diffuse B-cell lymphoma but could be generalized to other cancer types. The team for the first prototype included researchers (T.A.L., L.J.H.), experts in data architecture and query programming (D.E.B., D.N.), a project manager (S.P.), and the oncology leader (D.M.B.). The first PRISM prototype asked the user to specify the cancer type and stage, age range, sex, race/ethnicity, and COPS2 comorbidity score quartile. These variables were selected based on clinical experience. The user was then prompted to specify the treatments of interest. Using these inputs, PRISM selected similar patients from among all KPNC patients treated for diffuse large B-cell lymphoma. PRISM generated two screens with results: one with a survival curve for similar patients and one with a set of survival curves for all patients with diffuse B-cell lymphoma stratified by quartile of COPS2 comorbidity score. q y We conducted user testing of this prototype with six oncologists. All testers felt that it was potentially useful, could envision using it to prepare for consultations with patients, and said it was worth developing further. The tests also identified issues and concerns. A key concern was that all six testers said they would like to use PRISM to choose treat­ ments. Use of observational data to make treatment decisions is well documented to be fraught with complexity and potential bias due to confounding by indication and other problems [12, 13]. For this reason, we did not design PRISM to help users select treatments or conduct ad-hoc comparative effectiveness analyses. p p y In addition, users asked that PRISM automatically select similar patients based on the known characteristics of an index patient, rather than asking the user to manually enter these specifications. One user expressed concern that if printed survival curves were handed to a patient, they might be compared with survival data available in the litera­ ture or offered by outside medical groups, leading to confusion or loss of trust. Survival following diffuse large B-cell lymphoma is relatively good, and users wanted the next prototype to address a cancer with worse survival, for which prognostic information would be more central to patient counseling. Second Prototype: Pancreatic Cancer PRISM also generates a screen that compares the survival curve of similar patients with that of all patients with the same tumor type and stage. This is to help the oncologist explain why the patient’s prognosis might be different from published estimates for the general population, for example on the American Cancer Society website [17]. PRISM also generates screens with survival curves stratified by age and COPS2 comorbidity score. Training users to not use PRISM to select treatments. Because PRISM was not designed to evaluate counterfactual survival curves under various therapy decisions, attempts to use it for ad-hoc comparative effectiveness analyses are prone to confounding by indication and are at high risk of yielding erroneous causal inferences. Thus, we designed a special mod­ ule to train users to not use PRISM to conduct ad-hoc comparative effectiveness analyses to guide treatment selection. p y g The training module uses the example of autologous bone-marrow transplantation of breast cancer [18]. We selected this example because it received substantial media attention and is familiar to most oncologists. In the 1980s, research­ ers used the results of observational studies to claim that autologous bone-marrow transplantation increased survival for breast cancer patients [19, 20]. Based on these studies and the associated media attention, autologous bone-marrow transplantation became the standard of care. It was not until the early 2000s that randomized trials clearly demon­ strated that such transplantation was ineffective and harmful [21, 22]. The earlier findings that transplantation was associated with benefit stemmed from healthier patients with more comprehensive staging and less extensive disease having been selected to receive transplantation. Our training module, placed within the user introduction materials, is designed to show that using PRISM to make treatment decisions could lead to the same types of errors made by the proponents of autologous bone-marrow transplantation for breast cancer. User testing of the pancreatic cancer prototype. We have recently completed user testing of the second prototype’s user interface with five oncologists, two of whom were testers of the first prototype. All users could imagine using PRISM in clinical practice and were generally supportive of continued development. However, all identified specific problems with tool’s design. Their overall reactions ranged from enthusiastic to mildly skeptical. Second Prototype: Pancreatic Cancer We classified these into six tumor type subgroups, which had sample sizes ranging from 3,499 (for ductal adenocarcinoma) to 5 (for acinar cell carcinoma). We further divided these subgroups by cancer stage. For each of the possible treatment pathways, for tumor type/stage subgroups with 100 patients or more, we used proportional hazards regression modeling to identify variables to be used to personal­ ize prognosis. Constructing these models enabled us to identify predictors associated with survival for patients who had different treatment pathways, so that PRISM could use these variables to select the best similar-patient group and provide more personalized prognostic information than would be available from published literature. We tested Art. 2, page 4 of 9 Art. 2, page 4 of 9 Lieu et al: Developing a Prognostic Information System for Personalized Care in Real Time predictors that had been identified in previous studies of pancreatic cancer [14–16], conducted iterative modeling to select variables that were independently and significantly associated with survival, and constructed one model for each possible treatment pathway (e.g., no treatment; surgery followed by no treatment; surgery followed by first-line pal­ liative chemotherapy; first-line palliative chemotherapy alone; second-line or higher palliative chemotherapy, etc.). We also constructed models for patients who had not yet decided on specific treatment plans; these models included all patients, including those with and without the treatment. The variables used as predictors in the final models included age, sex, comorbidity score, weight loss of 5 percent or more the past 12 months, and laboratory tests (aspartate ami­ notransferase, CA 19–9, and percentage lymphocytes); not every predictor was used in every model. It is important to note that PRISM uses deterministic business rules to select similar patients from the KPNC popula­ tion, then creates a survival curve with 95 percent confidence limits for the similar patients. It does not calculate the survival curves by running a parametric statistical model. The primary purpose of our proportional hazards modeling was to select predictors to be used to personalize prognosis. Developing the user interface. The TPMG Technology Group team developed a professional-grade user interface that a physician could show to a patient during a clinic visit. The user interface is designed to increase patients’ understand­ ing of their survival outcomes and their confidence in decisions about their care. Second Prototype: Pancreatic Cancer PRISM is designed to offer physician- users simplicity of use as well as the ability to tailor inputs individually and view results in ways that inform and assist conversations with patients. PRISM asks the oncologist to input the patient’s medical record number, then auto-populates the patient’s age, sex, COPS2 comorbidity score, weight change in the past 90 days, and recent laboratory values. It then prompts the user to input tumor type, stage at diagnosis, treatments completed or in progress, and treatments planned. After the index patient is specified, PRISM selects similar patients and displays the ranges of their variables. For exam­ ple, for a 60-year-old index patient, we selected a default age range for similar patients of 55–65 years based on our descriptive analyses and proportional hazards models. The types of data gathered and processed by PRISM come from the VDW and other sources (Table 1). Some data sources are refreshed daily while others are refreshed less frequently. A special feature of PRISM is that it allows the user to modify the size of the similar-patients cohort by editing the ranges of selected matching variables (e.g., the age range). It also displays the sample size of the similar-patient group. Thus, PRISM enables the physician-user to trade off statistical precision (which is obtained by using a larger cohort) with After the index patient is specified, PRISM selects similar patients and displays the ranges of their variables. For exam­ ple, for a 60-year-old index patient, we selected a default age range for similar patients of 55–65 years based on our descriptive analyses and proportional hazards models. The types of data gathered and processed by PRISM come from the VDW and other sources (Table 1). Some data sources are refreshed daily while others are refreshed less frequently. A special feature of PRISM is that it allows the user to modify the size of the similar-patients cohort by editing the ranges of selected matching variables (e.g., the age range). It also displays the sample size of the similar-patient group. Thus, PRISM enables the physician-user to trade off statistical precision (which is obtained by using a larger cohort) with personalization (which is achieved by using a more precise definition of similarity between patients). The results screen displays a survival curve and 95 percent confidence interval for the similar-patient group that can be easily interpreted by the doctor and shared with the patient (Figure 2). Sources of mortality data are KPNC ­clinical and administrative data, Social Security Death Master files, California State death certificate data Second Prototype: Pancreatic Cancer Users suggested major areas for improvement, noting that they: (1) felt burdened when they had to input data they thought could be auto-populated, such as laboratory values; (2) became confused when they were presented with the COPS2 comorbidity score and the percent weight change within the last 90 days without clear documentation of how these data had been derived; (3) found the survival curves stratified by comorbidity and age less useful than those that showed similar patients and all patients with the same tumor type and stage; and (4) wanted clarification of the specific factors that made their patient’s predicted survival different from other patients with the same tumor type and stage. Our next steps are to refine PRISM’s user interface, algorithms, and back-end architecture, connect the user interface and back ends, and conduct at least one more round of user testing. Technical requirements include (1) creating a trans­ lation layer that enables the user interface to interact with the existing back-end VDW query tool data architecture; (2) Lieu et al: Developing a Prognostic Information System for Personalized Care in Real Time Art. 2, page 5 of 9 Table 1: Types of Data Processed by the Prognostic Information System (PRISM) Prototype for Pancreatic Cancer, with the Requirements for Timeliness of Capture. Second Prototype: Pancreatic Cancer * Generated by another KPNC department in the Integrated Data Repository and shared with the VDW. NA: Not applicable. * Generated by another KPNC department in the Integrated Data Repository and shared with the VDW. adding secure sign-on and addressing other risks; (3) identifying a site to host the user introduction and user guide; and (4) creating a place to store data input by the user. adding secure sign-on and addressing other risks; (3) identifying a site to host the user introduction and user guide; and (4) creating a place to store data input by the user. Validation of PRISM will use hypothetical cases to test a priori hypotheses about how survival curves should behave. In addition, we will compare the results of PRISM with the results of statistical analyses performed by the research team. We also will need to make decisions about how to handle situations in which the numbers of patients in a given subgroup become small. Once we are confident that PRISM is stable, valid, and user-friendly, we plan to introduce it to selected clinical leaders and early adopters for evaluation it in clinical practice. Second Prototype: Pancreatic Cancer Data type/variable Data source for index patient Data source for ­similar patients Lag time Comments Variables autopopulated by PRISM Age, sex Clarity VDW For Clarity, one day For VDW, one month COPS2 comorbidity score VDW* VDW* One month Weight change in last 90 days Laboratory results Clarity VDW For Clarity, one day For VDW, one month User can enter today’s weight or ­laboratory results, if available Year of pancreatic cancer diagnosis VDW KPNC Cancer Registry and VDW For VDW, one day For KPNC Cancer Registry, six months (with monthly updates) User can enter the ­diagnosis year for patients who do not have a diagnosis before their first visit Variables entered by the oncologist Tumor type Pathology KPNC Cancer Registry For KPNC Cancer Registry, six months (with monthly updates) Could be derived from pathology reports via natural language ­processing but ­difficult to do this in real time Stage at diagnosis Oncologist KPNC Cancer Registry NA Complex variable; may eventually become avail­ able more routinely via a structured staging tool Treatments ­completed, in ­progress, and planned Oncologist and patient VDW For VDW, one month Outcome variables Death Not applicable VDW Three months (with quarterly updates) Sources of mortality data are KPNC ­clinical and administrative data, Social Security Death Master files, California State death certificate data VDW: Virtual Data Warehouse. KPNC: Kaiser Permanente Northern California. NA: Not applicable. * Generated by another KPNC department in the Integrated Data Repository and shared with the VDW. Table 1: Types of Data Processed by the Prognostic Information System (PRISM) Prototype for Pancreatic Cancer, with the Requirements for Timeliness of Capture. their first visit Variables entered by the oncologist Tumor type Pathology KPNC Cancer Registry For KPNC Cancer Registry, six months (with monthly updates) Could be derived from pathology reports via natural language ­processing but ­difficult to do this in real time Stage at diagnosis Oncologist KPNC Cancer Registry NA Complex variable; may eventually become avail­ able more routinely via a structured staging tool Treatments ­completed, in ­progress, and planned Oncologist and patient VDW For VDW, one month Outcome variables Death Not applicable VDW Three months (with quarterly updates) Sources of mortality data are KPNC ­clinical d d i i t ti Variables entered by the oncologist Three months (with quarterly updates) Three months (with quarterly updates) VDW: Virtual Data Warehouse. KPNC: Kaiser Permanente Northern California. NA: Not applicable. Findings and Major Themes Successful development of the PRISM prototype has addressed seven dimensions described in a strategic innovation framework, including strategic alignment, insight about emerging trends and customer (physicians’ and patients’) needs, and leveraging and extending existing assets (Table 2) [23]. Specifically, this project’s progress has relied on (1) execu­ tive support and a clinical champion; (2) collaboration among experts from multiple disciplines; (3) starting with simple cases rather than ambitious ones; (4) extensive research experience with the Virtual Data Warehouse, related databases, and an existing query tool; and (5) following agile software development principles, especially iterative user testing. Art. 2, page 6 of 9 Lieu et al: Developing a Prognostic Information System for Personalized Care in Real Time Lieu et al: Developing a Prognostic Information System for Personalized Care in Real Time Figure 2: Results Screen Presented by the Prognostic Information System (PRISM). Executive support has been a key element in the development of PRISM because the substantial effort required from the research and technology teams could not have been invested without sponsorship and encouragement from these high-level leaders. The clinical champion has been essential for articulating the vision and guiding the team through key decisions to ensure PRISM’s fit into real-life workflows. He also recruited other oncologists to test PRISM, and he cultivated interest in PRISM among these colleagues. The PRISM team brings together experts from multiple disciplines, including oncology, computer software develop­ ment, user interface design, epidemiological methods, data science, and biostatistics. Bringing these experts into face- to-face meetings shortened the time needed for development and enabled the team to troubleshoot complex issues. By working with the oncologist as the product owner, the multidisciplinary team has assured that PRISM addresses the needs of end-users as best possible within existing data structures. The relatively simple use cases used for the first and second PRISM prototypes enabled the project team to learn in a focused fashion as we created a common understanding and common set of approaches. Use of simple cases also enabled more rapid development of prototypes and simplified user testing. Our team’s extensive experience with the VDW and other research databases has been another success factor. The VDW makes it possible to run analytic queries in seconds. The fact that our oncologists create the data in the EMR, which sub­ sequently populate the VDW and PRISM, gives them an inherent understanding of the information that PRISM produces. How Applied in PRISM Development Work is broken into small increments that help limit the initial planning and design. Several iterations of the user interface have been tested. 1. Iterative and incremental approaches The multidisciplinary team holds face-to-face meetings as needed to address the evolving requirements of the project. The oncologist and lead researcher serve as the product owners and are available whenever the developers have questions. 2. Efficient and face-to-face communication 2. Efficient and face-to-face communication The project plan changes each month to incorporate the activities needed to develop PRISM, given the currently available data sources and technological environment. The research ­component of the project has been reduced to make time for additional iterations of PRISM’s development. 3. Adaptive approaches with short feedback loops 4. Quality focus Quality assurance will be conducted through validation testing before PRISM is released for oncologists to use with patients. Safety and security needs are also being addressed through user training and software configuration. The project is organized by a multidisciplinary team of highly experienced individuals. The team has adjusted its approaches and the project plan at regular intervals to enhance the effectiveness of collaborative interactions and time invested. Our team has followed agile software development principles (Table 3), which emphasize close collaboration among end-users, developers, and researchers to assure the design satisfies the end-user; frequent demonstrations of PRISM and iterative development to enable the end-user to revise business requirements; face-to-face meetings; and considera­ tion for changing the schedule as needs evolve [24, 25]. For example, our initial plan was crafted by researchers who planned a 24-month timeline with 9 months of development and 15 months of research. Now that the research and technology teams have worked together, we realize that the project needs 15 months for development. We have reiter­ ated and clarified that the project’s focus is to produce a usable prototype and to produce knowledge that generalizes to the next phase of this tool. p Important questions remain about how this tool will be used in actual patient encounters. PRISM is one of many ongoing efforts to create tools for prognosis or prediction. Adoption of such tools in actual practice has remained lim­ ited, in part because they are sometimes developed without a clear picture of how they will influence real-life decision processes [26]. Experience in our setting suggests that prediction tools may succeed best when their development is initiated and promoted by clinicians [27, 28]. Dimension of Strategic Innovation 1. Managed innovation (combining tradi­ tional and non-traditional approaches) 2. Strategic alignment (building support) 3. Industry foresight (understanding emerging trends) 4. Consumer/customer insight (understanding articulated and ­unarticulated needs) PRISM leverages our skills in research, technology, and clinical care, and builds on areas of synergy among these disciplines. 5. Core technologies and ­competencies (leveraging and extending existing assets) The oncologists who are prospective users of PRISM express readiness to use it; there is cultural, process, and structural readiness to test and implement PRISM. 6. Organizational readiness (ability to take action) Each step in PRISM’s development has been managed to achieve a goal feasible within 6 to 12 months, with iterative adjustments as the team has learned about the needs that must be addressed to achieve implementation. 7. Disciplined implementation (­managing the path from inspiration to ­implementation) Table 3: Agile Software Development Concepts [24] Applied in Development of the Prognostic Information System (PRISM)*. Table 3: Agile Software Development Concepts [24] Applied in Development of the Prognostic Information System (PRISM)*. Agile Software Concept How Applied in PRISM Development 1. Iterative and incremental approaches Work is broken into small increments that help limit the initial planning and design. Several iterations of the user interface have been tested. 2. Efficient and face-to-face communication The multidisciplinary team holds face-to-face meetings as needed to address the evolving requirements of the project. The oncologist and lead researcher serve as the product owners and are available whenever the developers have questions. 3. Adaptive approaches with short feedback loops The project plan changes each month to incorporate the activities needed to develop PRISM, given the currently available data sources and technological environment. The research ­component of the project has been reduced to make time for additional iterations of PRISM’s development. 4. Quality focus Quality assurance will be conducted through validation testing before PRISM is released for oncologists to use with patients. Safety and security needs are also being addressed through user training and software configuration. 5. Self-organizing team of motivated individuals The project is organized by a multidisciplinary team of highly experienced individuals. The team has adjusted its approaches and the project plan at regular intervals to enhance the effectiveness of collaborative interactions and time invested. Findings and Major Themes Industry foresight (understanding emerging trends) The project is aligned with emerging trends in health care, in which data ­science and informatics are being used to personalize care for individual patients. 4. Consumer/customer insight (understanding articulated and ­unarticulated needs) PRISM is designed to meet the needs described by the oncologist leader of the team, as well as the oncologist users. It also addresses the objectives of ­executive sponsors to fully use our data to optimize patient care. 5. Core technologies and ­competencies (leveraging and extending i i ) PRISM leverages our skills in research, technology, and clinical care, and builds on areas of synergy among these disciplines. Findings and Major Themes Experience building the D3 query tool taught the team key skills for assembling PRISM’s back-end architecture. Art. 2, page 7 of 9 Lieu et al: Developing a Prognostic Information System for Personalized Care in Real Time Table 2: Dimensions of Strategic Innovation [23] Applied in Development of the Prognostic Information System (PRISM). Dimension of Strategic Innovation How Applied in PRISM Development 1. Managed innovation (combining tradi­ tional and non-traditional approaches) The process combines traditional statistical and research methods with modern technology development processes. 2. Strategic alignment (building support) The development work is being supported by multiple stakeholders including executives and teams from multiple disciplines. 3. Industry foresight (understanding emerging trends) The project is aligned with emerging trends in health care, in which data ­science and informatics are being used to personalize care for individual patients. 4. Consumer/customer insight (understanding articulated and ­unarticulated needs) PRISM is designed to meet the needs described by the oncologist leader of the team, as well as the oncologist users. It also addresses the objectives of ­executive sponsors to fully use our data to optimize patient care. 5. Core technologies and ­competencies (leveraging and extending existing assets) PRISM leverages our skills in research, technology, and clinical care, and builds on areas of synergy among these disciplines. 6. Organizational readiness (ability to take action) The oncologists who are prospective users of PRISM express readiness to use it; there is cultural, process, and structural readiness to test and implement PRISM. 7. Disciplined implementation (­managing the path from inspiration to ­implementation) Each step in PRISM’s development has been managed to achieve a goal feasible within 6 to 12 months, with iterative adjustments as the team has learned about the needs that must be addressed to achieve implementation. Table 3: Agile Software Development Concepts [24] Applied in Development of the Prognostic Information System (PRISM)* Table 2: Dimensions of Strategic Innovation [23] Applied in Development of the Prognostic Information System (PRISM). Dimension of Strategic Innovation How Applied in PRISM Development 1. Managed innovation (combining tradi­ tional and non-traditional approaches) The process combines traditional statistical and research methods with modern technology development processes. 2. Strategic alignment (building support) The development work is being supported by multiple stakeholders including executives and teams from multiple disciplines. 3. Funding Information g This work is supported by The Permanente Medical Group through its sponsorship of the TPMG Rapid Analytics Unit and through a Delivery Science Research Program funding award. Acknowledgements We are grateful for the support of the executives and technology leaders of The Permanente Medical Group, including Yi-Fen Irene Chen, MD, Patricia Conolly, MD, Judy Anderson, MBA, Philip Madvig, MD, Robert Pearl, MD, and Richard Isaacs, MD. This project was possible due to the leadership of Jamila Gul and contribution of Madhu Gandotra of our Strategic Programming Group, and Michael Oehrli and Charles Quesenberry, PhD, of the KPNC Cancer Registry. We appreciate the collaboration of the Division of Research IT Development and Application Services team (Joey Macapinlac, Michael Ford, Amelia Chen, Edison Uy, Jeffrey Lavarez, and Sherwin Dela Cruz) and colleagues from the TPMG Technology Group, including Eduardo Fernandez, Ivan Jerez, Wendi Robertson, and Shweta Srivastava. We are deeply grateful for the key contributions of the oncologists who have tested PRISM, the cancer researchers of our Divi­ sion of Research, and the patients of Kaiser Permanente. Conclusion Web-based tools can enable physicians to use EMR data to generate personalized prognostic information for clinical care in real time. The process of developing such tools benefits from strategic innovation and agile software develop­ ment methods. Progress can be accelerated by collaborations among researchers, technology specialists, and clinicians, and structures like the Virtual Data Warehouse. References 1. Wachter, R and Wehrwein, P. A Conversation With Robert Wachter, MD. Reality Bytes: Medicine’s Bumpy Ride Into the Digital Age. Manag Care. 2015; 24(5): 33–35. 1. Wachter, R and Wehrwein, P. A Conversation With Robert Wachter, MD. Reality Bytes: Medicine’s Bumpy Ride Into the Digital Age. Manag Care. 2015; 24(5): 33–35. g g ( ) 2. Centers for Disease Control and Prevention; National Center for Health Statistics. National Electronic Health Records Survey: 2015 State and National Electronic Health Record Adoption Summary Tables. In: 2017. 2. Centers for Disease Control and Prevention; National Center for Health Statistics. National Electronic Health Records Survey: 2015 State and National Electronic Health Record Adoption Summary Tables. In: 2017. y p y 3. Frankovich, J, Longhurst, CA and Sutherland, SM. Evidence-based medicine in the EMR era. N Engl J Med. 2011; 365(19): 1758–1759. DOI: https://doi.org/10.1056/NEJMp1108726 3. Frankovich, J, Longhurst, CA and Sutherland, SM. Evidence-based medici 2011; 365(19): 1758–1759. DOI: https://doi.org/10.1056/NEJMp1108726 3. Frankovich, J, Longhurst, CA and Sutherland, SM. Evidence-based medicine in the EMR era. N Engl J Med. 2011; 365(19): 1758–1759. DOI: https://doi.org/10.1056/NEJMp1108726 ( ) p g p 4. Patientslikeme. https://www.patientslikeme.com/. Accessed June 3, 2018. e. https://www.patientslikeme.com/. Accessed June 5. Smart Patients. https://www.smartpatients.com/. Accessed June 3, 2018. 6. Davenport, T and Ronanki, R. Artificial intelligence for the real world. Harvard Business Review; 201 7. Hudis, C and Goldberg, P. Hudis: ASCO needed collaborators to help CancerLinQ deliver faster on its mission. The Cancer Letter; 2017. https://cancerletter.com/articles/20180105_2/. Accessed Dec 21, 2017. 7. Hudis, C and Goldberg, P. Hudis: ASCO needed collaborators to help CancerLinQ deliver faster on its mission. The Cancer Letter; 2017. https://cancerletter.com/articles/20180105_2/. Accessed Dec 21, 2017. ; p // / / / , 8. Oehrli, MD and Quesenberry, CP. Kaiser Permanente Northern Calfornia Cancer Registry: 2016 Annual Report on Trends, Incidence, and Outcomes; 2017. Oakland, CA January 2017. 8. Oehrli, MD and Quesenberry, CP. Kaiser Permanente Northern Calfornia Cancer Registry: 2016 Annual Report on Trends, Incidence, and Outcomes; 2017. Oakland, CA January 2017. 9. Escobar, GJ, Gardner, MN, Greene, JD, Draper, D and Kipnis, P. Risk-adjusting hospital mortality using a comprehensive electronic record in an integrated health care delivery system. Med Care. 2013; 51(5): 446–453. DOI: https://doi.org/10.1097/MLR.0b013e3182881c8e 9. Escobar, GJ, Gardner, MN, Greene, JD, Draper, D and Kipnis, P. Risk-adjusting hospital mortality using a comprehensive electronic record in an integrated health care delivery system. Med Care. 2013; 51(5): 446–453. DOI: https://doi.org/10.1097/MLR.0b013e3182881c8e p g 10. How Applied in PRISM Development Although the oncologists who have tested PRISM have described how it could be useful in conversations with patients, empirical evaluation in clinician-patient interactions is needed to elu­ cidate its actual benefits and limitations. User acceptability testing with patients will be an important next step. Such Art. 2, page 8 of 9 Lieu et al: Developing a Prognostic Information System for Personalized Care in Real Time testing could compare alternative methods of offering the prognostic information, for example via one-year survival estimates versus survival curves. The methods used to create this tool are generalizable to other health care systems, especially those with VDWs. All components of the D3 tool and PRISM prototypes use widely available, industry standard application languages, oper­ ating environments, and analytical/query methods. Most of the data elements required exist in the VDW, which has files for tumor characteristics and chemotherapy, but specific information such as tumor type may need to be drawn from a cancer tumor registry. The cost of PRISM’s development to date is estimated at several hundred thousand dollars, almost exclusively for per­ sonnel effort. This is far less than the tens of millions of dollars reported for other oncology decision support systems in development [6]. However, PRISM is a simpler tool than others because it does not attempt to recommend a best treatment, but instead shows prognostic information for a specified course of treatment. In addition, the foundation for PRISM in our setting had already been built via our existing VDW data infrastructure and the D3 query tool. Competing Interests g The authors have no competing interests to declare. References Ng, D, Buzkov, D, Chimmula, S and Gul, J. Division of Research Data-on-Demand: The development process for a research data warehouse rapid-querying tool. J Patient-Centered Res Rev. 2016; 3: 203. DOI: https://doi. org/10.17294/2330-0698.1351 10. Ng, D, Buzkov, D, Chimmula, S and Gul, J. Division of Research Data-on-Demand: The development process for a research data warehouse rapid-querying tool. J Patient-Centered Res Rev. 2016; 3: 203. DOI: https://doi. org/10.17294/2330-0698.1351 11. Mane, K, Buzkov, D, Blank, J, Lieu, T and Horberg, M. Population insight tool: a novel visual interactive query interface to Virtual Data Warehouse. J Patient-Centered Res Rev. 2017; 4: 174. DOI: https://doi. org/10.17294/2330-0698.1522 ieu et al: Developing a Prognostic Information System for Personalized Care in Real Time Art. 2, page 9 of 9 Art. 2, page 9 of 9 12. Brookhart, MA, Sturmer, T, Glynn, RJ, Rassen, J and Schneeweiss, S. Confounding control in healthcare database research: challenges and potential approaches. Med Care. 2010; 48(6 Suppl): S114–120. DOI: https:// doi.org/10.1097/MLR.0b013e3181dbebe3 13. Hernan, MA and Robins, JM. Using Big Data to Emulate a Target Trial When a Randomized Trial Is N Am J Epidemiol, 2016; 183(8): 758–764. DOI: https://doi.org/10.1093/aje/kwv254 14. Deng, QL, Dong, S, Wang, L, et al. Development and Validation of a Nomogram for Predicting Survival in Patients with Advanced Pancreatic Ductal Adenocarcinoma. Sci Rep. 2017; 7(1): 11524. DOI: https://doi.org/10.1038/ s41598-017-11227-8 15. Katz, MH, Hu, CY, Fleming, JB, Pisters, PW, Lee, JE and Chang, GJ. Clinical calculator of conditional survival estimates for resected and unresected survivors of pancreatic cancer. Arch Surg. 2012; 147(6): 513–519. DOI: https://doi.org/10.1001/archsurg.2011.2281 16. Zhang, DX, Dai, YD, Yuan, SX and Tao, L. Prognostic factors in patients with pancreatic cancer. Exp Ther Med. 2012; 3(3): 423–432. DOI: https://doi.org/10.3892/etm.2011.412 ( ) p g 17. American Cancer Society. www.cancer.org. Accessed May 24, 2018. ancer Society. www.cancer.org. Accessed May 24, 20 18. Welch, HG and Mogielnicki, J. Presumed benefit: lessons from the American experience with marrow transplantation for breast cancer. BMJ. 2002; 324(7345): 1088–1092. DOI: https://doi.org/10.1136/ bmj.324.7345.1088 j 19. Antman, K and Gale, RP. Advanced breast cancer: high-dose chemotherapy and bone marrow aut Ann Intern Med, 1988; 108(4): 570–574. DOI: https://doi.org/10.7326/0003-4819-108-4-570 20. Rahman, ZU, Frye, DK, Buzdar, AU, et al. Impact of selection process on response rate and long-term survival of potential high-dose chemotherapy candidates treated with standard-dose doxorubicin-containing chemotherapy in patients with metastatic breast cancer. J Clin Oncol. 1997; 15(10): 3171–3177. DOI: https://doi.org/10.1200/ JCO.1997.15.10.3171 21. Submitted: 10 June 2018 Accepted: 23 January 2019 Submitted: 10 June 2018 Accepted: 23 January 2019 References Stadtmauer, EA, O’Neill, A, Goldstein, LJ, et al. Conventional-dose chemotherapy compared with high-dose chemotherapy plus autologous hematopoietic stem-cell transplantation for metastatic breast cancer. Philadelphia Bone Marrow Transplant Group. N Engl J Med. 2000; 342(15): 1069–1076. DOI: https://doi.org/10.1056/ NEJM200004133421501 22. Lippman, ME. High-dose chemotherapy plus autologous bone marrow transplantation for metastatic breast cancer. N Engl J Med. 2000; 342(15): 1119–1120. DOI: https://doi.org/10.1056/NEJM200004133421508 23. Palmer, D and Kaplan, S. A framework for strategic innovation. https://www.innovation-point.com/ Innovation%20White%20Paper.pdf. Accessed June 3, 2018. Innovation%20White%20Paper.pdf. Accessed June 3, 2018. 24. Agile Alliance. 12 Principles Behind the Agile Manifesto. https://www.agilealliance.org/agile101/12-principles- behind-the-agile-manifesto. Accessed June 3, 2018. . Agile Alliance. 12 Principles Behind the Agile Manifesto. https://www.agilealliance.org/agile101/12-p behind-the-agile-manifesto. Accessed June 3, 2018. 25. Anonymous. Agile software devevelopment. https://en.wikipedia.org/wiki/Agile_software_development Accessed May 21, 2018. 26. Shah, ND, Steyerberg, EW and Kent, DM. Big Data and Predictive Analytics: Recalibrating Expectations. JAMA; 2018. DOI: https://doi.org/10.1001/jama.2018.5602 27. Ballard, DW, Vemula, R, Chettipally, UK, et al. Optimizing Clinical Decision Support in the Electronic Health Record. Clinical Characteristics Associated with the Use of a Decision Tool for Disposition of ED Patients with Pulmonary Embolism. Appl Clin Inform. 2016; 7(3): 883–898. DOI: https://doi.org/10.4338/ACI-2016-05-RA-0073 28. Kuzniewicz, MW, Puopolo, KM, Fischer, A, et al. A Quantitative, Risk-Based Approach to the Management of Neonatal Early-Onset Sepsis. JAMA Pediatr. 2017; 171(4): 365–371. DOI: https://doi.org/10.1001/ jamapediatrics.2016.4678 How to cite this article: Lieu, TA, Herrinton, LJ, Buzkov, DE, Liu, L, Lyons, D, Neugebauer, R, Needham, T, Ng, D, Prausnitz, S, Stewart, K, Van Den Eeden, SK and Baer, DM 2019 Developing a Prognostic Information System for Personalized Care in Real Time. eGEMs (Generating Evidence & Methods to improve patient outcomes), 7(1): 2, pp. 1–9. DOI: https://doi.org/10.5334/ egems.266 Published: 25 March 2019 Copyright: © 2019 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/licenses/by/4.0/. OPE eGEMs (Generating Evidence & Methods to improve patient outcomes) is a peer-reviewed open access journal published by Ubiquity Press. eGEMs (Generating Evidence & Methods to improve patient outcomes) is a peer-reviewed open access journal published by Ubiquity Press.
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Colonizing Paradise: Landscape and Empire in the British West Indies, written by Jefferson Dillman
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375 375 book reviews Jefferson Dillman Colonizing Paradise: Landscape and Empire in the British West Indies. Tuscaloosa: University Alabama Press, 2015. x + 249 pp. (Cloth US$54.95) Jefferson Dillman Colonizing Paradise: Landscape and Empire in the British West Indies. Tuscaloosa: University Alabama Press, 2015. x + 249 pp. (Cloth US$54.95) Jefferson Dillman Colonizing Paradise: Landscape and Empire in the British West Indies. Tuscaloosa: University Alabama Press, 2015. x + 249 pp. (Cloth US$54.95) In this concise volume, Jefferson Dillman argues that the European perception of the Caribbean landscape evolved in stages, beginning with the didactic perspective of early Iberian explorers, and culminating in an English view of the Caribbean as pastoral and picturesque. The study runs from Columbus’s first letters to the abolition of slavery in the British West Indies. Chapter 1 focuses on fifteenth- to sixteenth-century Iberian perceptions, as reconstructed from letters and published writings. Dillman concludes that these initial views were shaped by the juxtaposition of what explorers, colonists, and critics saw as Edenic and Satanic qualities. Pulling out refer- ences to the biblical Eden from this literature, he argues that many of them felt that they had come close to finding the actual location of the Garden of Eden. Given their Catholic background, they saw in the temperate climate, abundant fresh water, verdure of the forests, and fertility of the soil a paradise identical to the Garden before the Fall and expulsion of humanity. By the middle of the sixteenth century the literal interpretation of the New World as the location of the Garden of Eden was tempered somewhat. Dillman’s analysis of texts by Bartolomé de Las Casas, José de Acosta, and others suggests that by this time the Edenic trope was used rhetorically rather than literally. Simultane- ously, the Iberians interpreted such things as dangerous animals, treacherous waters, and the prevalence of disease as manifestations of Satan. Although Dillon belabors this point somewhat, he convincingly argues that this juxta- position of the Edenic with the Satanic significantly influenced perceptions of the Caribbean. The remaining four chapters focus on English/British perceptions of the Caribbean landscape from the earliest published accounts to the end of slavery in the British West Indies. © james a. delle, 2017 | doi: 10.1163/22134360-09103043 This is an open access article distributed under the terms of the prevailing cc-by-nc license at the time of publication. Downloaded from Brill.com 10/24/2024 04:03:43AM via Open Access. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International Public License (CC-BY-NC 4.0). © james a. delle, 2017 | doi: 10.1163/22134360-09103043 This is an open access article distributed under the terms of the prevailing cc-by-nc license at the time of publication. Downloaded from via Open Access. This is an open acces of the Creative Commons Attribution-NonC This is an open access article distributed under the terms of the prevailing cc-by-nc license at the time of publication. via Open Access. This is an open acces of the Creative Commons Attribution-NonC 375 Dillman’s reading of the sixteenth- and seventeenth- century accounts suggests that English perceptions, influenced by the six- teenth-century Iberians, saw the New World, not as The Paradise, but rather a paradise, full of social and commercial potential, and “a place through which man’s story of the fall from grace and eventual redemption might be enacted” (p. 39). If the Spanish had failed to realize the full potential of this paradise it was because they fell into the grasp of Satan, devolving into debauchery and indolence. Dillman argues that the early English explorers built on the Edenic trope to promote stable, prosperous settlements. By the seventeenth century, Dillman argues, the hard realities of the English experiences in Virginia and the West Indies promoted a view of the Caribbean 376 book reviews as an abyss of immorality; by the late seventeenth century, it was seen as a fallen place in need of redemption. Seventeenth-century English colonists abused the abundance of their paradisiacal garden, descending into sin and sloth. Privateering, prostitution, drunkenness, and idleness reigned in a lawless landscape where pirates and maroons alike could thrive outside of the bounds of civilized society. By the early eighteenth century, there was a third phase, one dominated by the Enlightenment impulse to control the natural environment. Chapter 4 examines both natural history treatises and descriptions of botanical gardens established throughout the West Indies to show that during the eighteenth century the West Indian landscape was seen as redeemed and mastered—a place where great fortunes could at last be realized. The beauty of that landscape, of course, masked the ugly reality of enslaved labor. In the late eighteenth century and early nineteenth, enslaved people wereincorporatedintothelandscapeaspicturesqueandpastoral.Attheheight of the sugar boom, the West Indian landscape was viewed similarly to English rural landscapes, the natural beauty of swaying canes hiding the violence inherent in the social landscape. In a brief epilogue, Dillman ruminates on thenineteenth-centurylandscapeasitemergedafterabolition,focusingonthe scientific and touristic landscape tropes, which he defines as the Tropical. The initial and final stages of the process are less well developed than the middlecenturies.Theanalysisof theIberianwritersisoccasionallylaboredand redundant, and Dillman’s brief treatment of the postemancipation landscape in the epilogue lacks development. Nevertheless, Colonizing Paradise presents an important review of the landscape history of the (primarily) British West Indies, framed by an interesting thesis and boasting a first-rate bibliography. This is a book that any serious student of the Caribbean should read. James A. James A. Delle College of Arts and Sciences, Shippensburg University of Pennsylvania, Shippensburg PA 17257, U.S.A. JADelle@ship.edu 375 Delle College of Arts and Sciences, Shippensburg University of Pennsylvania, Shippensburg PA 17257, U.S.A. JADelle@ship.edu New West Indian Guide 91 (2017) 271–400 Downloaded from Brill.com 10/24/2024 04:03:43AM via Open Access. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International Public License (CC-BY-NC 4.0). http://creativecommons.org/licenses/by-nc/4.0 New West Indian Guide 91 (2017) 271–400 Downloaded from Brill.com 10/24/2024 04:03:43AM via Open Access. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International Public License (CC-BY-NC 4.0). http://creativecommons.org/licenses/by-nc/4.0
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https://link.springer.com/content/pdf/10.1140/epjc/s10052-023-12320-1.pdf
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Using entropy bounds to avoid the cosmological singularity and constrain cosmological particle production
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Using entropy bounds to avoid the cosmological singularity and constrain cosmological particle production Hao Yua, Jin Lib Physics Department, Chongqing University, Chongqing 401331, China Received: 7 June 2023 / Accepted: 1 December 2023 / Published online: 15 December 2023 © The Author(s) 2023 is commonly known as the Bekenstein bound, which is inde- pendent of the gravitational theory. It is worth mentioning that Unruh and Wald did not agree with the original deriva- tion of the Bekenstein bound [10,11]. They argued that the entropy bound of a black hole is not needed for the valid- ity of the generalized second law, provided that there exists buoyancy force of the thermal atmosphere near the black hole horizon. Subsequent studies show that, according to physi- cal processes, gravitational theories, the background space- time, etc., entropy bounds can take on different forms. In 1995, Susskind argued that by applying the generalized sec- ond law to a transformation that a system is converted to a black hole, one can derive the spherical entropy bound as S ≤k A/(4l2 p), where A is a properly defined area enclos- ing the system [6,12]. Subsequently, inspired by the work of Fischler and Susskind [13], Bousso proposed a covari- ant entropy bound (which is also called Bousso bound) S[L(B)] ≤A(B)/4, where A is the area of the bound- ary B [14]. The covariant entropy bound can be applied to any space-time including strong gravitational systems, and it adheres to general covariance, but it is only applica- ble to general relativity. In Ref. [15], the authors provided two methods to demonstrate the validity of the covariant entropy bound and put forward a stronger entropy bound. In the same year that the covariant entropy bound was pro- posed, Brustein and Veneziano proposed a causal entropy bound [16]. The subsequent year, Verlinde proposed the Bekenstein-Verlinde bound, the Bekenstein-Hawking bound and the Hubble bound [17]. For more research on entropy bounds, one can refer to Refs. [18–25]. Abstract In this work, we study the applications of entropy bounds in two toy cosmological models with particle pro- duction (annihilation), i.e., a radiation-dominated universe and a dust-dominated universe. We consider the co-moving volume and the volume covered by the particle horizon of a given observer as the thermodynamic systems satisfying entropy bounds. For the Bekenstein bound and the spherical entropy bound, it is found that the cosmological singularity can be avoided and cosmological particle production needs to be truncated in some special cases. Eur. Phys. J. C (2023) 83:1136 https://doi.org/10.1140/epjc/s10052-023-12320-1 Regular Article - Theoretical Physics Using entropy bounds to avoid the cosmological singularity and constrain cosmological particle production Our study can be extended to other cosmological models with particle production. a e-mail: yuhaocd@cqu.edu.cn b e-mail: cqujinli1983@cqu.edu.cn (corresponding author) 1 Introduction Blackholethermodynamics[1–4],whichhasledtothedevel- opment of the holographic principle [5–7], holds signifi- cant potential for comprehending quantum gravity. In 1972, Bekenstein found that black holes could have entropy [1], which triggered the research enthusiasm on black hole ther- modynamics. In these studies, entropy plays a fundamental role in connecting black holes with thermodynamics. How- ever, for nearly a decade after Bekenstein found that black holes have entropy, few researchers considered the relation- ship between the entropy of black holes and the entropy of other gravitational systems. It was not until 1981, when Bekenstein studied the generalized second law (of thermo- dynamics) for black holes [8], that he first proposed that the generalized second law implies the entropy of any weakly gravitating matter system in asymptotically flat space should satisfy a bound S ≤2πkE R/(¯hc), where E is the total mass- energy of the system and R is the radius of the smallest sphere that fits around the system [9]. This entropy bound Although the concept of entropy bounds originated from black hole research, it also carries significant implications in cosmology. Some models of the Big Bang theory predict that there exists a cosmological singularity (initial singularity or Big Bang singularity) before the Big Bang, which contained all the energy and space-time of the universe. While the Big 12 3 3 Eur. Phys. J. C (2023) 83 :1136 1136 Page 2 of 15 Bang theory aligns with cosmological observations and has garnered acceptance among many physicists, the notion of the initial singularity in the universe has faced criticism. As a result, some cosmological models and theories have been proposed to explain or avoid the cosmological singularity, such as the cyclic model of the universe, multiverse, loop quantum gravity, etc. In 1989, Bekenstein found that the cos- mological singularity is thermodynamically irrational [26] from the perspective of the entropy bound proposed by him- self [9]. Recently, Powell et al. proposed a re-examination of Bekenstein’s approach in a radiation-dominated universe and also verified that the Bekenstein bound can be a feasible alternative to avoiding the cosmological singularity [27]. In addition to the application of entropy bounds to the initial singularity of the universe, entropy bounds may also assist in deducing the shape of the universe. 2 Cosmological particle production and entropy The continuous production and annihilation of particles in our universe is an undeniable reality, so the cosmological model with particle production (annihilation) is more consis- tent with the real universe. The mechanisms and applications of particle production in cosmology have long been a sub- ject of interest for physicists (see a recent review [34] and references therein). In the 1960s, Parker laid the foundation for the micromechanism of particle production in the context of cosmology by employing quantum field theory in curved space-time [35–38]. Then, particle production in different background space-time has been investigated successively [39–42]. A few years ago, Harko discovered that, from a thermodynamic point of view, gravitational induced parti- cle production can happen in non-minimal coupling theories [43,44], which provides a new mechanism for cosmological particle production. Currently, a majority of studies indicate that the production and annihilation of particles in an expand- ing universe have a profound theoretical foundation and they can not be ignored, particularly during the early stages of the universe. Regarding the applications of particle production in cosmology, they include multiple aspects, such as the avoid- ance the cosmological singularity [45–47], the explanation of entropy production in the universe [45–49], the acceleration of the universe’s expansion [47,50–54], and the initiation of inflation [55,56]. In this section, we discuss cosmological particle produc- tion and entropy in the context of a homogeneous and isotropicuniverse,whichcanbedescribedbytheFriedmann– Lematˆıre–Robertson–Walker metric: ds2 = −dt2 + a2(t)  dr2 1 −˜k r2 + r2dθ2 + r2sin2θ dφ2  . (1)  (1) For simplicity, we consider that the universe is spatially flat, i.e., ˜k = 0. Here, a(t) is the scale factor. In this work, all the components of the universe are regarded as ideal fluids, so the energy-momentum tensor of all the ideal fluids is given as For simplicity, we consider that the universe is spatially flat, i.e., ˜k = 0. Here, a(t) is the scale factor. In this work, all the components of the universe are regarded as ideal fluids, so the energy-momentum tensor of all the ideal fluids is given as (2) Tμν = (p + ρ)uμuν + p gμν, (2) where ρ and p represent the total energy density and pres- sure of the ideal fluids, respectively. The four-velocity of the ideal fluids satisfies uνuν = −1. 1 Introduction For a closed universe, a contradiction arises between the Fischler-Susskind bound and the positive curvature, which means that the Fischler- Susskind bound requires the shape of the universe to be non- closed [13]. For more cosmological applications of various entropy bounds, one can refer to Refs. [25,28–33]. bounds in cosmology. The constraint on cosmological par- ticle production is of significance for cosmology because it could provide us with truncations for certain interactions in the context of cosmology. Previously, the (generalized) sec- ondlawofthermodynamicswascommonlyemployedtocon- strain cosmological particle production, but the results were not entirely satisfactory as it can only constrain the sign of the particle production rate [49,57]. The paper is organized as follows. Section 2 provides a brief review of cosmological particle production and the corresponding entropy. In Sect. 3, we discuss the Beken- stein bound and the spherical entropy bound in a radiation- dominated universe with particle production. We focus on the cosmological singularity and the constraint on cosmolog- ical particle production inside the co-moving volume and the volume covered by the particle horizon of a given observer. Then, in Sect. 4, we study similar content in a dust-dominated universe with particle production. The last part, Sect. 5, is conclusions and discussions. 123 1 In this work, we do not consider the non-extensive statistical entropy, such as Tsallis entropy [58]. Some studies indicate that, compared to classical statistics, non-extensive statistics may be more applicable to a gravitational system [58–60]. 2 Cosmological particle production and entropy If the particle number in the system is non- conserved, the entropy of the system will be affected by the production (increase) or annihilation (decrease) of particles. We label the current particle number as N0 and set the current radius of the system to R0. If the entropy of these particles is extensive1 in a homogeneous and isotropic gravitational system (such as the cosmological model we study), then the entropy of the system at time t can be written as 2 In this work, we are not concerned about the mechanism by which matter was produced (annihilated) at the beginning of the universe. 3 Entropy bounds and particle production in a radiation-dominated universe St = s(nt) R3 t , (6) St = s(nt) R3 t , (6) In 1989, Bekenstein studied the particle horizon of a given observer in a radiation-dominated universe and he found that the cosmological singularity is thermodynamically impos- sible by considering the Bekenstein bound [26]. However, in Bekenstein’s Friedmann model, there is no cosmologi- cal particle production inside the co-moving volume, which may be inappropriate for the early universe because non- relativistic particles were not decoupled from other matter during that period. In this section, we extend Bekenstein’s Friedmann model by introducing particle production into the radiation-dominated universe and discuss the relationship among the cosmological singularity, particle production and entropy bounds. where nt = Nt/Vt = Nt/R3 t is the particle number density and s(nt) is the entropy per volume at time t. The radius Rt of the system at time t is dependent on the way the universe is expanding. The particle number Nt at time t is given by Nt = N0 exp  t t0  dt  . (7) (7) If the system is isolated, one can constrain the evolution of the system by the second law of thermodynamics: If the system is isolated, one can constrain the evolution of the system by the second law of thermodynamics: dSt dt = ds(nt) dn   nt −3 nt Rt dRt dt  + 3s(nt) R2 t dRt dt > 0. (8) (8) As a toy model, we simplify the radiation-dominated uni- verse as a universe containing only photons. The production of photons can be ascribed to the coupling between photons and the background space-time [43,44] (or the running vac- uum [49,70,71]). Since we do not know how to define the entropy of the background space-time, the total entropy of the universe is considered to be only dependent on photons. We assume that the number of photons per unit volume is On the other hand, if the system possesses area entropy (such as the system covered by the apparent horizon [7,61–69]), then the system can be constrained by the generalized second law of thermodynamics [1,2,8]: dSt dt + dSA dt > 0. (9) (9) The area entropy SA of the system is usually proportional to the area of the system, but the coefficient is related to the gravitational theory. 2 Cosmological particle production and entropy For general relativity, the Friedmann equations are given by H2 = 8πG 3 ρ, (3) ˙H + H2 = −4πG 3 (ρ + 3p) . (4) (3) (4) Now, we focus on a spherical system in the universe with a radius of R. According to the purpose of research, the phys- ical interpretations of R can be multiple, such as the scale factor, the particle horizon, the apparent horizon, the radius of the visible universe, etc. Assuming that there are N parti- cles in the system, we can define the particle production rate Now, we focus on a spherical system in the universe with a radius of R. According to the purpose of research, the phys- ical interpretations of R can be multiple, such as the scale factor, the particle horizon, the apparent horizon, the radius of the visible universe, etc. Assuming that there are N parti- cles in the system, we can define the particle production rate In this work, we study entropy bounds in the cosmolog- ical model with particle production (annihilation). We use entropy bounds to judge whether the cosmological singular- ity is thermodynamically rational in a radiation-dominated universe and a dust-dominated universe. Moreover, entropy bounds offer a potential means to constrain cosmological particle production, which is a novel application of entropy as 123 Eur. Phys. J. C (2023) 83 :1136 Page 3 of 15 1136  = dN dt 1 N .  = dN dt 1 N . with entropy bounds. Since entropy bounds do not require the corresponding system to be closed, we can study cos- mological particle production in any system. On the other hand, we will also examine the effect of cosmological par- ticle production on the cosmological singularity in the view of entropy bounds.2 The entropy bounds employed in this work are the Bekenstein bound [9] and the spherical entropy bound [6,7,12]. In order to obtain analytical solutions of the Friedmann equations, each of the toy cosmological models we consider only contains a species of matter. (5) Usually, if R is the scale factor and there is no interaction between these particles and other matter (or the background space-time), we have  = 0. For other cases of R, even if there is no interaction,  is generally nonzero due to the evolution of R. 3.1 Co-moving volume where we can set a0 = 1 (m) and T0 ∼2.7 K. For 0 ≤t < t0 and  > 0, there must be a critical time tc corresponding to the equality sign of inequality (20). When t < tc, inequality (20) will be violated, which means that the Bekenstein bound requires the initial time of the universe to be nonzero. However, due to the existence of , the scale factor does not satisfy a(t = 0) = 0, so a nonzero initial time does not imply a nonzero initial volume. In the later discussion, we will see that, for  > 0, the universe naturally has no initial singularity according to the solution the scale factor. Therefore, in this case we do not need the Bekenstein bound to avoid the cosmological singularity. We will focus on the effect of the Bekenstein bound on the initial volume of the universe. Regarding this, we can see detailed examples later. If R is the scale factor, according to Eqs. (11) and (13), the entropy of photons inside the co-moving volume is given as S = 2π4k 45ζ(3) N0 exp  3  t t0  ˙T T + ˙a a  dt  . (14) (14) If  = 0, then S is a constant. We first consider the Bekenstein bound, which requires the entropy of any weakly gravitating matter system to satisfy [9] S ≤2πk ¯hc E R, (15) (15) where E is the total mass-energy including any rest mass and R is the radius of a sphere that can enclose the given system. For the photons inside the co-moving volume, we have E = π2k4 15c3¯h3 a3T 4, so the Bekenstein bound can be expressed as where E is the total mass-energy including any rest mass and R is the radius of a sphere that can enclose the given system. For the photons inside the co-moving volume, we have E = π2k4 15c3¯h3 a3T 4, so the Bekenstein bound can be expressed as For 0 ≤t < t0 and  ≤0, since the right-hand side of inequality (20) increases with the decrease of t, the Beken- stein bound does not help to avoid the cosmological singu- larity. 2π4k 45ζ(3) N0 exp  3  t t0  ˙T T + ˙a a  dt  ≤2πk ¯hc π2k4 15c3¯h3 a4T 4. 3 Entropy bounds and particle production in a radiation-dominated universe 2 3 ≤πk ¯hc a0T0, 2 3 ≤πk ¯hc a0T0, (17) Since the entropy of photons per unit volume is given by (17) s = 4π2k4 45c3¯h3 T 3, (12) (12) set a0 = 1 (m) and T0 ∼2.7 K, then this inequality is tenable. Therefore, for a radiation-dominated universe without parti- cleproduction,ifthecurrenttemperatureoftheuniverseisthe same as our real universe, applying the Bekenstein bound to the co-moving volume can not avoid the cosmological sin- gularity. Moreover, based on inequality (17), one can find that if T0 < 2 ∗10−4 K, the entropy of photons inside the co-moving volume will conflict with the Bekenstein bound. the entropy of photons inside the spherical system with radius R is S = s R3 = 4π2k4 45c3¯h3 T 3R3 = 2π4k 45ζ(3) N = 2π4k 45ζ(3) N0 exp  t t0  dt  , (13) (13) g When  ̸= 0, a T will evolve over time. We can rewrite Eq. (11) as where N0 is the current number of photons in the system. When R and  are given, we can calculate the entropy of photons in the system at any time. Next, we consider that R is the scale factor (i.e., the system is the co-moving volume) or the particle horizon of a given observer (i.e., the system is the volume covered by the particle horizon). As t approaches to zero, the production (annihilation) of photons may influ- ence the cosmological singularity from the perspective of entropy bounds. Moreover, as t increases, entropy bounds may require that there exists a truncation for the production of photons.  = 3d(aT ) dt 1 aT . (18)  = 3d(aT ) dt 1 aT . (18) Then, one can obtain Then, one can obtain exp  t t0  dt  =  aT a0T0 3 . (19) (19) Substituting it into inequality (16) to cancel a Substituting it into inequality (16) to cancel aT and N0 yields Substituting it into inequality (16) to cancel aT and N0 yields 2 3 ≤πk ¯hc a0T0 exp 1 3  t t0  dt  , (20) (20) 3.1 Co-moving volume 3 Entropy bounds and particle production in a radiation-dominated universe Owing to inequality (8) [or inequal- ity (9)], the particle production rate  in the universe can not be arbitrary. Therefore, the (generalized) second law of ther- modynamicsisameansofconstrainingcosmologicalparticle production (or the interaction between different substances in the context of cosmology). The area entropy SA of the system is usually proportional to the area of the system, but the coefficient is related to the gravitational theory. Owing to inequality (8) [or inequal- ity (9)], the particle production rate  in the universe can not be arbitrary. Therefore, the (generalized) second law of ther- modynamicsisameansofconstrainingcosmologicalparticle production (or the interaction between different substances in the context of cosmology). n = 2k3ζ(3) π2c3¯h3 T 3, (10) (10) where c is the light speed, k is the Boltzmann constant, ζ(n) is the Riemann zeta function. If there is no coupling between photons and the background space-time (i.e., there is no par- ticle production in the co-moving volume), the temperature T of photons should be proportional to a−1. For a spheri- cal system with radius R in the universe, with Eq. (5) and N = n R3, one can obtain the production rate of photons in the system is given as However, for a general radius R, the system is not isolated, so it may be not appropriate to use the (generalized) second law of thermodynamics to constrain cosmological particle production. In this work, we omit the area entropy of the system and try to constrain cosmological particle production  = 1 n dn dt + 1 R3 dR3 dt = 3  ˙T T + ˙R R  . (11) (11) 2 In this work, we are not concerned about the mechanism by which matter was produced (annihilated) at the beginning of the universe. 3 Eur. Phys. J. C (2023) 83 :1136 1136 Page 4 of 15 2 3 ≤πk ¯hc a0T0, (17) where k ∼1.38∗10−23 J/K and ¯hc ∼3.16∗10−26 J·m. If we set a0 = 1 (m) and T0 ∼2.7 K, then this inequality is tenable. Therefore, for a radiation-dominated universe without parti- cleproduction,ifthecurrenttemperatureoftheuniverseisthe same as our real universe, applying the Bekenstein bound to the co-moving volume can not avoid the cosmological sin- gularity. Moreover, based on inequality (17), one can find that if T0 < 2 ∗10−4 K, the entropy of photons inside the co-moving volume will conflict with the Bekenstein bound. 3.1 Co-moving volume However, the latter is the volume of a sphere that can enclose the given system, which is related to the total mass-energy of the sys- tem. With regard to the issue, it is not the topic of this work, so we do not discuss it any further here. ground space-time must be truncated at some point. It is worth noting that the result seems to defy our physical intu- ition. With the increase of the scale factor and the annihilation of photons, the entropy and energy of photons inside the co- moving volume will be reduced synchronously. However, the reduction of the energy is faster than that of the entropy, so the critical condition of the Bekenstein bound (i.e., S = 2πk ¯hc E R) will appear and then be broken as t increases. It is different from the critical condition of the Bekenstein bound for com- pact objects, which only occurs in the case of the black hole and will not be violated as the mass of the black hole changes. The difference stems from the selection of the volume of the system. The former is the co-moving volume of the universe, which is affected by the background space-time. However, the latter is the volume of a sphere that can enclose the given system, which is related to the total mass-energy of the sys- tem. With regard to the issue, it is not the topic of this work, so we do not discuss it any further here. When  > c, we have aT a0T0 3 > a2 a2 0 for t > t0. As long as t is large enough, inequality (22) will be violated. Therefore, to meet the spherical entropy bound there should be a truncation for the production of photons at some point. Moreover, for  > c > 0, as we mentioned earlier, there is naturally no cosmological singularity due to the solution of the scale factor, but the spherical entropy bound might modify the initial volume of the universe. When  < c and t > t0, we have aT a0T0 3 < a2 a2 0 and then inequality (22) must be true. Therefore, the spherical entropyboundcannotconstraintheproduction(annihilation) rate of photons. However, if  < c and t < t0, we have aT a0T0 3 > a2 a2 0 andthentherecouldexistalowerboundforthe scale factor, which can avoid the cosmological singularity. 3.2 Particle horizon 2π4k 45ζ(3) N0 exp  t t0  dt  ≤k a2 4l2p . (22) (22) We will now take the volume covered by the particle horizon of a given observer as the thermodynamic system satisfying entropy bounds. For convenience, the production rate of pho- tons inside the particle horizon will be characterized by the production rate of photons inside the co-moving volume. In an identical universe, the difference between the two parti- cle production rates depends on the evolution of the universe. Therefore, the subsequent calculations are also characterized by the production rate of photons inside the co-moving vol- ume. When  = 0, since the scale factor is monotonically increasing, once a2 ≥ 8π4l2 p 45ζ(3) N0, the spherical entropy bound When  = 0, since the scale factor is monotonically increasing, once a2 ≥ 8π4l2 p 45ζ(3) N0, the spherical entropy bound ζ( ) will not be violated. Therefore, a =  8π4l2 p 45ζ(3) N0 1/2 is the will not be violated. Therefore, a =  8π4l2 p 45ζ(3) N0 / is the   lower bound of the scale factor, which means that the spher- ical entropy bound could avoid the cosmological singularity in a radiation-dominated universe without particle produc- tion. We start from the solution of the energy density of photons in the presence of particle production. According to the laws of thermodynamics for open systems, the energy density of photons satisfies [45–47] When  ̸= 0, using Eq. (19) to eliminate , we can obtain 2π4k 45ζ(3) N0  aT a0T0 3 ≤k a2 4l2p . (23) (23) d(ρa3) + p da3 = ρ + p n d(na3). (25) (25) In order to judge whether this inequality is true, we need to figure out the relationship between a and T by solving Eq. (19). Here, we can discuss some issues qualitatively in the absence of the solutions of Eq. (19). Reviewing inequal- ity (22), one can find that if  = 2 a da dt > 0, we have exp  t t0  dt = aT a0T0 3 = a2 a2 0 . Taking aT a0T0 3 = a2 a2 0 and 3 In order to judge whether this inequality is true, we need to figure out the relationship between a and T by solving Eq. (19). Here, we can discuss some issues qualitatively in the absence of the solutions of Eq. 3.1 Co-moving volume Next, we consider the spherical entropy bound, which is given as In this section, we have not provided the solutions of Eq. (19), so there is only qualitative analysis. Next, we will discuss some similar issues in detail in the presence of ana- lytical solutions. S ≤k A 4l2p . (21) S ≤k A 4l2p . (21) Here, A is the area of the system and lp is the Planck length. For the photons inside the co-moving volume (A = a2), the spherical entropy bound can be expressed as 3.1 Co-moving volume (16) For t0 ≤t and  ≥0, the right-hand side of inequal- ity (20) increases with t. Based on the previous analysis, the Bekenstein bound is always true. Therefore, the photons inside the co-moving volume can be continuously produced and so the particle production rate  can not be limited by the Bekenstein bound. (16) When  = 0, the energy density of photons inside the co-moving volume is given by ρ = ρ0 a4 (the Friedmann equations) or ρ = π2k4 15c3¯h3 T 4 (thermodynamic state functions for black-body photons), so aT = a0T0 is a constant. Taking N0 = 2k3ζ(3) π2c3¯h3 a3 0T 3 0 into inequality (16), then the Bekenstein bound can be simplified as For t0 ≤t and  < 0, as t increases, the entropy of pho- tons inside the co-moving volume will violate the Bekenstein bound. Therefore, photons in the universe can not keep anni- hilating and the interaction between photons and the back- bound can be simplified as 123 Eur. Phys. J. C (2023) 83 :1136 Page 5 of 15 1136 1136 If a0 = 1 (m) and T0 ∼2.7 K, this inequality is true and independent of the evolution of the scale factor. We can set c = 2 a da dt as a critical particle production rate. Note that c is a function of time not a point. ground space-time must be truncated at some point. It is worth noting that the result seems to defy our physical intu- ition. With the increase of the scale factor and the annihilation of photons, the entropy and energy of photons inside the co- moving volume will be reduced synchronously. However, the reduction of the energy is faster than that of the entropy, so the critical condition of the Bekenstein bound (i.e., S = 2πk ¯hc E R) will appear and then be broken as t increases. It is different from the critical condition of the Bekenstein bound for com- pact objects, which only occurs in the case of the black hole and will not be violated as the mass of the black hole changes. The difference stems from the selection of the volume of the system. The former is the co-moving volume of the universe, which is affected by the background space-time. 3.2 Particle horizon When  = 0, we have a ∼t1/2. As for  ̸= 0, to get the analytical solution of the scale factor, we have to presuppose a specific form of . We can analyze qualitatively the properties of the solution of the scale factor for different ’s. If  = 0 results in a = b t1/2 (where a(t0) = a0 and a(0) = 0), it can be expected that  > 0 corresponds to a > b t1/2 (where a(t0) = a0 and a(0) > 0) and  < 0 corresponds to a < b t1/2 (where a(t0) = a0 and a(0) < 0). Note that a(0) > 0 (i.e.,  > 0) means that the beginning (t = 0) of the universe is not a singularity [45–47]. Moreover, a(0) < 0 (i.e.,  < 0) indicates that the singularity (i.e., a(t) = 0) of the universe appears at the non-beginning (t ̸= 0) of the universe (see Fig. 1). In this section, we must be cautious about these two strange situations because the particle hori- zon may be unnormal, which should be avoided. Next, we discuss these issues in detail. where G0 = 8 3πG with G the Newtonian constant. When  = 0, we have a ∼t1/2. As for  ̸= 0, to get the analytical solution of the scale factor, we have to presuppose a specific form of . We can analyze qualitatively the properties of the solution of the scale factor for different ’s. If  = 0 results in a = b t1/2 (where a(t0) = a0 and a(0) = 0), it can be expected that  > 0 corresponds to a > b t1/2 (where a(t0) = a0 and a(0) > 0) and  < 0 corresponds to a < b t1/2 (where a(t0) = a0 and a(0) < 0). Note that a(0) > 0 (i.e.,  > 0) means that the beginning (t = 0) of the universe is not a singularity [45–47]. Moreover, a(0) < 0 (i.e.,  < 0) indicates that the singularity (i.e., a(t) = 0) of the universe appears at the non-beginning (t ̸= 0) of the universe (see Fig. 1). In this section, we must be cautious about these two strange situations because the particle hori- zon may be unnormal, which should be avoided. Next, we discuss these issues in detail. Fig. 3.2 Particle horizon However, as a toy model, we actually only need an analytical solution of the scale factor increasing monotonically over time. Incorporating the matter (or the background space-time) interact- ing with photons into the Friedmann equations would only complicate the analytical solution and would not be beneficial for the subsequent research. 3 Note that the reason why the number of photons inside the co-moving volume is not conserved is due to the interaction between photons and other matter (or the background space-time). Therefore, ignoring the matter (or the background space-time) interacting with photons is a rough approximation. However, as a toy model, we actually only need an analytical solution of the scale factor increasing monotonically over time. Incorporating the matter (or the background space-time) interact- ing with photons into the Friedmann equations would only complicate the analytical solution and would not be beneficial for the subsequent research. 3.2 Particle horizon (19). Reviewing inequal- ity (22), one can find that if  = 2 a da dt > 0, we have exp  t t0  dt = aT a0T0 3 = a2 a2 0 . Taking aT a0T0 3 = a2 a2 0 and 3 With  = dN dt 1 N , N = na3, and p = 1 3ρ, we have With  = dN dt 1 N , N = na3, and p = 1 3ρ, we have dρ da + 4 a ρ −4 3ρ  dt da = 0. (26) (26) Therefore, the solution of the energy density of photons can be expressed as N0 = 2k3ζ(3) π2c3¯h3 a3 0T 3 0 into inequality (23) yields ρ = ρ0 a4 0 a4 exp 4 3  t t0  dt  . (27) 4k3π2 45c3¯h3 a3 0T 3 0 ≤a2 0 4l2p . (24) 4k3π2 45c3¯h3 a3 0T 3 0 ≤a2 0 4l2p . (24) (27) 123 3 1136 Page 6 of 15 Eur. Phys. J. C (2023) 83 :1136 Fig. 1 Plot of the scale factor (31). In the schematic diagram, we have set a(t0) = a0 = 1, t0 = 1, and b1 = 1 with g = 0. We study three sets of g: g = 1 2 ( > 0, b1 = 3 4 and d1 = 1 4) marked with the black solid line, g = 0 ( = 0, b1 = 1 and d1 = 0) marked with the red dashed line, and g = −1 2 ( < 0, b1 = 3 2 and d1 = −1 2) marked with the green dash-dotted line, respectively. These three lines intersect at the point (t0, a0). When  > 0 and t = 0, the scale factor is larger than zero. When  < 0, the minimum value of time is larger than zero For  = 0 and a0 = 1, it degenerates into the standard solution ρ = ρ0 a−4. Since the universe is dominated by photons, we can ignore other matter when we solve the Friedmann equations.3 Thus, taking Eq. (27) into the Friedmann equations, we finally get a2 −a2 0 = 2(G0ρ0) 1 2 a2 0  t t0 exp 2 3  t′ t0  dt dt′, (28) where G0 = 8 3πG with G the Newtonian constant. 3.2 Particle horizon Therefore, we can not easily estimate the effect of  on the lower bound of the scale of the universe on the basis of inequality (30). With a specific , we can analyze the issues further. The particle horizon of a given observer in the radiation- dominated universe is given as RH =  t ts c a(t′)dt′, (29) (29) where ts ≥0 is the time at which the observer starts observing at r = 0. Since a(t) > 0, RH is a monoton- ically increasing function of t. For a = b t1/2, we have RH = 2b−1c t1/2 −t1/2 s . In Ref. [26], based on the par- ticle horizon of a given observer in a radiation-dominated universe and the Bekenstein bound, Bekenstein pointed out that the cosmological singularity is not thermodynamically possible (see Refs. [26,27] for more details). We set  = g t , where g > 0 (g < 0) represents particle production (annihilation). Then, taking it into Eq. (28), the solution of the scale factor can be expressed as Now, let us analyze how  affects the cosmological sin- gularity. For a system covered by the particle horizon of a given observer, the Bekenstein bound can be expressed as a2 = b1t 2 3 g+1 + d1, (31) (31) 4π2k4 45c3¯h3 T 3R3 H ≤2πk ¯hc π2k4 15c3¯h3 T 4R4 H. (30) where b1 = 2(G0ρ0) 1 2 3a2 0 3+2gt −2g 3 0 > 0 and d1 = −2(G0ρ0) 1 2 2 (30) 3a2 0 3+2gt0 + a2 0. We consider that g > 0, g = 0, and g < 0 correspondtod1 > 0,d1 = 0,andd1 < 0,respectively.From Fig. 1, one can find that when  < 0, the value of the scale factor at t = 0 will be negative by extending the green dash- dotted line, which may lead to confusion when we calculate the particle horizon. In order to avoid such a nuisance, the lower bound of the integral in Eq. (29) should not be smaller than the minimum value of time (which ensures that the scale factor is non-negative). 3 Note that the reason why the number of photons inside the co-moving volume is not conserved is due to the interaction between photons and other matter (or the background space-time). Therefore, ignoring the matter (or the background space-time) interacting with photons is a rough approximation. 3.2 Particle horizon 1 Plot of the scale factor (31). In the schematic diagram, we have set a(t0) = a0 = 1, t0 = 1, and b1 = 1 with g = 0. We study three sets of g: g = 1 2 ( > 0, b1 = 3 4 and d1 = 1 4) marked with the black solid line, g = 0 ( = 0, b1 = 1 and d1 = 0) marked with the red dashed line, and g = −1 2 ( < 0, b1 = 3 2 and d1 = −1 2) marked with the green dash-dotted line, respectively. These three lines intersect at the point (t0, a0). When  > 0 and t = 0, the scale factor is larger than zero. When  < 0, the minimum value of time is larger than zero When  = 0, there is a lower bound for the scale of the universe determined by the Bekenstein bound [26,27]. Since  influences the solution of the scale factor (which deter- mines the particle horizon and the temperature of photons), it may modify the lower bound of the scale of the universe. From the previous analysis, a positive-definite  will lead to a bigger scale factor (the scale factor given by Eq. (28) should satisfy a > b t1/2) and a smaller particle horizon RH < 2b−1c t1/2 −t1/2 s . However, a positive-definite  will also cause the temperature of photons to be higher. Therefore, we can not easily estimate the effect of  on the lower bound of the scale of the universe on the basis of inequality (30). With a specific , we can analyze the issues further. When  = 0, there is a lower bound for the scale of the universe determined by the Bekenstein bound [26,27]. Since  influences the solution of the scale factor (which deter- mines the particle horizon and the temperature of photons), it may modify the lower bound of the scale of the universe. From the previous analysis, a positive-definite  will lead to a bigger scale factor (the scale factor given by Eq. (28) should satisfy a > b t1/2) and a smaller particle horizon RH < 2b−1c t1/2 −t1/2 s . However, a positive-definite  will also cause the temperature of photons to be higher. Then, the particle horizon can be expressed as Labelling all the posi- tive coefficients in T RH as a positive-definite parameter M0, T RH can be written as T RH = M0 t g 3 +1 d1 t g 3 0 2F1 ×  1, 2g + 9 4g + 6; 2g + 6 2g + 3; −b1 d1 t 2g 3 +1  , (34) (34) where M0 = c  15c3¯h3ρ0a4 0 π2k4 1/4 . For a given g, T RH gen- erally can be simplified. However, since the hypergeometric function is complicated, for a given g, it is more reasonable to calculate T RH starting from Eq. (31). Next, we consider the three cases of g adopted in Fig. 1. . For a given g, T RH gen-   erally can be simplified. However, since the hypergeometric function is complicated, for a given g, it is more reasonable to calculate T RH starting from Eq. (31). Next, we consider the three cases of g adopted in Fig. 1. When  = 0 and ts = 0, T RH = 2M0b −3 2 1 is a constant (see the red dashed line in Fig. 2). In this case, one can prove that inequality (30) is tenable if a0 = 1 (m) and T0 ∼2.7 K, so the cosmological singularity can not be avoided. Note that ts = 0 just corresponds to a special observer. If there are other observers who deny the rationality of the cosmo- logical singularity, the universe should not have the initial singularity. When  = 0 and ts > 0, one can find that T RH = 2M0b −3 2 1  1 −t 1 2s · t−1 2  increases with t and has a maximum value. When t →ts, we have T RH →0, which inevitablycausesinequality(30)tofail.Therefore,foragiven ts, t must satisfy Next, we analyze the constraint of the Bekenstein bound on the production of photons. One can find, from the right panel in Fig. 2, that with the increase of t, whatever the value of  is, T RH is always increasing. Only when  = 0 and ts = 0, T RH is a constant, for which the Bekenstein bound is tenable. Recalling inequality (30), the larger T RH, the easier it is satisfied. Since T RH increases with t, the Bekenstein bound usually will not be violated in the late universe. Then, the particle horizon can be expressed as Then, the particle horizon can be expressed as (35) RH = c d1 t  b1t 2g 3 +1 + d1 2F1 ×  1, 2g + 9 4g + 6; 2g + 6 2g + 3; −b1 d1 t 2g 3 +1  −c d1 ts  b1t 2g 3 +1 s + d1 2F1 ×  1, 2g + 9 4g + 6; 2g + 6 2g + 3; −b1 d1 t 2g 3 +1 s  , (32) We set tc as the critical time corresponding to the equality sign of the above inequality. Then, a(tc) characterizes the critical scale (i.e., lower bound of the scale) of the universe determined by the Bekenstein bound. If ts can approach to zero, the critical value (i.e., lower bound) of the scale factor can also approach to zero. However, if there is a minimum scale for time (such as the Planck–Wheeler time), then a(tc) can not approach to zero [26,27]. We will not dig and delve what value ts should take here. We just show that the Beken- stein bound could require a (nonzero) critical value for the scale factor in the radiation-dominated universe without par- ticle production ( = 0), so the cosmological singularity can be avoided. (32) where 2F1(a, b, c, z) is a hypergeometric function. For con- venience, we can assume that, for any , we always have F(t) =  c a(t)dt and F(ts) = 0, which will not alter our subsequent analysis and conclusion. Then, the second line in the above equation is vanishing. When  ̸= 0, in order to study the effect of  on the critical value of the scale factor, we first analyze the critical time for  ̸= 0. For convenience, we set ts as the minimum value of time (see Fig. 1). Note that here the minimum value of time depends on the solution of the scale factor and has no connection with the Bekenstein bound. The “minimum value of time” determined by the Bekenstein bound is called criticaltime.Weplottheschematicdiagramof T RH inFig.2. From the left panel in Fig. 2, we can find that when t ≪t0, if there exists production of photons, T RH (see the black solid line) will be smaller than the one in the case of  = 0 (see the red dashed line). Then, the particle horizon can be expressed as According to inequality (30), if the critical time for  = 0 is tc, then the critical time for  > 0 would be tc1 > tc. Moreover, from Fig. 1, the scale factor for  > 0 is larger than that for  = 0, so tc1 > tc means that the critical value of the scale factor for  > 0 is larger than that for  = 0. In other words, the production of photons causes the critical scale of the universe to be larger. As we mentioned earlier, for  > 0, the solution of the scale factor naturally removes the cosmological singularity (i.e., a(t = 0) > 0, see Fig. 1). Here, the lower bound of the scale factor determined by the Bekenstein bound is unquestionably larger than a(t = 0). As for  < 0, it is difficult to tell what the critical time is, because it could be tc2 < tc or may not exist. The former means that the critical value of the scale factor for  < 0 is smaller than that for  = 0, and the latter means the cosmological singularity can appear. With  = g t , ρ = π2k4 15c3¯h3 T 4, Eqs. (27) and (30), the tem- perature of photons is given as With  = g t , ρ = π2k4 15c3¯h3 T 4, Eqs. (27) and (30), the tem- perature of photons is given as T 4 = 15c3¯h3ρ0a4 0 π2k4 b1t 2 3 g+1 + d1 2  t t0 4g/3 . (33) (33) Reviewing inequality (30), to study the influence of  on the cosmological singularity, we only need to figure out the impact of the parameter g on T RH. Labelling all the posi- tive coefficients in T RH as a positive-definite parameter M0, T RH can be written as Reviewing inequality (30), to study the influence of  on the cosmological singularity, we only need to figure out the impact of the parameter g on T RH. 3 Eur. Phys. J. C (2023) 83 :1136 Page 7 of 15 1136 Then, the particle horizon can be expressed as t ≥  1 − ¯hc 3πk M b 3 2 1 −2 ts > 0. t ≥  1 − ¯hc 3πk M0 b 3 2 1 −2 ts > 0. (35) Then, the particle horizon can be expressed as Then, the particle horizon can be expressed as Therefore, when we consider the entropy of pho- tons in a system covered by the particle horizon in the late g y s T RH = 2M0b −3 2 1  1 −t 1 2s · t−1 2    maximum value. When t →ts, we have T RH →0, which inevitablycausesinequality(30)tofail.Therefore,foragiven ts, t must satisfy ts, t must satisfy 12 3 Eur. Phys. J. C (2023) 83 :1136 1136 Page 8 of 15 Fig. 2 Plot of T RH (34). In the schematic diagram, we have set t0 = 1, M0 = 1 and b1 = 1 with g = 0. We study three sets of g: g = 1 2 ( > 0, b1 = 3 4 and d1 = 1 4) marked with the black solid line, g = 0 ( = 0, b1 = 1 and d1 = 0) marked with the red dashed line, and g = −1 2 ( < 0, b1 = 3 2 and d1 = −1 2) marked with the green dash-dotted line, respectively b1 = 1 and d1 = 0) marked with the red dashed line, and g = −1 2 ( < 0, b1 = 3 2 and d1 = −1 2) marked with the green dash-dotted line, respectively b1 = 1 and d1 = 0) marked with the red dashed line, and g = −1 2 ( < 0, b1 = 3 2 and d1 = −1 2) marked with the green dash-dotted line, respectively Fig. 2 Plot of T RH (34). In the schematic diagram, we have set t0 = 1, M0 = 1 and b1 = 1 with g = 0. We study three sets of g: g = 1 2 ( > 0, b1 = 3 4 and d1 = 1 4) marked with the black solid line, g = 0 ( = 0, b1 = 1 and d1 = 0) marked with the red dashed line, and g = −1 2 ( < 0, b1 = 3 2 and d1 = −1 2) marked with the green dash-dotted line, respectively by the particle horizon in the radiation-dominated universe, the cosmological singularity can be avoided. radiation-dominated universe, the Bekenstein bound can not provide effective constraint on the production of photons (i.e., the Bekenstein bound is easily satisfied). Then, the particle horizon can be expressed as The interac- tion between photons and other matter (or the background space-time) does not need to be truncated. When  ̸= 0, we can still set  = g t . Based on the pre- vious calculations, if g = 1 2, inequality (36) can be reduced as as c3 M3 0 k 4l2p  t t0 −1/2 R−1 H  3 4t4/3 + 1 4 −3/2 ≥1, (38) Now,weconsiderthesphericalentropyboundforasystem covered by the particle horizon of a given observer. We still assume that a0 = 1 (m), T0 ∼2.7 K, and ts is the minimum value of time. With Eq. (27) and ρ = π2k4 15c3¯h3 T 4, the spherical entropy bound can be written as (38) where RH is given by Eq. (32) with ts = 0 (the minimum value of time, see Fig. 1). If g = −1 2, inequality (36) is reduced as S = 4π2k4 45c3¯h3 T 3R3 H = M3 0 c3 exp  t t0  dt  R3 H a3 ≤kR2 H 4l2p , (36) S = 4π2k4 45c3¯h3 T 3R3 H = M3 0 c3 exp  t t0  dt  R3 H a3 ≤kR2 H 4l2p , c3 M3 0 k 4l2p  t t0 1/2 R−1 H  3 2t2/3 −1 2 −3/2 ≥1, (39) (39) (36) where M0 = c  15c3¯h3ρ0a4 0 π2k4 1/4 . where RH is given by Eq. (32) with ts = 3−3/2 (the minimum value of time, see Fig. 1).   When  = 0, considering a = t1/2 and RH = 2c t1/2 −t1/2 s , inequality (36) can be simplified as In order to compare the critical times (the lower bounds of time) for the three cases, we can set c3 M3 0 k 8c l2p = 1 and t0 = 1. Then, we label uniformly the left-hand sides of these three inequalities as a function f (t). The evolutions of f (t) over time for the three cases are plotted in Fig. 3. From Fig. 3, one can find that when there exists production of photons ( > 0, see the black solid line), f (t) decreases monotonically with time, so the corresponding critical time does not exist. c3 M3 0 k 8c l2p ⎛ ⎝1 −t 1 2s t 1 2 ⎞ ⎠ −1 t ≥1. 4.1 Co-moving volume For the co-moving volume, we have N0 = n0a3 0 in Eq. (40) and  is the production rate of dust inside the co-moving volume. Comparing Eq. (40) with Eq. (14), one can find that if σ(t) is a constant, the entropy evolution of the dust-dominated universe is similar to that of the radiation- dominated universe. Therefore, an evolving σ(t) has more general properties in characterizing the entropy evolution of the universe. Moreover, the solutions of the Friedmann equa- tions for the dust-dominated universe are different from the ones for the radiation-dominated universe, which could also lead to different conclusions for similar situations. Then, the particle horizon can be expressed as There are of f (t): t t0 −1/2 R−1 H 3 4 t4/3+ 1 4 −3/2 , and t t0 1/2 R−1 H 3 2 t2/3−1 2 −3/2 . espond to g = 1 2 1 = 3 4 and d1 = 1 4) with the black solid line, = 0, b1 = 1 and marked with the red ne, and g = −1 2 1 = 3 2 and d1 = −1 2) with the green ed line, respectively one obtained by  = 0. According to Fig. 1, the scale or  < 0 is smaller than that for  = 0, so the critical h i f  0 b ll h h f volume and the volume covered by the particle horizon of a given observer. They correspond to g = 1 2 3 1 2 ( > 0, b1 = 3 4 and d1 = 1 4) marked with the black solid line, volume and the volume covered by the particle horizon of a given observer. than the one obtained by  = 0. According to Fig. 1, the scale factor for  < 0 is smaller than that for  = 0, so the critical scale of the universe for  < 0 must be smaller than that for  = 0. Moreover, since f (t) increases monotonically with time, the spherical entropy bound can not constrain the anni- hilation of photons in the radiation-dominated universe. It is worth mentioning that these results are based on ts being the minimum value of time. As for other values of ts, different results may arise, which we will not continue to discuss in this work. Then, the particle horizon can be expressed as (37) (37) Since t > ts ≥0, for a given nonzero ts, when t →ts, the left-hand side of the above inequality will approach to infin- ity. As t increases, it reaches the minimum at t = 9 4ts and then monotonically increases to infinity as t →∞. In this case, it is not easy to determine the lower bound of time (critical time) according to inequality (37). However, when ts = 0, we can find that inequality (37) requires a nonzero lower bound for time (see the red dashed line in Fig. 3). As long as there exists an observer requiring a nonzero lower bound for time, the lower bound of time must be nonzero. Therefore, considering the spherical entropy bound in a system covered In other words, even if t →0, inequality (38) still holds. Therefore, in this case the spherical entropy bound can not modify the critical scale of the universe (which depends on the solution of the scale factor). On the other hand, since f (t) decreases monotonically with time, there should be a trunca- tion for the production of photons (see the black solid line). When there exists annihilation of photons ( < 0, see the green dash-dotted line), f (t) increases monotonically with time. We find that the critical time for such case is smaller Page 9 of 15 1136 Eur. Phys. J. C (2023) 83 :1136 Fig. 3 Plot of f (t). There are three sets of f (t): f (t) = t t0 −1/2 R−1 H 3 4 t4/3+ 1 4 −3/2 , f (t) = t, and f (t) = t t0 1/2 R−1 H 3 2 t2/3−1 2 −3/2 . They correspond to g = 1 2 ( > 0, b1 = 3 4 and d1 = 1 4) marked with the black solid line, g = 0 ( = 0, b1 = 1 and d1 = 0) marked with the red dashed line, and g = −1 2 ( < 0, b1 = 3 2 and d1 = −1 2) marked with the green dash-dotted line, respectively than the one obtained by  = 0. According to Fig. 1, the scale factor for  < 0 is smaller than that for  = 0, so the critical volume and the volume covered by the particle horizon of a given observer. lot of f (t). 4 Entropy bounds and particle production in a dust-dominated universe For p > 1, the Bekenstein bound does not hold at any time (see inequality (49) and the red dotted line), and thus this situation can not happen inside the co-moving volume of the dust-dominated universe. (44) Note that although there is no apparent  in the inequality, the solution of the scale factor depends on . Therefore,  and σm(t) jointly determine the cosmological singularity, and  canbeconstrainedbytheBekensteinboundforagivenσm(t). With an analytical solution of the scale factor, we can discuss these issues in detail. Note that although there is no apparent  in the inequality, the solution of the scale factor depends on . Therefore,  and σm(t) jointly determine the cosmological singularity, and  canbeconstrainedbytheBekensteinboundforagivenσm(t). With an analytical solution of the scale factor, we can discuss these issues in detail. When g = 2 ( > 0) and p = 3 (see the red solid line), since the Bekenstein bound holds at the beginning (t = 0) of the universe, it can not avoid the cosmological singularity. Note that for  > 0 the scale factor is naturally nonzero at the beginning (t = 0) of the universe. So, there is still no ini- tial singularity and the Bekenstein bound can not modify the critical scale of the universe. But over time, the production of dust will break the Bekenstein bound, so it requires a trunca- tion for the interaction between dust and other matter (or the background space-time). When g = 2 ( > 0) and p = 3 5 (see the black solid line), the Bekenstein bound is tenable. Therefore, it can not avoid the cosmological singularity and constrain the production of dust. Since we have absorbed the potential energy of dust into its rest mass, the (equivalent) pressure of dust can be approx- imately equal to zero and the (equivalent) energy density of dust satisfies dρ da + 3 a ρ −ρ  dt da = 0. (45) (45) The solution of the above equation can be expressed as The solution of the above equation can be expressed as ρ = ρ0a3 0 a3 exp  t t0  dt  . (46) (46) When g = −3 ( < 0) and p = 3 (see the red dashed line), the Bekenstein bound is invalid at any time, so it is an unreasonable case. 4 Entropy bounds and particle production in a dust-dominated universe C (2023) 83 :1136 p t ≤ 2πkc ¯h 3/2 × 3 2(G0ρ0) 1 2 a 3 2 0 t −g 2 0 2 g + 2  t 2+g 2 −t 2+g 2 0  + a 3 2 0  . (4 σ(t) ≤2πkc ¯h m a. (43) If σ(t) is a constant, the Bekenstein bound remains robust in the late dust-dominated universe due to the nonzero lower bound of m. Therefore, the production (annihilation) of dust in the late universe will not be limited by the Bekenstein bound. In addition, the existence of a lower bound for the scalefactor depends ontheevolutions ofm, σ(t) andthescale factor itself. If inequality (43) does not hold when a is below a certain threshold, then the threshold is the lower bound of the scale factor determined by the Bekenstein bound, which avoids the cosmological singularity. However, if inequal- ity (43) holds for all values of a, the cosmological singularity can not be avoided. (48) For convenience, we can set 2πkc ¯h = 1, 3 2(G0ρ0) 1 2 = 1, a0 = 1, and t0 = 1. Then, the Bekenstein bound is reduced to 0 ≤ 2 g + 2t 2+g 2 + g g + 2 −p t. (49) (49) We can set the right-hand side of the above inequality as a new function G(t), which is plotted in Fig. 4 with some rep- resentative values of the parameters (g, p). From Fig. 4, we can judge whether the universe has the initial singularity for different situations and obtain the constraint of the Beken- stein bound on the production of dust. For different values of the parameters (g, p), the results are totally different. For a general σ(t), since it can be independent of the scale factor, it is convenient to absorb m into σ(t) as a new param- eter. Next, we set σm(t) = σ(t)/m signifying the entropy of dust per unit mass, so inequality (43) can be re-expressed as σm(t) ≤2πkc ¯h a. (44) When g = 0 ( = 0), as long as p ≤1, the Bekenstein bound is always valid (see inequality (49) and the black dot- ted line), so it can not avoid the cosmological singularity. 4 Entropy bounds and particle production in a dust-dominated universe In the late 1980s, Prigogine proposed a cosmological model that suggests particle production as a solution to the initial singularity and the entropy problem [45–47]. In his cosmo- logical model, the entropy of the universe is expressed as the product of the particle number and the specific entropy (the average entropy of a single particle). If there exists particle production in the universe, the entropy inside the co-moving volume will keep increasing and so the entropy problem can be solved. In this section, we consider a similar toy cosmo- logical model, i.e., a dust-dominated universe. The entropy of dust in any system can be expressed as We first consider the Bekenstein bound, which, for the dust inside the co-moving volume, is given as S = σ(t)N0 exp  t t0  dt  ≤2πk ¯hc E a. (41) (41) For convenience, the potential energy of dust can be inte- grated into the rest mass of a single particle. In this case, the energy of dust inside the co-moving volume can be written as as E = N · mc2 = N0 exp  t t0  dt  · mc2, (42) S = σ(t)N = σ(t)N0 exp  t t0  dt  , (40) (42) (40) where m is the equivalent mass of a single particle. The lower bound of m corresponds to the rest mass of a single particle. Since the temperature of the universe is changing continu- ously, m also varies with time. Theoretically, as a →0, m could be infinite. So the Bekenstein bound is reduced as where σ(t) is the specific entropy of dust and  is the pro- duction rate of dust in the system. Next, we study the effect of the production of dust on the cosmological singularity in light of entropy bounds and the constraint of entropy bounds on the production of dust. We still focus on the co-moving where m is the equivalent mass of a single particle. The lower bound of m corresponds to the rest mass of a single particle. Since the temperature of the universe is changing continu- ously, m also varies with time. Theoretically, as a →0, m could be infinite. So the Bekenstein bound is reduced as 123 3 1136 Page 10 of 15 Eur. Phys. J. Eur. Phys. J. C (2023) 83 :1136 Fig. 4 Plot of G(t). There are six sets of the parameters (g, p): g = 2 ( > 0) and p = 3 marked with the red solid line; g = 2 ( > 0) and p = 3 5 marked with the black solid line; g = 0 ( = 0) and p = 3 marked with the red dotted line; g = 0 ( = 0) and p = 3 5 marked with the black dotted line; g = −3 ( < 0) and p = 3 marked with the red dashed line; g = −3 ( < 0) and p = 3 5 marked with the black dashed line h i l i hi h i h ihil i f d b k 3 1 re are (g, p): line; id line; d line; tted p = 3 ed line; 3 5 shed For convenience, we can set k 4l2p = 1, 3 2(G0ρ0) 1 2 = 1, N0 = 1, a0 = 1, and t0 = 1. Then, it could be further simplified as horizontal axis, which requires the annihilation of dust to be cut off in the late universe. Here we can briefly summarize the above discussion as follows. When  ≤0, the Bekenstein bound can not avoid the cosmological singularity, but there could exist a trunca- tion for the annihilation of dust. When  > 0, there is no cosmological singularity due to the solution of the scale fac- tor, and the Bekenstein bound can not affect the critical value of the scale factor (the critical scale of the universe). As for whether there is a truncation for the production of dust, it depends on the values of the parameters (g, p). All of these results are based on σm(t) = (p t) 2 3 (p > 0) and  = g t . For other forms of σm(t) and , the results could be completely different. 0 ≤ 2 g + 2t 2+g 2 + g g + 2 −p 1 2 t 3g 4 . (52) (52) Similarly, we set the right-hand side of the above inequality as a new function G1(t), which is plotted in Fig. 5 with some representative values of the parameters (g, p). Comparing Fig. 5 with Fig. 4, one can find that when  > 0 (see the solid lines), the results are similar. Eur. Phys. J. C (2023) 83 :1136 There- fore, we will not rehash the discussion on the case. When  = 0 (see the dotted lines), there exists a nonzero critical time (0 < p1/2 ≤tc), which can be obtained directly from inequality (52) with g = 0. Since the evolution of the scale factor starts from a(t = 0) = 0 for  = 0, the cosmolog- ical singularity can be avoided, which is different from the previous case (see the dotted lines in Fig. 4). Apparently, as the parameter p grows, the critical time will be larger and so the critical scale of the universe will be larger. When  < 0 (see the dashed lines), there exists a nonzero critical time. However, the nonzero critical time usually can not avoid the cosmological singularity because a(t = 0) < 0 for  < 0. Only when the critical time is larger than the minimum value of time, the cosmological singularity can be avoided. In addi- tion, there does not exist a truncation for the annihilation of dust. Again, these results are based on σ(t) = (p t) 2 3 (p > 0) and  = g t . For other forms of σ(t) and , one may get dif- ferent results. If we use the spherical entropy bound to discuss the cos- mological singularity and the constraint on the production of dust inside the co-moving volume, we need the entropy of dust inside the co-moving volume to satisfy S = σ(t)N0 exp  t t0  dt  ≤ka2 4l2p . (50) (50) We can still set σ(t) = (p t) 2 3 (p > 0) and  = g t . Note that the spherical entropy bound does not involve the equivalent mass of a single particle, so we do not have to define the entropy of dust per unit mass. The spherical entropy bound can be expressed as (p t)2/3N0  t t0 g ≤ k 4l2p × 3 2(G0ρ0) 1 2 a 3 2 0 t −g 2 0 2 g + 2  t 2+g 2 −t 2+g 2 0  + a 3 2 0 4/3 . (51) 4 Entropy bounds and particle production in a dust-dominated universe When g = −3 ( < 0) and p = 3 5 (see the black dashed line), there exists a nonzero critical time. However, a nonzero critical time does not signify a nonzero critical scale of the universe. For  < 0, according to the solutionofthescalefactor(seeFig.1),onlywhenthenonzero critical time is larger than the minimum value of time, the cosmological singularity can be avoided. By incorporating the nonzero critical time into the solution of the scale factor, it is found that the scale factor is negative, which means that the Bekenstein bound can not prevent the appearance of the cosmological singularity. With the increase of t, there will be a new intersection between the black dashed line and the Taking it into the Friedmann equations, one can get the solu- tion of the scale factor: a 3 2 −a 3 2 0 = 3 2(G0ρ0) 1 2 a 3 2 0  t t0 exp 1 2  t′ t0  dt dt′. (47) (47) Reviewing inequality (44), in order to obtain the upper bound (which is related to the constraint on the production of dust) and the lower bound (which is related to the cosmo- logical singularity) of the scale factor, we need to presup- pose specific forms of σm(t) and . We take σm(t) = (p t) 2 3 (p > 0 to guarantee that the entropy of the system is always increasing) and  = g t as an example to illustrate the issues in detail. In this case, inequality (44) can be expressed as Page 11 of 15 1136 Eur. Phys. J. C (2023) 83 :1136 4.2 Particle horizon At last, we consider the volume covered by the particle hori- zon of a given observer in the dust-dominated universe. The solution of the scale factor is also given by Eq. (47). We still (51) 12 3 Eur. Phys. J. C (2023) 83 :1136 1136 Page 12 of 15 Fig. 5 Plot of G1(t). There are six sets of the parameters (g, p): g = 2 ( > 0) and p = 3 marked with the red solid line; g = 2 ( > 0) and p = 3 5 marked with the black solid line; g = 0 ( = 0) and p = 3 marked with the red dotted line; g = 0 ( = 0) and p = 3 5 marked with the black dotted line; g = −3 ( < 0) and p = 3 marked with the red dashed line; g = −3 ( < 0) and p = 3 5 marked with the black dashed line Fig. 6 Plot of G2(t). There are six sets of the parameters (g, p): g = 2 ( > 0) and p = 3 marked with the red solid line; g = 2 ( > 0) and p = 3 5 marked with the black solid line; g = 0 ( = 0) and p = 3 marked with the red dotted line; g = 0 ( = 0) and p = 3 5 markedwith the black dotted line; g = 0 ( = 0); g = −3 ( < 0) and p = 3 marked with the red dashed line; g = −3 ( < 0) and p = 3 5 marked with the black dashed line the black dotted line; g = 0 ( = 0); g = −3 ( < 0) and p = 3 marked with the red dashed line; g = −3 ( < 0) and p = 3 5 marked with the black dashed line Fig. 6 Plot of G2(t). There are six sets of the parameters (g, p): g = 2 ( > 0) and p = 3 marked with the red solid line; g = 2 ( > 0) and p = 3 5 marked with the black solid line; g = 0 ( = 0) and p = 3 marked with the red dotted line; g = 0 ( = 0) and p = 3 5 markedwith Fig. 4.2 Particle horizon 6 Plot of G2(t). There are six sets of the parameters (g, p): g = 2 ( > 0) and p = 3 marked with the red solid line; g = 2 ( > 0) and p = 3 5 marked with the black solid line; g = 0 ( = 0) and p = 3 marked with the red dotted line; g = 0 ( = 0) and p = 3 5 markedwith the black dotted line; g = 0 ( = 0); g = −3 ( < 0) and p = 3 marked with the red dashed line; g = −3 ( < 0) and p = 3 5 marked with the black dashed line set  = g t , c = 1, 3 2(G0ρ0) 1 2 = 1, a0 = 1, and t0 = 1. So, the particle horizon can be expressed as where 2F1(a, b, c, z) is a hypergeometric function. Accord- ing to our previous assumption, for any , if F(t) =  c a(t)dt and F(ts) = 0, then the Bekenstein bound (replacing the scale factor in inequality (44) with RH) can be expressed as where 2F1(a, b, c, z) is a hypergeometric function. Accord- ing to our previous assumption, for any , if F(t) =  c a(t)dt and F(ts) = 0, then the Bekenstein bound (replacing the scale factor in inequality (44) with RH) can be expressed as RH = (g + 2)t g 3  2t g 2 +1 + g g + 2 2F1 ×  1, g + 8 3g + 6; g + 4 g + 2; −2t g 2 +1 g  −(g + 2)ts g 3    2t g 2 +1 s + g g + 2 2F1 × ⎛ ⎝1, g + 8 3g + 6; g + 4 g + 2; −2t g 2 +1 s g ⎞ ⎠, (53) 0 ≤(g + 2)t g 3  2t g 2 +1 + g g + 2 × 2F1  1, g + 8 3g + 6; g + 4 g + 2; −2t g 2 +1 g  −(p t) 2 3 . (54) (54) Here, we have set 2πkc ¯h = 1 and σm(t) = (p t) 2 3 (p > 0). Here, we have set 2πkc ¯h = 1 and σm(t) = (p t) 2 3 (p > 0). 4.2 Particle horizon The right-hand side of the above inequality is defined as a new function G2(t), which is plotted in Fig. 6 with some representative values of the parameters (g, p). In all cases, ts is the corresponding minimum value of time. Here, we have set 2πkc ¯h = 1 and σm(t) = (p t) 2 3 (p > 0). The right-hand side of the above inequality is defined as a new function G2(t), which is plotted in Fig. 6 with some representative values of the parameters (g, p). In all cases, ts is the corresponding minimum value of time. (53) 123 3 Eur. Phys. J. C (2023) 83 :1136 Page 13 of 15 1136 From Fig. 6, we can find that when  > 0 (see the solid lines), there are two possible consequences. For smaller val- ues of p (such as g = 2 and p = 3 5, the black solid line), there existsatruncationfortheproductionofdustduetotheBeken- stein bound (see the black solid line in the right panel), and the cosmological singularity can be avoided by the Beken- stein bound (see the black solid line in the left panel). In this case, the Bekenstein bound will make the critical scale of the universe bigger. For larger values of p (such as g = 2 and p = 3, the red solid line), the Bekenstein bound can not be satisfied, so this situation can not happen inside the volume covered by the particle horizon. When  = 0 (see the dotted lines), there is only one possible consequence. The entropy of dust inside the volume covered by the particle horizon is con- sistent with the Bekenstein bound at the beginning (t = 0) of the universe, and then it will break the Bekenstein bound at some point (i.e., the special critical time). Therefore, in this case, the Bekenstein bound can not avoid the cosmological singularity but it provides an upper bound for time (i.e., the special critical time). In this work, we can not offer a defini- tive explanation for the upper bound of time. When  < 0 (see the dashed lines), it is found that the critical time is larger than the minimum value (ts = 4 9) of time (at which the scale factor is vanishing). Therefore, the cosmological singularity can be avoided. 5 Conclusions and discussions The Bekenstein bound, proposed over 30 years ago, has led to the emergence of multiple definitions of entropy bounds. Although these entropy bounds are primarily established in the context of black hole research, their applications in cos- mology have also been widely studied, especially in relation to the initial singularity and entropy of the universe. The entropy resulting from particle production in the universe is typically constrained by the (generalized) second law of thermodynamics and the law of thermal equilibrium, but the results in practice are not satisfactory [49,57]. In this work, we use entropy bounds (the Bekenstein bound and the spher- ical entropy bound) to constrain the entropy of the universe in two toy cosmological models with particle production. By doing so, we aim to constrain the production of the corre- sponding particles. Additionally, we investigate the impact of particle production on the cosmological singularity in light of entropy bounds. For the spherical entropy bound, the entropy of dust inside thevolumecoveredbytheparticlehorizonofagivenobserver needs to satisfy S = σ(t)N0 exp  t t0  dt  R3 H a3 ≤kR2 H 4l2p . (55) (55) In this case, we do not need to define the entropy of dust per unit mass. Note that since  is the particle production rate of dust inside the co-moving volume, there is an extra dimensionless factor R3 H a3 on the left-hand side of the above inequality. Similarly, we set σ(t) = (p t) 2 3 (p > 0) and  = g t . With the previous parameter settings, the spherical entropy bound can be reduced as In this case, we do not need to define the entropy of dust per unit mass. Note that since  is the particle production rate of dust inside the co-moving volume, there is an extra dimensionless factor R3 H a3 on the left-hand side of the above inequality. Similarly, we set σ(t) = (p t) 2 3 (p > 0) and  = g t . 4.2 Particle horizon Comparing the critical time for p = 3 (see the red dashed line in the right panel) and the one for p = 3 5 (see the black dashed line in the left panel), one can find that as p increases, the critical scale of the universe will be larger. In addition, we can find that the annihilation of dust can not be truncated in the late universe. The right-hand side of the above inequality is defined as a new function G3(t), which is plotted in Fig. 7 with some representative values of the parameters (g, p). In all cases, ts is still the corresponding minimum value of time. From the upper left panel in Fig. 7, it can be found that when  > 0, there exists a critical time and a truncation for the production of dust due to the spherical entropy bound. But, the critical time is later than the time corresponding to the truncation, which seems strange. Here, we can deem that the truncation is non-physical and the universe began at the critical time. In this case, the universe has no initial singularity and the production of dust can not be cut off in the late universe. But, if we accept the existence of the truncation, then the universe originated from the minimum value (t = 0) of time, but has no initial singularity due to the solution of the scale factor. Therefore, the spherical entropy bound can not modify the critical scale of the universe. The production of dust can only occur in the early universe and beyond the critical time. According to the upper right panel and lower panel in Fig. 7, when  ≤0, the cosmological singularity can be avoided by the spherical entropy bound. And there is no truncation for the annihilation of dust. Finally, we emphasize again that these results are based on the specific assumptions about the model and the parameters. The results may be completely different for other models and parameters. 5 Conclusions and discussions With the previous parameter settings, the spherical entropy bound can be reduced as 0 ≤  2 g + 2t 2+g 2 + g g + 2 2 −(p t) 2 3 · tg · (g + 2)t g × 3  2t g 2 +1 + g g + 2 2F1 ×  1, g + 8 3g + 6; g + 4 g + 2; −2t g 2 +1 g  . (56) g For the two toy cosmological models we study, the cos- mological singularity can be avoided by considering entropy bounds in certain special cases, such as the system covered by the particle horizon in the radiation-dominated universe with  > 0 constrained by the Bekenstein bound (see Fig. 2). In addition, the production (annihilation) of particles needs (56) 123 12 3 Eur. Phys. J. C (2023) 83 :1136 1136 Page 14 of 15 Fig. 7 Plot of G3(t). There are six sets of the parameters (g, p). The upper left panel corresponds to g = 2 ( > 0), where p = 3 is marked with the red solid line and p = 3 5 is marked with the black solid line. The upper right panel corresponds to g = 0 ( = 0), where p = 3 is marked with the red dotted line and p = 3 5 is marked with the black dotted line. The lower panel corresponds to g = −3 ( < 0), where p = 3 is marked with the red dashed line and p = 3 5 is marked with the black dashed line marked with the red dotted line and p = 3 5 is marked with the black dotted line. The lower panel corresponds to g = −3 ( < 0), where p = 3 is marked with the red dashed line and p = 3 5 is marked with the black dashed line Fig. 7 Plot of G3(t). There are six sets of the parameters (g, p). The upper left panel corresponds to g = 2 ( > 0), where p = 3 is marked with the red solid line and p = 3 5 is marked with the black solid line. 5 Conclusions and discussions The upper right panel corresponds to g = 0 ( = 0), where p = 3 is to be truncated in certain special cases due to entropy bounds, suchastheco-movingvolumeinthedust-dominateduniverse with  < 0 and p = 3 5 constrained by the Bekenstein bound (see Fig. 4). There are also some cases that consistently fail to satisfy specific entropy bounds, and so they can not happen in the corresponding cosmological model, such as the system covered by the particle horizon in the dust-dominated uni- verse with  > 0 and p = 3 constrained by the Bekenstein bound (see Fig. 6). Conversely, some cases always satisfy specific entropy bounds, which means that the corresponding entropy bounds do not play much of a role, such as the co- moving volume in the dust-dominated universe with  > 0 and p = 3 5 constrained by the spherical entropy bound (see Fig. 5). Overall, it is crucial to acknowledge that different situations can yield various possible outcomes regarding the cosmological singularity and the truncation of particle pro- duction (annihilation). It should be emphasized that these results are mainly dependent on the selection of the system in the universe and the entropy bound. research, which can also avoid the emergence of various pos- sible results for different entropy bounds. To sum up, our methods are basically applicable to all cosmological models with particle production, but it is still a preliminary attempt. Further studies are necessary to determine how to implement these research findings in the real universe, select an appro- priate system in the universe, and identify the genuinely prac- tical entropy bound. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grants no. 11873001, and no. 12047564) and the Postdoctoral Science Foundation of Chongqing (Grant no. cstc2021jcyj-bsh0124). Data Availability Statement The manuscript has associated data in a data repository. 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Quantifying annual spatial consistency in chick-rearing seabirds to inform important site identification
Biological conservation
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Quantifying annual spatial consistency in chick-rearing seabirds to inform important site identification 95060, USA k Centre d’Etudes Biologiques de Chiz´e, CNRS La Rochelle Universit´e, 79360 Villiers en Bois, France l Department of Natural Resource Sciences, McGill University, 21111 Lakeshore Rd, Ste Anne-de-Bellevue, QC H9X3V9, Canada m CESAM, Departamento de Biologia Animal, Faculdade de Ciˆencias, Universidade de Lisboa, Rua Ernesto Vasconcelos, 1749-016 Lisboa, Portugal n School of Environmental Sciences, University of Liverpool, Liverpool L69 3GP, UK o University of Roehampton, Holybourne Avenue, London SW15 4JD, UK p School of Biology, University of Leeds, Irene Manton Building, Leeds LS2 9JT, UK q Faculty of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura-machi, Japan r School of Biology, University of Leeds, Irene Manton Building, Leeds LS2 9JT, UK s Korea Polar Research Institute (KOPRI), 26, Songdomirae-ro, Yeonsu-gu, Incheon 21990, Republic of Korea t National Institute of Polar Research, Tachikawa, Tokyo 190-8518, Japan u Graduate School of Environmental Studies, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan v ƗRSPB Centre for Conservation Science, RSPB, 7.3.1 Cameron House, White Cross Estate, Lancashire LA1 4XF, UK w Department of Fisheries, Wildlife, and Conservation Sciences, Oregon State University, Hatfield Marine Science Center, 2030 Marine Science Dr., Newport, OR, USA x RSPB Centre for Conservation Science, Royal Society for the Protection of Birds, RSPB North Scotland Office, Etive House, Beechwood Park, Inverness IV2 6AL, UK y University of Coimbra, MARE – Marine and Environmental Sciences Centre, Department of Life Sciences, Calçada Martim de Freitas, 3000-456 Coimbra, Portugal z National Institute of Water & Atmospheric Research, PO Box 8602, Riccarton, Christchurch 8011, New Zealand aa Atmosphere and Ocean Research Institute, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa City, Chiba Prefecture 277-8564, Japan ab Life Sciences, University of Roehampton, Whiteland Campus, London SW154JD, UK ac National Institute of Polar Research, Tachikawa, Tokyo 190-8518, Japan ad National Institute of Water & Atmospheric Research, 301 Evans Bay Parade, Hataitai, Wellington, New Zealand 6021 ae Marine Mammal Institute, Oregon State University, 2030 SE Marine Science Dr., Newport, OR 97365, USA af Faculty of Fisheries Sciences, Hokkaido University, Minato-cho 3-1-1, Hakodate 041-8611, Japan ag Ligue de la Protection des Oiseaux Fonderies Royales CS90263 17305 Rochefort France British Antarctic Survey, Natural Environment Research Council, High Cross, Madingley Road, Cambridge CB3 0ET, UK RSPB Centre for Conservation Science, Royal Society for the Protection of Birds, The David Attenborough Building, Pembroke Street, Cambridge CB2 3QZ s Korea Polar Research Institute (KOPRI), 26, Songdomirae-ro, Yeonsu-gu, Incheon 21990, Republic of Korea National Institute of Water & Atmospheric Research, PO Box 8602, Riccarton, Christchurch 8011, New Zealand aa Atmosphere and Ocean Research Institute, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa City, Chiba Prefecture 277-8564, Japan ab Lif S i U i i f R h Whi l d C L d SW154JD UK National Institute of Polar Research, Tachikawa, Tokyo 190-8518, Japan ad National Institute of Water & Atmospheric Research, 301 Evans Bay Parade, Hataitai, Wellington, New Zealand 6021 ae Marine Mammal Institute Oregon State University 2030 SE Marine Science Dr Newport OR 97365 USA ad National Institute of Water & Atmospheric Research, 301 Evans Bay Parade, Hataitai, Wellington, New Zealand 6 ae Marine Mammal Institute, Oregon State University, 2030 SE Marine Science Dr., Newport, OR 97365, USA af Faculty of Fisheries Sciences, Hokkaido University, Minato-cho 3-1-1, Hakodate 041-8611, Japan ty of Fisheries Sciences, Hokkaido University, Minato-cho 3-1-1, Hakodate 04 ag Ligue de la Protection des Oiseaux, Fonderies Royales, CS90263, 17305 Rochefort, France * Corresponding author at: cE3c Center for Ecology, Evolution and Environmental Changes & CHANGE - Global Change and Sustainability Institute, Department of Animal Biology, Faculdade de Ciˆencias, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal. Available online 9 March 2023 0006-3207/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.biocon.2023.109994 Received 25 March 2022; Received in revised form 13 February 2023; Accepted 24 February 2023 Biological Conservation 281 (2023) 109994 Contents lists available at ScienceDirect Biological Conservation journal homepage: www.elsevier.com/locate/biocon i An update to this article is included at the end Biological Conservation 281 (2023) 109994 Contents lists available at ScienceDirect Biological Conservation journal homepage: www.elsevier.com/locate/biocon i An update to this article is included at the end i An update to this article is included at the end Quantifying annual spatial consistency in chick-rearing seabirds to inform important site identification Martin Beal a,b,*, Paulo Catry a, Richard A. Phillips c, Steffen Oppel d, John P.Y. Arnould e, Maria I. Bogdanova f, Mark Bolton g, Ana P.B. Carneiro b, Corey Clatterbuck h,i, Melinda Conners j,k, Francis Daunt f, Karine Delord k, Kyle Elliott l, Aymeric Fromant e, Jos´e Pedro Granadeiro m, Jonathan A. Green n, Lewis Halsey o, Keith C. Hamer p, Motohiro Ito q, Ruth Jeavons r, Jeong-Hoon Kim s, Nobuo Kokubun t, Shiho Koyama u, Jude V. Lane r,v,1, Won Young Lee s, Sakiko Matsumoto u, Rachael A. Orben w, Ellie Owen x, Vitor H. Paiva y, Allison Patterson l, Christopher J. Pollock r, Jaime A. Ramos y, Paul Sagar z, Katsufumi Sato aa, Scott A. Shaffer h, Louise Soanes ab, Akinori Takahashi ac, David R. Thompson ad, Lesley Thorne k, Leigh Torres ae, Yutaka Watanuki af, Susan M. Waugh ag, Henri Weimerskirch k, Shannon Whelan l, Ken Yoda u, Jos´e C. Xavier y, Maria P. Dias b,ah,1 Marine and Environmental Sciences Centre/ARNET – Aquatic Research Network, Ispa – Instituto Universit´ario de Ciˆencias Psicol´ogicas, Sociais e da Vida, ugal MARE – Marine and Environmental Sciences Centre/ARNET – Aquatic Research Network, Ispa – Instituto Universit´ario de Ciˆencias Psicol´ogicas, Sociais e isboa, Portugal MARE Marine and Environmental Sciences Centre/ARNET Aquatic Research Network, Ispa Instituto Universitario de Ciencias Psicologicas, Sociais e da Vida, Lisboa, Portugal b BirdLife International, The David Attenborough Building, Pembroke Street, Cambridge CB2 3QZ, UK c British Antarctic Survey, Natural Environment Research Council, High Cross, Madingley Road, Cambridge CB3 0ET, UK d RSPB Centre for Conservation Science, Royal Society for the Protection of Birds, The David Attenborough Building, Pembroke Street, Cambridge CB2 3QZ, UK e School of Life and Environmental Sciences, Deakin University, 221 Burwood Highway, Burwood, VIC 3125, Australia f UK Centre for Ecology & Hydrology, Bush Estate, Penicuik EH26 0QB, UK g RSPB Centre for Conservation Science, Royal Society for the Protection of Birds, 10, Albyn Terrace, Aberdeen AB10 1YP, UK h Biological Sciences, San Jose State University, One Washington Square, San Jose, CA 95192-0100, USA i California State Water Resources Control Board Office of Information Management and Analysis, 1001 I Street, 19th Floor, Sacramento, CA 95814, USA j Long Marine Laboratory, University of California, Santa Cruz, 100 Shaffer Rd., Santa Cruz, CA. 1. Introduction important sites (e.g., three years for KBAs; KBA Standards and Appeals Committee, 2020). However, whether such universal thresholds are appropriate for different species and environmental contexts is uncer­ tain. Given the substantial financial and labor costs involved in tracking animals in remote locations, understanding whether multi-year sam­ pling is necessary to identify stable sites of importance can help ensure that the potential of available tracking data for informing conservation is realized (Canessa et al., 2015; Williams et al., 2020). To date, few studies have compared annual consistency in population-level space use between seabird species, limiting our understanding of the relative importance of multi-year sampling for informing conservation planning (but see Arcos et al., 2012; Carpenter-Kling et al., 2020; Evans et al., 2021). The accurate estimation of the spatial distributions of animal pop­ ulations is important for understanding patterns of resource use and demographic change, as well as for informing biodiversity conservation and management (Hays et al., 2019). The at-sea distributions of many species of marine megafauna have in recent decades been revealed using data from animal-borne tracking devices (Bernard et al., 2021; Hussey et al., 2015). To ensure population-level inferences are robust and spatial management is properly targeted, it is vital to consider how representative is a tracking dataset of the movements of the entire population across both space and time (Shimada et al., 2020). In the marine realm, tracking data have been used widely to inform conservation and management (Davies et al., 2021; Hays et al., 2019; Hindell et al., 2020). Population-level spatial distributions derived from tracking data have contributed to assessments of the impacts of threats at sea, such as incidental mortality (bycatch) in fisheries, overfishing, and resource extraction (Clay et al., 2019; Garthe et al., 2017; Gr´emillet et al., 2016; Queiroz et al., 2019). Sites contributing to the global persistence of species, such as Key Biodiversity Areas (KBAs), can now be identified for a wide diversity of marine taxa using tracking data and novel analytical tools (Beal et al., 2021). It is important to consider, however, that if key sites are identified using tracking samples that do not fully encompass natural variability in space use, the resulting bor­ ders may not adequately represent the areas on which a population depends, potentially increasing exposure to risks elsewhere (Lovvorn et al., 2014). Quantifying annual spatial consistency in chick-rearing seabirds to inform important site identification E-mail address: martinbeal88@gmail.com (M. Beal). Available online 9 March 2023 0006-3207/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Biological Conservation 281 (2023) 109994 M. Beal et al. ah cE3c Center for Ecology, Evolution and Environmental Changes & CHANGE - Global Change and Sustainability Institute, Department of Animal Biology, Faculdade de Ciˆencias, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal A R T I C L E I N F O Keywords: Biotelemetry Animal tracking Area-based conservation Protected areas Marine spatial planning Key biodiversity areas Sampling effort Spatial consistency Animal tracking has afforded insights into patterns of space use in numerous species and thereby informed area- based conservation planning. A crucial consideration when estimating spatial distributions from tracking data is whether the sample of tracked animals is representative of the wider population. However, it may also be important to track animals in multiple years to capture changes in distribution in response to varying envi­ ronmental conditions. Using GPS-tracking data from 23 seabird species, we assessed the importance of multi-year sampling for identifying important sites for conservation during the chick-rearing period, when seabirds are most spatially constrained. We found a high degree of spatial overlap among distributions from different years in most species. Multi-year sampling often captured a significantly higher portion of reference distributions (based on all data for a population) than sampling in a single year. However, we estimated that data from a single year would on average miss only 5 % less of the full distribution of a population compared to equal-sized samples collected across three years (min: −0.3 %, max: 17.7 %, n = 23). Our results suggest a key consideration for identifying important sites from tracking data is whether enough individuals were tracked to provide a representative es­ timate of the population distribution during the sampling period, rather than that tracking necessarily take place in multiple years. By providing an unprecedented multi-species perspective on annual spatial consistency, this work has relevance for the application of tracking data to informing the conservation of seabirds. 1 Present address. 1. Introduction i Here, we analyzed movement tracks from 23 seabird species to investigate the importance of annual variability in space use for area- based conservation. We analyzed the distributions of each species dur­ ing chick-rearing, as this is the time of year when seabirds are most spatially restricted and therefore when area-based management mea­ sures can be particularly effective (Oppel et al., 2018). Indeed, tracking data collected during chick-rearing are often used to identify priority areas for conservation (Dias et al., 2018; Handley et al., 2021; Heerah et al., 2019). To estimate the average degree of consistency across years for each species, we quantified the spatial similarity between distribu­ tions from different years, and explored whether taxonomy, type of foraging habitat, and latitude could explain inter-specific variation therein. We investigated the influence of sampling regimes on important site identification by performing two resampling procedures. First, we held the number of tracked individuals constant and quantified the de­ gree to which tracking across multiple years can provide a fuller rep­ resentation of the population distribution compared to a sample from a single year. Second, for each species, we varied both the number of tracks and the number of years to understand the contribution of each sampling level to the population distribution. By assessing the relevance of spatial consistency across years for identifying important sites for seabirds differing widely in morphology and lifestyle, our results inform the design of future tracking studies and support ongoing efforts to improve area-based conservation planning at sea. The importance of tracking sufficient individuals to capture a stable picture of population-level space use has received considerable attention (Gutowsky et al., 2015; Hindell et al., 2003; Shimada et al., 2020; Soanes et al., 2013). However, another aspect pertinent to assessing the repre­ sentativeness of a tracking sample is the potential for a population to change distribution from year to year. A number of studies have inves­ tigated the importance of annual (among-year) variability in seabird foraging areas, reporting both minimal and substantial shifts (Bogda­ nova et al., 2014; Cerveira et al., 2020; Fromant et al., 2021; Meier et al., 2015; Osborne et al., 2020). Global standards for identifying important sites for biodiversity, such as the KBA Standard, account for annual variation by setting minimum thresholds for the number of years of distribution data that are required from the population to delineate Table 1 Table 1 Summary of GPS-tracking data used in this study to analyze annual consistency in the space use of seabirds. ‘n years’ indicates the number of years in which tracking data was collected, over a range of study years (i.e., ‘Year range’). ‘n birds’ refers to the median number of individuals tracked per year and ‘n trips’ to the median number of foraging trips recorded per year, with the ranges shown in parentheses. Species are arranged alphabetically by taxonomic order (Charadriiformes to Suliformes) and family. 2. Materials and methods We compiled Global Positioning System (GPS) tracking data using 2 M. Beal et al. Biological Conservation 281 (2023) 109994 several selection criteria to ensure comparability across species and sufficient sample sizes to allow for rigorous testing of sampling effects (Table 1). We solicited datasets meeting the following criteria: at least four years of GPS data with a minimum of 10 birds tracked in each year, where all birds were tracked from the same breeding colony and during the chick-rearing stage (alias ‘chick-provisioning’). In total, we collated tracking data for 23 species, representing seven of 14 families, and four of seven orders of seabirds. Collated tracking data came from 3 to 6 different breeding years for each colony, and sampled years were 1–17 years apart (median difference 3 years; Table 1). For two species, Aus­ tralasian gannet (Morus serrator) and little penguin (Eudyptula minor), we had sufficient data from two colonies; for all other species, we analyzed data from a single colony (Table 1). When producing popula­ tion distributions (see Population distribution estimation section), we treated each species-colony dataset separately, and then took the mean across colonies when comparing species-level metrics (e.g., annual overlap). Although all datasets were from breeding adults during chick- rearing, the relative timing in terms of the age of the brood (i.e., whether adults were brooding chicks or not) differed in some cases between species (see Table S2 for breeding-stage coverage). Nevertheless, for 104 out of 106 annual datasets (98 %), tracking was initiated during the first half of the chick-rearing period. 2.2. Tracking data standardization We cleaned and filtered tracking datasets for each species and study colony to improve comparability. First, we applied speed filters, with thresholds set to include only biologically realistic travel speeds (Table S1; Adams and Flora, 2010; Baylis et al., 2019). Then, using the R package track2KBA (Beal et al., 2021), we split tracks from individual birds into discrete foraging trips, which we defined as periods of a minimum duration spent outside a spatial buffer around the breeding Table 1 Beal et al. Biological Conservation 281 (2023) 109994 larger than the maximum range, or under-smooth the data and result in no overlapping use-areas. To achieve a similar degree of smoothing for each species, and thereby make overlap estimates comparable, we calculated the reference smoothing parameter (href), which reflects the number of positions and their spatial variance in the X and Y directions (i.e., longitude and latitude) and is a typical smoother used for identi­ fying important sites for biodiversity (Beal et al., 2021). To check for smoothing values that were outliers (and thereby result in inflated or underestimated distributions), we fitted a second-order polynomial function to smoothing value vs. species rank, ordered by foraging range (calculated as the median of the maximum distance from the colony for each trip). Next, for species with an href value that deviated >5 km from the value predicted by the model, the prediction was used instead of the actual values to set the smoothing parameter systematically, i.e., relative to the ranked scale of movement of each species (Fig. S2). We assessed whether the parameter selection process imposed a pattern on the re­ sults by including the smoothing parameter in a linear mixed-model framework along with the various factors that might explain annual consistency as predictors (see Section 2.4.1, Fig. S7). colony. Tracking data for each population were inspected to set an appropriate buffer radius that would exclude GPS-locations in the vi­ cinity of the colony, likely representing periods spent at the nest, resting on land, or rafting nearby (Fig. S3). To further standardize comparisons and reduce the effect of extended tracking of some species and not others, we only analyzed data from trips initiated within the first two weeks of device deployment for each individual bird. We used a threshold of two weeks to reduce any effects of advancing season on space use and to minimise the likelihood of including data for the same individual when its breeding stage or status had changed (e.g., as it transitioned from early to late chick-rearing). Tracking data were originally collected at varying sampling intervals (Fig. S1), so to improve comparability, we regularized data to a 10 min interval via linear interpolation using the R package adehabitatLT (Cal­ enge, 2006). Table 1 Common name Scientific name Family Order Breeding site Year range n years n birds n trips Common murre Uria aalge Alcidae Charadriiformes Colonsay, Scotland 2011–2014 3 12 (10–16) 35 (21–45) Thick-billed murre Uria lomvia Alcidae Charadriiformes Coats Island, Canada 2010–2019 5 55 (18–55) 136 (45–252) Black-legged kittiwake Rissa tridactyla Laridae Charadriiformes Bempton Cliffs, England 2010–2015 4 14 (13–16) 34 (22–44) Wandering albatross Diomedea exulans Diomedeidae Procellariiformes Bird Island, South Georgia 2002–2019 5 25 (11–34) 30 (12–40) Laysan albatross Phoebastria immutabilis Diomedeidae Procellariiformes Midway Atoll, United States 2013–2018 4 11 (10–25) 12 (11–27) Black-footed albatross Phoebastria nigripes Diomedeidae Procellariiformes Midway Atoll, United States 2013–2018 3 13 (12−23) 13 (13–26) Buller’s albatross Thalassarche bulleri Diomedeidae Procellariiformes The Snares, New Zealand 2008–2011 4 20 (11−23) 20 (11–27) Grey-headed albatross Thalassarche chrysostoma Diomedeidae Procellariiformes Bird Island, South Georgia 2009–2020 4 28 (24–30) 29 (25–33) Black-browed albatross Thalassarche melanophris Diomedeidae Procellariiformes Bird Island, South Georgia 2009–2020 5 27 (24–30) 33 (29–59) Cory’s shearwater Calonectris borealis Procellariidae Procellariiformes Selvagem Grande, Portugal 2009–2018 6 25 (12–41) 35 (22–89) Cape Verde shearwater Calonectris edwardsii Procellariidae Procellariiformes Raso Islet, Cabo Verde 2015–2019 4 28 (12–34) 149 (102–160) Streaked shearwater Calonectris leucomelas Procellariidae Procellariiformes Awashima Island, Japan 2013–2016 4 40 (26–42) 211 (129–243) Common diving- petrel Pelecanoides urinatrix Procellariidae Procellariiformes Procellariiformes Kanowna Island, Australia 2017–2020 4 13 (11–38) 25 (13–76) Westland petrel Procellaria westlandica Procellariidae Procellariiformes Punakaiki, New Zealand 2012–2017 3 14 (12–16) 20 (18–26) Little penguin Eudyptula minor Spheniscidae Sphenisciformes Gabo Island, Australia 2011–2016 5 28 (20–52) 32 (21–54) Little penguin Eudyptula minor Spheniscidae Sphenisciformes London Bridge, Australia 2011–2015 5 27 (10–43) 34 (11–77) Chinstrap penguin Pygoscelis antarcticus Spheniscidae Sphenisciformes King George Island, South Shetland 2006–2014 3 18 (11–19) 18 (11–19) European shag Gulosus aristotelis Phalacrocoracidae Suliformes Isle of May, Scotland 2003–2014 6 22 (10−33) 198 (22–314) Pelagic cormorant Urile pelagicus Phalacrocoracidae Suliformes Middleton Island, United States 2006–2018 4 20 (14–22) 166 (84–273) Northern gannet Morus bassanus Sulidae Suliformes Bass Rock, Scotland 2015–2019 5 21 (13−31) 109 (34–139) Australasian gannet Morus serrator Sulidae Suliformes Point Danger, Australia 2012–2018 4 20 (10–28) 36 (10–56) Australasian gannet Morus serrator Sulidae Suliformes Pope’s Eye, Australia 2011–2015 4 19 (14–38) 94 (19–179) Masked booby Sula dactylatra Sulidae Suliformes Dog Island, Anguilla 2014–2016 3 22 (19–68) 87 (69–137) Brown booby Sula leucogaster Sulidae Suliformes Dog Island, Anguilla 2012–2015 4 29 (19–41) 127 (67–189) Red-footed booby Sula sula Sulidae Suliformes Europa Island, French Scattered Islands 2003–2013 4 23 (11–37) 29 (11–116) 3 M. Table 1 Although many year-datasets were of a higher temporal resolution, we used a standard 10 min interval to ensure that most datasets (102 of 105 [98 %]) had a ratio of interpolated to raw points below 2:1. European shag (Gulosus aristotelis) and pelagic cormorant (Urile pelagicus) perform short foraging trips and therefore interpolation to 10 min would result in too few location estimates (<10) to run kernel density estimation for >5 % of all foraging trips; to avoid losing these shorter-duration trips, we instead interpolated data for these species to 5 min intervals. For each species and colony, we produced single-year population distributions by averaging together, with equal weighting, trip UDs from all birds tracked in each year (Fig. 1, Fig. S3). If multiple trips were available for each individual, we randomly selected a single trip UD per individual and year. This process of selecting a trip per individual was re-iterated in each analysis to maximize usage of available information while accounting for potential pseudoreplication (see Overlap analyses section for details; Lascelles et al., 2016). We then generated multi-year reference distributions (hereafter the ‘reference distribution’) for each species by averaging together single-year distributions across years (range 3 to 6 single years). We averaged single-year distributions (i.e., took the mean of grid cell probability densities), rather than combine all trip UDs from across the multi-year dataset, to avoid years with higher sample sizes contributing disproportionately to the shape of the refer­ ence distribution (Fig. 1). 2.3. Population distribution estimation To estimate space use, we used kernel density estimation (KDE) to derive utilization distributions (UD) for each foraging trip (hereafter referred to as ‘trip UDs’), which is a method often used when analyzing tracking data to inform conservation (Beal et al., 2021; Lascelles et al., 2016; Soanes et al., 2016). Determining an appropriate smoothing parameter, or bandwidth, is an important step in KDE, as it determines the scale at which the data points are smoothed. When comparing spe­ cies that move at similar scales, authors often recommend using the same parameter value, to avoid introducing differences as an artifact of processing (Carneiro et al., 2020). However, the movement scales of the species in this study ranged from <5 km to >700 km in terms of maximum range from the colony. Therefore, applying a standard smoothing value across species would either over-smooth data at a scale 2.4. Overlap analyses 2.4.1. Annual consistency To estimate the degree of annual consistency in space use for each 2.4. Overlap analyses 2.4.1. Annual consistency To estimate the degree of annual consistency in space use for each 2.4.1. Annual consistency Fig. 1. Utilization distributions (UDs; % kernel density estimations) of Cory’s Shearwaters from Selvagem Grande Island, Portugal (yellow diamond) tracked during the chick-rearing period in different years, (A) all years combined, and (B) years separate. The multi-year reference distribution (A) is the average of the single-year UDs (B), based on a single trip from each bird. Each single-year distribution is the average of the trip UDs of individual birds. Values in the bottom right-hand corner represent the number of birds tracked in each year. This visualization represents one iteration of the re-sampling process used to avoid pseudoreplication while maximizing use of available data. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Fig. 1. Utilization distributions (UDs; % kernel density estimations) of Cory’s Shearwaters from Selvagem Grande Island, Portugal (yellow diamond) tracked during the chick-rearing period in different years, (A) all years combined, and (B) years separate. The multi-year reference distribution (A) is the average of the single-year UDs (B), based on a single trip from each bird. Each single-year distribution is the average of the trip UDs of individual birds. Values in the bottom right-hand corner represent the number of birds tracked in each year. This visualization represents one iteration of the re-sampling process used to avoid pseudoreplication while maximizing use of available data. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 4 M. Beal et al. Biological Conservation 281 (2023) 109994 latitude (centroid of at-sea locations). The full model specification and results of this analysis are located in the Supplementary Materials. latitude (centroid of at-sea locations). The full model specification and results of this analysis are located in the Supplementary Materials. population, we calculated the spatial overlap between all pairwise combinations of single-year distributions (utilization distributions derived from all tracked individuals in each year) (Table 2). Overlap was calculated using the Bhattacharyya’s Affinity (BA) and Volume of Intersection (VI) indices, which both provide probabilistic measures of UD similarity, ranging from 0 (no overlap) to 1 (identical UDs) (Fieberg and Kochanny, 2005). We report the mean BA index values for the main analysis, as this is the recommended index for comparing UD similarity (Fieberg and Kochanny, 2005). 2.4.1. Annual consistency We then calculated the mean overlap for each pairwise comparison across iterations (Fig. S4), and plotted mean annual overlap by species, family and foraging habitat to illustrate intra- and inter-specific variation in consistency. We classified species as foraging in predominantly ‘shelf’, ‘oceanic’ or ‘mixed’ habitat by inspecting the distribution of foraging trips of each population overlaid on bathymetry (see Supplement for details and Table S1 for classification). i = m −2 to ensure that many more than m combinations of trip UDs were possible. For example, if a species had four years of data, with yearly sample sizes of 10, 12, 14, and 16 birds, then the third highest year- sample would be m = 12 individuals, and n = 10 trip UDs would be drawn per iteration, allowing for a total of 66 unique combinations of trip UDs to be drawn. The third-highest sample size was used (as opposed to the first- or second-highest) to ensure that at least three different years could contribute data when drawing data from a single year. In each iteration, we calculated the percentage of the 50 % and 95 % UD areas of the reference distribution covered by sample UDs of the same level (i.e., 50 % and 95 % sample UDs). For this analysis, we calculated simple spatial overlap (i.e., direc­ tional measure of the percent spatial coverage of the reference distri­ bution by a sample distribution) to ensure the effect sizes were interpretable in terms of area, which is more useful for the practicalities of management than probability values (e.g., BA or VI indices). Sample distributions were generated and overlap calculated over 100 iterations, wherein trip UDs were randomly re-sampled in each iteration. We used Tukey HSD post-hoc tests (of ANOVAs) to test whether sample distri­ butions based on data drawn from a single year covered a different amount of the reference distribution compared with distributions based on samples from two or three years. 2.4.1. Annual consistency As VI integrates over the minimum probability density of each cell between the two UDs being compared, overlap values are generally lower than BA (Kochanny et al., 2009). We used the mean VI overlap values to validate whether the relative dif­ ferences between species were sensitive to the chosen index. 2.4.2. Single-year vs. multi-year sampling To test whether sampling across years provides more robust esti­ mates of population distributions than sampling in a single year, we compared the spatial coverage of reference distributions (i.e. distribu­ tions of each population estimated using data from all individuals and years) with samples of equal size (i.e., the same number of individuals) drawn from varying numbers of years (Table 2). For this analysis, we limited comparisons to a maximum of three years to ensure compara­ bility across species. We iteratively re-sampled n trip UDs from one, two, or three years, and averaged them together to form a sample distribu­ tion. The number n was determined by the third-highest number of in­ dividuals tracked in any given year (m) for each species, and was set at n = m −2 to ensure that many more than m combinations of trip UDs were possible. For example, if a species had four years of data, with yearly sample sizes of 10, 12, 14, and 16 birds, then the third highest year- sample would be m = 12 individuals, and n = 10 trip UDs would be drawn per iteration, allowing for a total of 66 unique combinations of trip UDs to be drawn. The third-highest sample size was used (as opposed to the first- or second-highest) to ensure that at least three different years could contribute data when drawing data from a single year. In each iteration, we calculated the percentage of the 50 % and 95 % UD areas of the reference distribution covered by sample UDs of the same level (i.e., 50 % and 95 % sample UDs). The trip UDs contributing to each single-year distribution were randomly re-sampled across 100 iterations (i.e., one trip selected per individual per iteration), to ensure that different trips from a given in­ dividual would be included, and pairwise annual overlap calculated. For datasets with fewer than 50 unique combinations of trip UDs, unique sets of UDs were determined for each iteration (e.g., with only 1 trip per bird, only 1 iteration was run). Table 2 Analysis workflow table illustrating the three analyses of spatial overlap conducted in this study. Each column corresponds to an analysis based on the overlap of spatial distributions of 23 species of GPS-tracked seabirds during the chick-rearing period. In each analysis, pairwise spatial overlap was calculated between distributions using either a probabilistic (Bhattacharyya’s Affinity and Volume of Intersection) or Euclidean (% coverage) metric of overlap. In the Annual consistency analysis, single-year distributions of each population were compared, each of which included foraging trips from all birds tracked in each year. In the Single-year vs. multi-year sampling analysis, sample distributions were created by drawing varying numbers of trips from the full tracking sample and then overlaid on the multi-year reference distribution of each population to assess the degree of coverage. In N tracks vs. N years, both the number of trips and number of years was varied when forming sample distributions, which were again compared to the reference distribution (based on data from all birds and years). In all three analyses, spatial distributions (single-year, sample, and reference) were estimated by randomly selecting a single foraging trip from each individual bird, running kernel density estimation with the selected trips, and repeating this process over 100 iterations. p g p Annual consistency Single-year vs. multi-year sampling N tracks vs. 2.4.1. Annual consistency We report the mean model effect size (i.e., average difference in the percentage of the reference distri­ bution covered by two and three years of data) for species-years, which represents the predicted spatial information gained by sampling a i We fitted a linear mixed-effects model using the R package lme4 (Bates et al., 2015) to evaluate which predictor variables explained significant amounts of variation in mean overlap among years, with variables either representing (1) experimental design choices (i.e., smoothing parameter definition, time lags), or (2) biological factors (habitat type, latitude, taxonomy). As we lacked sufficient data to properly control for phylogeny in this analysis, the purpose was not to predict distributional consistency across seabirds in general, but to explain the variation in our dataset. We specified random intercepts for each species and site to account for foraging patterns specific to species or locations, and evaluated the following fixed factors: smoothing parameter, lag (time difference between single-year distributions), taxonomic family, foraging habitat (oceanic, shelf, mixed), and foraging Table 2 N years Aim Quantify spatial similarity of population distributions across years Test how well samples drawn from differing numbers of years capture the full population distribution Illustrate relative contribution of sample size and study duration to capturing the full population distribution Overlap index Bhattacharyya’s Affinity (BA); Volume of Intersection (VI) % spatial coverage % spatial coverage What is being overlapped Single-year distribution Single-year distribution Sample distribution Multi-year reference distribution Sample distribution Multi-year reference distribution N trips underlying distributions 1 per bird, for all birds per year 1 per bird, for all birds per year 1 per bird through third- lowest sample size among years 1 per bird for all birds 1 per bird through lowest sample size among years 1 per bird for all birds N years underlying distributions 1 1 Varies; 1, 2 or 3 All years Varies: 1, 2, …all years All years Method of inference Linear mixed-model to assess contribution of design choices and biological factors to spatial consistency estimates ANOVA (and post-hoc tests) to test for differences in amount of full (reference) distribution captured by samples drawn from differing number of years Visualize the contribution of changing the number of birds and number of years to capturing the full (reference) distribution Linear mixed-model to assess contribution of design choices and biological factors to spatial consistency estimates 5 M. Beal et al. Biological Conservation 281 (2023) 109994 sizes (i.e., 1 to n birds) and from differing numbers of years (i.e., 1 to Nyears), as this indicates whether annual variability influences the esti­ mation of population distributions. population across years. 2.4.3. Number of tracks vs. number of years 2.4.3. Number of tracks vs. number of years We varied both the sample size (i.e., number of birds tracked) and the number of years from which the sample was drawn to illustrate the relative importance of each sampling level for capturing the multi-year reference distribution (Table 2). We used the same re-sampling pro­ cedure as in Single-year vs. multi-year sampling to calculate percentage of overlap between sample and reference distributions, and iterated the process 100 times at each sample size to include different trip UD combinations. The maximum number of birds drawn for a species was again capped at n = m −2, but in this case m was set at the smallest sample size available among year-samples to be able to visualize the relationship up to the maximum number of years available. For example, for a species with four years of data and yearly sample sizes of 10, 12, 14, and 16 birds, sub-samples up to 8 birds would be iteratively selected, averaged together, and overlapped with the reference distribution. Both here and in Single-year vs. multi-year sampling the proximity to 100 % coverage of the reference distribution is a measure of sample represen­ tativeness. However, the focus of these analyses is rather on the differ­ ence in coverage of the full distribution provided by samples of varying We visualized the percent coverage relationship for each species and site to provide guidance on the information gained by tracking pop­ ulations across a varying number of years. We present three examples from species which showed contrasting degrees of consistency and provide the results for the remaining species in the Supplement. 3. Results 3.1.1. Annual consistency Estimates of annual consistency measured by BA overlap among single-year distributions were generally high (Fig. 2A), with 22 of 23 species falling between 0.69 (mean, SD 0.1) for Pelagic Cormorant (Urile pelagicus), and 0.93 (mean, SD 0.02) for Chinstrap Penguin (Pygoscelis antarcticus); Common Diving Petrel (Pelecanoides urinatrix) was an outlier, with a mean consistency estimate of 0.56 (SD 0.27) (Fig. 2A). As 6 Fig. 2. Annual spatial consistency of 23 species of seabirds from 7 families during the chick- rearing period. Consistency was estimated as the degree of spatial overlap between pairwise combinations of single-year utilization distribu­ tions using the Bhattacharyya’s Affinity (BA) index. (A) Species were ranked according to their mean level of annual consistency. Dots represent the mean overlap across 100 iterations of re-sampling for each pairwise year compari­ son, in each of which a single foraging trip was selected per individual to derive each single- year population distribution. Boxes represent the distribution of overlaps calculated across all pairwise combinations of years. The numbers above each box indicate the number of years of tracking data from each species and population. Values for little penguin and Australasian gannet are based on tracking data from two populations (each with same number of years tracked). (B) Estimates of annual spatial consis­ tency aggregated by family and predominant foraging habitat of each species. Box height in (B) relates to the variation in annual consistency in at-sea distributions within and between spe­ cies in each family, with the number of species indicated above the box. M. Beal et al. Biological Conservation 281 (2023) 109994 expected, overlap values calculated using the VI index were lower, with species-level mean consistency estimates ranging from 0.35 (SD 0.22) to 0.74 (SD 0.06) (Fig. S5). The species ranks were very similar between the BA and VI indices, with a significant positive correlation in species ranks ordered by mean overlap (Spearman rank correlation: S = 60, rho = 0.97, p < 0.001, Fig. S6). single year or multiple years differed in the percent coverage of the multi-year reference distribution. We found that multi-year sample distributions covered a significantly higher percentage of reference distributions in 17 and 16 species (n = 23 species) for 95 % and 50 % UD areas, respectively (Fig. 3, Table S4). 3.1.1. Annual consistency For 95 % sample UDs, the mean difference in coverage across species was 5.0 % and ranged from a species mean of −0.3 % in masked boobies (Sula dactylatra) to 17.7 % in common diving petrels. For 50 % sample UDs, the mean difference in coverage was 6.1 %, and ranged from −1.0 % in common murre (Uria aalge) to 21.7 % in common diving petrels (Table S4). Three species, wandering albatross (Diomedea exulans), common murre and streaked shearwater (Calonectris leucomelas) showed no significant difference in coverage between single-year and multi-year samples for either 95 % or 50 % sample UDs, and in six other species, Laysan albatross (Phoebastria immutabilis), Buller’s albatross (Thalassarche bulleri), chinstrap penguin (Pygoscelis antarcticus), Westland petrel (Procellaria westlandica), masked booby (S. dactylatra) and red-footed booby (S. sula), there were only differences for either the 95 % or the 50 % sample UDs (Fig. 3, Tables S4–S5), but not both. Our GLMM exploring which factors explained variation in annual consistency showed that the smoothing parameter did not affect the population-level estimate of consistency (Fig. S7, Table S3). Time lag between years was negatively related to consistency; however, the effect size was marginal relative to within- and among-species variation at the time scales (1–4 years) analyzed for most species (Fig. S8). Despite differences between families in terms of consistency (Phalacrocoracidae significantly lower than Spheniscidae, Table S3), within-family varia­ tion (i.e., combination of inter- and intra-specific components) was greater than between-family variation (Fig. 2B). There was no effect of the predominant foraging habitat type on annual consistency (Fig. 2B, Table S3). Foraging latitude was positively related to consistency; however, the effect size was also marginal and the latitudinal range covered within most families was limited (Supplementary Methods, Fig. S9, Table S3). 3.2. Single-year vs. multi-year sampling For all 23 species, increasing the number of birds tracked increased the coverage of the multi-year reference distribution (Fig. 4, see sup­ plementary Figs. S10–S31 for all species). Sample distributions based on the smallest sample size available among years for each species covered Using ANOVA post-hoc tests, we assessed whether distributions derived from samples of the same number of individuals drawn from a Fig. 3. Comparison of the degree to which tracking samples from differing numbers of years capture full multi-year population distributions for 23 seabird species. For each species, a certain number of foraging trips, indicated by the number above the x-axis, were re-sampled 100 times. In each run, a sample distribution was derived, by averaging together the selected trip utilization distributions, and overlapped with a reference distribution made from all tracked birds and years of tracking data. Tukey HSD post-hoc tests of ANOVAs were used to test whether sample distributions from a single year (N year = 1) differed in their proportional cover of the reference distribution compared to samples drawn from multiple years (N year = 2 or 3); bold text at the top of each panel indicates where at least one test comparison was significant (1–2 y or 1–3 y), with the value indicating the mean effect size between comparisons. Species were ranked according to the mean effect size for the 50 % utilization distribution. Panels correspond to the coverage for 95 % (upper) and 50 % (lower) utilization distributions. Fig. 3. Comparison of the degree to which tracking samples from differing numbers of years capture full multi-year population distributions for 23 seabird species. For each species, a certain number of foraging trips, indicated by the number above the x-axis, were re-sampled 100 times. In each run, a sample distribution was derived, by averaging together the selected trip utilization distributions, and overlapped with a reference distribution made from all tracked birds and years of tracking data. Tukey HSD post-hoc tests of ANOVAs were used to test whether sample distributions from a single year (N year = 1) differed in their proportional cover of the reference distribution compared to samples drawn from multiple years (N year = 2 or 3); bold text at the top of each panel indicates where at least one test comparison was significant (1–2 y or 1–3 y), with the value indicating the mean effect size between comparisons. 3.2. Single-year vs. multi-year sampling By re-sampling the number of tracks (i.e., one per bird) and the number of years from which tracks were selected, the functional relationship between sample size and annual sampling was estimated for each species. The process of re-sampling tracks was iterated 100 times, and in each iteration the percentage to which the 95 % (top row) and 50 % (bottom row) probability areas of the resulting sample distribution covered the same quantile areas of a multi-year reference distribution was calculated. Reference distributions were the average of all single-year distributions derived using the full samples for each species. Points signify the mean spatial coverage across iterations, and bars denote the standard deviation. a mean of 62.8 % (min = 45.9 %, max = 79.6 %, n = 23) and 66.3 % (min = 40.3 %, max = 86.6 %, n = 23) of reference distributions based on all tracks for a species, for 95 % and 50 % UDs respectively. Addi­ tionally, for many species, the relationship between sample size and coverage appeared to begin levelling off, indicating that the sample sizes in our datasets captured a large portion of the population-level spatial distribution (Figs. S10–S31). Increasing the number of years in which the population was tracked added spatial information for most species (in addition to that gained by tracking more birds), but lacked a marked gain in some (Figs. S10–S31). As indicated by the estimates of average spatial overlap between years (Fig. 2A), the relative importance of sampling across years differed among species, with the benefit of multi- year samples most apparent for common diving petrels, and negligible for Northern gannets (M. bassanus), highlighted here to show the vari­ ation among species (Fig. 4) among the populations studied here. We found that tracking seabirds in multiple years generally improved estimates of population-level distri­ butions, although in most cases there was only a marginal loss of in­ formation about space use by the population if tracking data were only available for one year. These results indicate that, tracking samples deemed representative of the wider population during the sampling period can be useful for informing the area-based conservation of sea­ birds, even if only collected in a single year. 3.2. Single-year vs. multi-year sampling Species were ranked according to the mean effect size for the 50 % utilization distribution. Panels correspond to the coverage for 95 % (upper) and 50 % (lower) utilization distributions. Fig. 3. Comparison of the degree to which tracking samples from differing numbers of years capture full multi-year population distributions for 23 seabird species. For each species, a certain number of foraging trips, indicated by the number above the x-axis, were re-sampled 100 times. In each run, a sample distribution was derived, by averaging together the selected trip utilization distributions, and overlapped with a reference distribution made from all tracked birds and years of tracking data. Tukey HSD post-hoc tests of ANOVAs were used to test whether sample distributions from a single year (N year = 1) differed in their proportional cover of the reference distribution compared to samples drawn from multiple years (N year = 2 or 3); bold text at the top of each panel indicates where at least one test comparison was significant (1–2 y or 1–3 y), with the value indicating the mean effect size between comparisons. Species were ranked according to the mean effect size for the 50 % utilization distribution. Panels correspond to the coverage for 95 % (upper) and 50 % (lower) utilization distributions. 7 M. Beal et al. Biological Conservation 281 (2023) 109994 Fig. 4. Sampling effects on estimation of population-level distributions for three species of seabirds (left to right: common diving-petrel, grey-headed albatross, Northern gannet). By re-sampling the number of tracks (i.e., one per bird) and the number of years from which tracks were selected, the functional relationship between sample size and annual sampling was estimated for each species. The process of re-sampling tracks was iterated 100 times, and in each iteration the percentage to which the 95 % (top row) and 50 % (bottom row) probability areas of the resulting sample distribution covered the same quantile areas of a multi-year reference distribution was calculated. Reference distributions were the average of all single-year distributions derived using the full samples for each species. Points signify the mean spatial coverage across iterations, and bars denote the standard deviation. Fig. 4. Sampling effects on estimation of population-level distributions for three species of seabirds (left to right: common diving-petrel, grey-headed albatross, Northern gannet). 4.1. Effects of study duration Using GPS-tracking data, we estimated the spatial consistency of 23 seabird species during the chick-rearing period to assess the relevance of sampling across years for identifying important sites for conservation at the population level. Most species showed a similarly high degree of average consistency in their distributions between years. We also found that the number of individuals tracked had a large effect on the esti­ mation of population distributions, supporting previous findings in seabirds (Gutowsky et al., 2015; Soanes et al., 2013; Thaxter et al., 2017). By contrast, the time lag between years, taxonomic family, and latitude explained only small amounts of variation in annual consistency 3.2. Single-year vs. multi-year sampling When using tracking data to identify important sites for seabirds, our results suggest that a key consideration is whether the sample of tracked birds is representative of the population distribution during the sam­ pling period. If the tracking data available for a population are limited to one or two years, we further recommend using independent information to assess whether conditions were typical of the region and season. Important sites identified from data collected during periods of abnormal conditions have relevance for conservation, as they may represent places used when regular feeding areas are unprofitable (Bogdanova et al., 2014). However, in such cases, tracking data from additional years will likely be needed to also identify areas used under typical environmental conditions. 4.2. Temporal and spatial scale The datasets we analyzed were collected in 3 to 6 different years, sometimes in sequential years and in other cases collected >10 years apart (Table 1, Fig. S4). Climatic cycles, such as the El Ni˜no-Southern Oscillation, often operate at a decadal scale and are known to affect the spatial distributions of marine predators and their prey (Ballance et al., 2006; Philander, 1983). Therefore, it is possible that the sample of years for which we had tracking data for our study populations were 8 Biological Conservation 281 (2023) 109994 M. Beal et al. roughly every decade. Although standard thresholds, such as the KBA ‘three-year rule’, are designed to ensure temporal robustness of sites, more flexible solutions might better enable sites to be identified where they are needed, not just in countries where there is funding for long- term studies. For example, instead of requiring a certain number of years of tracking data be used, site identification guidelines could incorporate estimates of the representativeness of tracking samples at the population level, or the vulnerability of a population to extreme weather events such as heatwaves. Existing tools, such as the R packages track2KBA and SDLfilter, can be used to assess the representativeness of a tracking sample for the period in which it was collected (Beal et al., 2021; Shimada et al., 2020). Further, when the available tracking data are limited to one or two years, data on breeding success, or climato­ logical and oceanographic information could be used to evaluate whether conditions were anomalous. In practice, this could range from basing assessments on expert judgement to combining various data sources in an integrated analytical framework (Ventura et al., 2021). insufficient to fully capture distributional changes in response to envi­ ronmental cycles. To identify links between at-sea distribution, envi­ ronmental conditions, and demographic responses, it is necessary to monitor populations for the length of a generation, at minimum (Ven­ tura et al., 2021). Indeed, as tracking studies begin to extend over decadal scales, they are revealing responses of populations to shifts in climatic conditions (Bogdanova et al., 2014; Clark et al., 2021; Wei­ merskirch et al., 2014). Although such long-term monitoring is clearly useful and desirable, conservation is often constrained financially, by the duration of opportunities to implement effective management, and by the imminent nature of threats (Bolam et al., 2019). 4.4. Recommendations for future studies We analyzed tracking data from seabirds collected during the chick- rearing phase; therefore, a useful extension of this work would be to analyze distributions during other phases, including the pre-laying, in­ cubation and non-breeding periods, when the movements of many species are less constrained (Phillips et al., 2017). Lower movement constraints outside chick-rearing mean birds can travel further in search of food, potentially reducing spatial consistency between years. If spatial consistency varies across the annual cycle, then differing sample sizes of tracked birds would be needed to achieve representativeness, and ulti­ mately to identify important sites (Carneiro et al., 2020). Globally, tracking effort in seabirds is biased toward large-bodied and high-latitude species (Bernard et al., 2021; Mott and Clarke, 2018). Therefore, it is also important to include under-studied taxa (e.g., storm-petrels and terns) and regions (e.g., the tropics) in future analyses of spatial consistency in seabirds. The combination of datasets we analyzed contained insufficient species contrasts within and across taxonomic families to properly account for phylogenetic effects, limiting our ability to robustly identify general predictors of annual consistency in seabirds. As multi-year tracking data become available for more species, life-history classes and stages in the annual cycle, it will become possible to examine the effects of ecological drivers and seasonality in movement patterns in conjunction with intrinsic factors such as sex, age, status and breeding population (Carneiro et al., 2020; Jovani et al., 2016; Phillips et al., 2017). Finally, as tracking studies begin capturing decadal scales of movements, it will be important to identify the degree to which important at-sea sites remain relevant in the face of long-term climatic shifts (Weimerskirch et al., 2014). Spatial scale is a key parameter to consider in analyses of overlap (Winner et al., 2018). Here, we determined appropriate scales of anal­ ysis using a standard approach recommended for identifying important sites for biodiversity (i.e., the ‘href’ method; Beal et al., 2021), adapted to ensure comparability among species. Nevertheless, it is important to recognize that our results correspond to species-specific scales, and are thereby not universal reflections of the spatial consistency of seabirds in general. For analyses aimed at identifying important sites for a single population, other factors should also be considered, such as the size of local management units, potentially requiring the use of different smoothing parameter values or grid cell sizes than those we employed (Soanes et al., 2015). 4.4. Recommendations for future studies Further, for investigations focused on ecological questions, analyses at finer scales may uncover subtle shifts in space use at the population level (Evans et al., 2021; Warwick-Evans et al., 2016). 4.2. Temporal and spatial scale As such, prag­ matic approaches are often necessary to help decide how much tracking data are sufficient to inform decision-making (see Section 4.3). i A number of studies conducted at timescales comparable to those explored here identified shifts in space use at the population level in response to oceanographic variability (Bogdanova et al., 2014; Evans et al., 2021; Osborne et al., 2020). Indeed, the dataset we analyzed from common diving petrels was used to illustrate the effects of a marine heatwave on breeding success and at-sea distributions, explaining the low consistency and large effect of adding years of data we report for this species (Fromant et al., 2021). This specific case illustrates how tracking data collected over just a few years can provide important information about the areas used during stressful climatic events, when effective protection may be particularly important for populations (Bogdanova et al., 2014). In contrast, for black-legged kittiwakes we found only small changes in space use across four years for a population in England, during the same period in which a population in the Gulf of Alaska shifted their distribution to largely new areas in response to a local heatwave (Osborne et al., 2020). These contrasting patterns within the same species suggest that the spatial consistency of seabird populations can vary more due to local environmental conditions than taxonomy or general foraging behavior. Acknowledgements Baylis, A.M.M., Tierney, M., Orben, R.A., Warwick-Evans, V., Wakefield, E., Grecian, W. J., Trathan, P., Reisinger, R., Ratcliffe, N., Croxall, J., Campioni, L., Catry, P., Crofts, S., Boersma, P.D., Galimberti, F., Granadeiro, J.P., Handley, J., Hayes, S., Hedd, A., Brickle, P., 2019. Important At-Sea areas of colonial breeding marine predators on the southern patagonian shelf. Sci. Rep. 9 (1), 8517. https://doi.org/ 10.1038/s41598-019-44695-1. We thank all the fieldworkers involved in tracking data collection and collation. We recognize the many USFWS volunteers who contrib­ uted assistance in the field to monitor the North Pacific albatrosses, including but not limited to: Hoku Cody, Megan Dalton, Ann Humphrey, Hope Ronco, and Amanda Gladics. We thank Elizabeth Mackley, Chris Bell, Mara Nydegger, Alice MacMillan, Guy Anderson, David Aitken, Ruth Brown, AJ Bellamy, Katherine Snell, Emily Scragg and Tessa Coledale for data collection. Some of the fieldwork was undertaken with permission from the Government of Anguilla. We thank Sir Hew Hamilton-Dalrymple and the Scottish Seabird Centre, North Berwick for access to Bass Rock. This project has received funding from the Euro­ pean Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 766417. This communication reflects only the authors’ views, and the Research Ex­ ecutive Agency of the European Union is not responsible for any use that may be made of the information it contains. Funding and project support includes: FCT-Portugal through projects UIDB/04292/2020 and UIDP/ 04292/2020, granted to MARE and through the project LA/P/0069/ 2020 granted to the Associate Laboratory ARNET; Falklands Islands Government; the Ecosystems component of the British Antarctic Survey Polar Science for Planet Earth Programme, funded by the Natural Environment Research Council (NERC); Sea World Research and Rescue Foundation Inc.; Holsworth Wildlife Research Endowment; Winifred Violet Scott Trust; USFWS, NOAA, BOEM (M14AC00026), and Papah¯anaumoku¯akea Marine National Monument; UK-SCaPE pro­ gramme delivering National Capability (NE/R016429/1) funded by NERC; French Polar Institute (IPEV n◦109, resp H Weimerskirch); R´eserve Naturelle Nationale des Terres Australes Françaises and the Zones Atelier Antarctique (LTSER France, CNRS-INEE); GulfWatch Alaska; Natural Sciences and Engineering Research Council of Canada; Canada Research Chair in Arctic Ecology; BirdLife Australia, Deakin University; UK Darwin Plus (DPLUS0007); Leverhulme Early Career research grant (‘The curious case of asynchronous breeding in tropical seabirds’). The authors report no competing interests. Supplementary data to this article can be found online at https://doi. org/10.1016/j.biocon.2023.109994. Appendix A. Supplementary data The authors report no competing interests. References All non-positional data derivatives produced in this study are either available in the Supplementary Materials or are available in the associated Zenodo repository (https://doi.org/10.5281/zenodo.7692668). The tracking data used in these analyses may be downloaded via the Seabird Tracking Database (www.seabirdtracking.org), under the following accession codes: 1343, 1344, 1345, 1347, 1440, 1628, 460, 462, 1387, 1885, 1341, 1342, 1472, 1653, 1660, 1222, 1857, 1858, 1449, 1819, 927, 928, 930, 1144, 1145, 1147, 1148, 936, 938, 937, 618, 619, 620, 1383, 1382, 1173, 1174, or requested directly from the authors. Adams, J., Flora, S., 2010. Correlating seabird movements with ocean winds: linking satellite telemetry with ocean scatterometry. Mar. Biol. 157 (4), 915–929. https:// doi.org/10.1007/s00227-009-1367-y. Arcos, J.M., B´ecares, J., Villero, D., Brotons, L., Rodríguez, B., Ruiz, A., 2012. Assessing the location and stability of foraging hotspots for pelagic seabirds: an approach to identify marine important bird areas (IBAs) in Spain. Biol. Conserv. 156, 30–42. https://doi.org/10.1016/j.biocon.2011.12.011. l g j Ballance, L.T., Pitman, R.L., Fiedler, P.C., 2006. Oceanographic influences on seabirds and cetaceans of the eastern tropical Pacific: a review. Prog. Oceanogr. 69 (2), 360–390. https://doi.org/10.1016/j.pocean.2006.03.013. Bates, D., M¨achler, M., Bolker, B., Walker, S., 2015. Fitting linear mixed-effects models using lme4. J. Stat. Softw. 67 (1), 1–48. https://doi.org/10.18637/jss.v067.i01. i CRediT authorship contribution statement M.Be., S.O., R.A.P., P.C. and M.P.D. conceived the idea for the study; M.Be. developed the original methods, analysed the data and led the writing of the manuscript. J.P.Y.A., M.Bog., M.Bol., A.P.B.C., P.C., C.C., M.C., F.D., K.D., M.P.D., K.E., A.F., J.P.G., J.A.G., L.H., K.C.H., M.I., R.J., J.H.K., N.K., S.K., J.V.L., W.Y.L., S.M., R.A.O., E.O., V.H.P., A.P., R.A.P., C.J.P., J.A.R., P.S., K.S., S.A.S., L.S., A.T., D.R.T., L.Th., L.To., Y.W., S.M. W., H.W., S.W., K.Y., J.C.X., collected data and contributed to data pre- processing. All authors contributed critically to manuscript drafts and gave final approval for publication. 5. Conclusion As area-based management tools are being increasingly integrated into global marine conservation policy (De Santo, 2018), the criteria used to designate sites as important for biodiversity are becoming standardized across countries and taxa (e.g., the KBA program; IUCN, 2016). Our results for chick-rearing seabirds suggest that setting stan­ dard thresholds of the number of years of distribution data needed to identify sites offers little advantage in many cases, particularly where a representative sample of individuals has been tracked in typical condi­ tions. Given the high expense involved in tracking marine animals in remote locations, the requirement to track a population in three years may be impractical, and ultimately to the detriment of conservation if important sites might otherwise have been identified from data from one or two years, and protective measures put in place sooner. i We found high average levels of annual spatial consistency across a broad variety of seabirds during the chick-rearing period. In addition, we show that a vital consideration when applying tracking data to questions of area-based conservation is whether the sample of tracked individuals is representative at the population level. Our findings indi­ cate that tracking chick-rearing seabirds across years improves the estimation of at-sea spatial distributions. However, in most cases the information gain we found was marginal, suggesting that representative samples collected in one or two years are also useful for the identifica­ tion of important sites. When only one or two years of tracking data are available for important site identification, we further recommend using independent evidence to assess whether conditions were typical of the region and time of year. This work has relevance for the use of tracking data to inform marine area-based conservation, as the identification of important sites for seabirds is an integral step in the process of assessing threats at sea and designing an effective network of marine protected Sites identified for their importance to biodiversity are meant to be re-assessed over time (e.g., every 8–12 years for KBAs). This adds to the costs involved in identifying important sites by requiring three years of data be collected over a relatively short period and the process repeated 9 M. Beal et al. 5. Conclusion Biological Conservation 281 (2023) 109994 Department for Business, Energy & Industrial Strategy (BEIS) Offshore Energy Strategic Environmental Assessment research programme and the Natural Environment Research Council; the Japan Society for the Promotion of Science (13J06234, 20310016, JP16H06541, JP16H01769, JP21H05294); Korea Polar Research Institute (PG21040); Japanese Antarctic Program for Joint Research with Foreign Countries; European Regional Development Fund through its Atlantic Area Pro­ gramme; Marine Scotland; NatureScot; Natural England; Joint Nature Conservation Committee; Royal Society for the Protection of Birds; MAVA Foundation (MAVA17022), through the project Alcyon – Con­ servation of seabirds from Cabo Verde; LIFE + Berlenga (LIFE13 NAT/ PT/000458); New Zealand Department of Conservation; New Zealand Ministry for Primary Industries; National Science Foundation (award number 79804); and Museum of New Zealand. Department for Business, Energy & Industrial Strategy (BEIS) Offshore Energy Strategic Environmental Assessment research programme and the Natural Environment Research Council; the Japan Society for the Promotion of Science (13J06234, 20310016, JP16H06541, JP16H01769, JP21H05294); Korea Polar Research Institute (PG21040); Japanese Antarctic Program for Joint Research with Foreign Countries; European Regional Development Fund through its Atlantic Area Pro­ gramme; Marine Scotland; NatureScot; Natural England; Joint Nature Conservation Committee; Royal Society for the Protection of Birds; MAVA Foundation (MAVA17022), through the project Alcyon – Con­ servation of seabirds from Cabo Verde; LIFE + Berlenga (LIFE13 NAT/ PT/000458); New Zealand Department of Conservation; New Zealand Ministry for Primary Industries; National Science Foundation (award number 79804); and Museum of New Zealand. 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Conserv. 281 (2023) 109994] Xavier y, Maria P Dias b,ag a MARE – Marine and Environmental Sciences Centre/ARNET – Aquatic Research Network, Ispa – Instituto Universit´ario de Ciˆencias Psicol´ogicas, Sociais e da Vida, Lisboa Portugal a MARE – Marine and Environmental Sciences Centre/ARNET – Aquatic Research Network, Ispa – Instituto Universit´ario de Ciˆencias Psicol´ogicas, Sociais e da Vida, Li b P l a MARE – Marine and Environmental Sciences Centre/ARNET – Aquatic Research Network, Ispa – Instituto Universit´ario de Ciˆencias Psicol´ogicas, Sociais e da Vida Lisboa, Portugal f , g g, , g Q , c British Antarctic Survey, Natural Environment Research Council, High Cross, Madingley Road, Cambridge CB3 0ET, UK d d RSPB Centre for Conservation Science, Royal Society for the Protection of Birds, The David Attenborough Building, Pembroke Street, Cambridge C f gy y gy g RSPB Centre for Conservation Science, Royal Society for the Protection of Birds, 10, Albyn Terrace, Aberdeen AB10 1YP, UK h y g Resources Control Board Office of Information Management and Analysis, 1001 I Street, 19th Floor, Sacramento, CA 95814, USA i California State Water Resources Control Board Office of Information Management and Analysis, 1001 I Street, 19th Floor, Sacramento, CA f ffi f f g y , , , , , j Long Marine Laboratory, University of California, Santa Cruz, 100 Shaffer Rd., Santa Cruz, CA. Corrigendum to “Quantifying annual spatial consistency in chick-rearing seabirds to inform important site identification” [Biol. Conserv. 281 (2023) 109994] Corrigendum to “Quantifying annual spatial consistency in chick-rearing seabirds to inform important site identification” [Biol. Conserv. 281 (2023) 109994] Martin Beal a,b,*, Paulo Catry a, Richard A. Phillips c, Steffen Oppel d, John P.Y. Arnould e, Maria I. Bogdanova f, Mark Bolton g, Ana P.B. Carneiro b, Corey Clatterbuck h,i, Melinda Conners j,k, Francis Daunt f, Karine Delord l, Kyle Elliott m, Aymeric Fromant e, Jos´e Pedro Granadeiro n, Jonathan A. Green o, Lewis G. Halsey p, Keith C. Hamer q, Motohiro Ito r, Ruth Jeavons q, Jeong-Hoon Kim s, Nobuo Kokubun t, Shiho Koyama u, Jude V. Lane q,v, Won Young Lee s, Sakiko Matsumoto u, Rachael A. Orben w, Ellie Owen x, Vitor H. Paiva y, Allison Patterson m, Christopher J. Pollock q, Jaime A. Ramos y, Paul Sagar z, Katsufumi Sato aa, Scott A. Shaffer h, Louise Soanes ab, Akinori Takahashi t, David R. Thompson ac, Lesley Thorne k, Leigh Torres ad, Yutaka Watanuki ae, Susan M. Waugh af,b, Henri Weimerskirch l, Shannon Whelan m, Ken Yoda u, Jos´e C. Xavier y, Maria P Dias b,ag vid R. Thompson ac, Lesley Thorne k, Leigh Torres ad, Yutaka Watanuki ae, f b l Susan M. Waugh af,b, Henri Weimerskirch l, Shannon Whelan m, Ken Yoda u, Jos´e C. Available online 21 March 2023 0006-3207/© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Update https://doi.org/10.1016/j.biocon.2023.110025 DOI: Biological Conservation 281 (2023) 110025 https://doi.org/10.1016/j.biocon.2023.110025 Corrigendum to “Quantifying annual spatial consistency in chick-rearing seabirds to inform important site identification” [Biol. Conserv. 281 (2023) 109994] 95060, USA l Centre d'Etudes Biologiques de Chiz´e, CNRS UMR 7372 - La Rochelle Universit´e, 79360 Villiers en Bois, France m Department of Natural Resource Sciences, McGill University, 21111 Lakeshore Rd, Ste Anne-de-Bellevue, QC H9X3V9, Canada p f , y, , , Q 9 9, n CESAM, Departamento de Biologia Animal, Faculdade de Ciˆencias, Universidade de Lisboa, Rua Ernesto Vasconcelos, 1749-016 Lisboa o School of Environmental Sciences, University of Liverpool, Liverpool L69 3GP, UK p University of Roehampton, Holybourne Avenue, London SW15 4JD, UK y f p y q School of Biology, University of Leeds, Irene Manton Building, Leeds LS2 9JT, UK r Faculty of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura-machi, Japan s Korea Polar Research Institute (KOPRI), 26, Songdomirae-ro, Yeonsu-gu, Incheon 21990, Republic of Korea e (KOPRI), 26, Songdomirae-ro, Yeonsu-gu, Incheon 21990, Republic of K t National Institute of Polar Research, Tachikawa, Tokyo 190-8518, Japan u Graduate School of Environmental Studies, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan v ƗRSPB Centre for Conservation Science, RSPB, 7.3.1 Cameron House, White Cross Estate, Lancashire LA1 4XF, UK i w Department of Fisheries, Wildlife, and Conservation Sciences, Oregon State University, Hatfield Marine Science Center, 2030 Marine Science Dr., Newport, OR, USA x RSPB Centre for Conservation Science, Royal Society for the Protection of Birds, RSPB North Scotland Office, Etive House, Beechwood Park, Inverness IV2 6AL, UK y University of Coimbra, MARE – Marine and Environmental Sciences Centre / ARNET – Aquatic Research Network, Department of Life Sciences, Calçada Martim de Freitas, 3000-456 Coimbra, Portugal w Department of Fisheries, Wildlife, and Conservation Sciences, Oregon State University, Hatfield Marine Science Center, 2030 Marine Science Dr., Newport, OR, USA x RSPB Centre for Conservation Science, Royal Society for the Protection of Birds, RSPB North Scotland Office, Etive House, Beechwood Park, Inverness IV2 6AL, UK y University of Coimbra, MARE – Marine and Environmental Sciences Centre / ARNET – Aquatic Research Network, Department of Life Sciences, Calçada Martim de Freitas, 3000-456 Coimbra, Portugal z National Institute of Water & Atmospheric Research, PO Box 8602, Riccarton, Christchurch 8011, New Zealand aa Atmosphere and Ocean Research Institute, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa City, Chiba Prefectur National Institute of Water & Atmospheric Research, PO Box 8602, Riccarton, Christchurch 8011, New Zealand a Atmosphere and Ocean Research Institute, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa City, Chiba Prefecture 2 ab Life Sciences, University of Roehampton, Whiteland Campus, London SW154JD, UK DOI of original article: https://doi.org/10.1016/j.biocon.2023.109994. Corrigendum to “Quantifying annual spatial consistency in chick-rearing seabirds to inform important site identification” [Biol. Conserv. 281 (2023) 109994] q School of Biology, University of Leeds, Irene Manton Building, Leeds LS2 9JT, UK r Faculty of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura- machi, Japan s School of Biology, University of Leeds, Irene Manton Building, Leeds LS2 9JT, UK t Korea Polar Research Institute (KOPRI), 26, Songdomirae-ro, Yeonsu-gu, Incheon 21990, Republic of Korea u National Institute of Polar Research, Tachikawa, Tokyo 190-8518, Japan v Graduate School of Environmental Studies, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan a MARE – Marine and Environmental Sciences Centre/ARNET – Aquatic Research Network, Ispa – Instituto Universit´ario de Ciˆencias Psicol´ogicas, Sociais e da Vida, Lisboa, Portugal a MARE – Marine and Environmental Sciences Centre/ARNET – Aquatic Research Network, Ispa – Instituto Universit´ario de Ciˆencias Psicol´ogicas, Sociais e da Vida, Lisboa, Portugal w ƗRSPB Centre for Conservation Science, RSPB, 7.3.1 Cameron House, White Cross Estate, Lancashire LA1 4XF, UK b BirdLife International, The David Attenborough Building, Pem­ broke Street, Cambridge CB2 3QZ, UK x Department of Fisheries, Wildlife, and Conservation Sciences, Oregon State University, Hatfield Marine Science Center, 2030 Marine Science Dr., Newport, OR, USA c British Antarctic Survey, Natural Environment Research Council, High Cross, Madingley Road, Cambridge CB3 0ET, UK y RSPB Centre for Conservation Science, Royal Society for the Pro­ tection of Birds, RSPB North Scotland Office, Etive House, Beechwood Park, Inverness IV2 6AL, UK d RSPB Centre for Conservation Science, Royal Society for the Pro­ tection of Birds, The David Attenborough Building, Pembroke Street, Cambridge CB2 3QZ, UK z University of Coimbra, MARE – Marine and Environmental Sciences Centre / ARNET – Aquatic Research Network, Department of Life Sci­ ences, Calçada Martim de Freitas, 3000-456 Coimbra, Portugal e School of Life and Environmental Sciences, Deakin University, 221 Burwood Highway, Burwood, VIC 3125, Australia f aa National Institute of Water & Atmospheric Research, PO Box 8602, Riccarton, Christchurch 8011, New Zealand f UK Centre for Ecology & Hydrology, Bush Estate, Penicuik EH26 0QB, UK ab Atmosphere and Ocean Research Institute, University of Tokyo, 5- 1-5 Kashiwanoha, Kashiwa City, Chiba Prefecture 277-8564, Japan g RSPB Centre for Conservation Science, Royal Society for the Pro­ tection of Birds, 10, Albyn Terrace, Aberdeen AB10 1YP, UK h h Biological Sciences, San Jose State University, One Washington Square, San Jose, CA 95192-0100, USA i ac Life Sciences, University of Roehampton, Whiteland Campus, London SW154JD, UK ad National Institute of Polar Research, Tachi­ kawa, Tokyo 190-8518, Japan i California State Water Resources Control Board Office of Informa­ tion Management and Analysis, 1001 I Street, 19th Floor, Sacramento, CA 95814, USA ae National Institute of Water & Atmospheric Research, 301 Evans Bay Parade, Hataitai, Wellington, New Zealand 6021 f af Marine Mammal Institute, Oregon State University, 2030 SE Ma­ rine Science Dr., Newport, OR 97365, USA j Long Marine Laboratory, University of California, Santa Cruz, 100 Shaffer Rd., Santa Cruz, CA. Corrigendum to “Quantifying annual spatial consistency in chick-rearing seabirds to inform important site identification” [Biol. Conserv. 281 (2023) 109994] DOI of original article: https://doi.org/10.1016/j.biocon.2023.109994. g p g j * Corresponding author at: MARE – Marine and Environmental Sciences Centre/ARNET – Aquatic Research Network, Ispa – Instituto Universit´ario de Ciˆencias Psicol´ogicas, Sociais e da Vida, Lisboa, Portugal. E mail address: martinbeal88@gmail com (M Beal) E-mail address: martinbeal88@gmail.com (M. Beal). Available online 21 March 2023 0006-3207/© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Biological Conservation 281 (2023) 110025 M. Beal et al. ac National Institute of Water & Atmospheric Research, 301 Evans Bay Parade, Hataitai, Wellington 6021, New Zealand ad Marine Mammal Institute, Oregon State University, 2030 SE Marine Science Dr., Newport, OR 97365, USA ac National Institute of Water & Atmospheric Research, 301 Evans Bay Parade, Hataitai, Wellington 6021, New Zealand ad Marine Mammal Institute, Oregon State University, 2030 SE Marine Science Dr., Newport, OR 97365, USA g y p ae Faculty of Fisheries Sciences, Hokkaido University, Minato-cho 3-1-1, Hakodate 041-8611, Japan f ae Faculty of Fisheries Sciences, Hokkaido University, Minato-cho 3-1-1, Hakodate 041-8611, Ja af Ligue de la Protection des Oiseaux, Fonderies Royales, CS90263, 17305 Rochefort, France af Ligue de la Protection des Oiseaux, Fonderies Royales, CS90263, 17305 Rochefort, France ag cE3c Center for Ecology, Evolution and Environmental Changes & CHANGE - Global Change and Sustainability Institute, Department of Animal Biology, Facul Ciˆ i U i id d d Li b C G d 1749 016 Li b P t l ag cE3c Center for Ecology, Evolution and Environmental Changes & CHANGE - Global Change and Sustainability Institute, Department of Animal Biology, Faculdade de Ciˆencias, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal ag cE3c Center for Ecology, Evolution and Environmental Changes & CHANGE - Global Change and Sustainability Institute, Department of A Ciˆencias, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal cE3c Center for Ecology, Evolution and Environmental Changes & CHANGE Global Change an Ciˆencias, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal o School of Environmental Sciences, University of Liverpool, Liver­ pool L69 3GP, UK The authors regret missing and incorrect affiliation information, which should be like: p University of Roehampton, Holybourne Avenue, London SW15 4JD, UK Martin Beal a,b,*, Paulo Catry a, Richard A. Phillips c, Steffen Oppel d, John P.Y. Arnould e, Maria I. Bogdanova f, Mark Bolton g, Ana P.B. Corrigendum to “Quantifying annual spatial consistency in chick-rearing seabirds to inform important site identification” [Biol. Conserv. 281 (2023) 109994] Carneiro b, Corey Clatterbuck h,i, Melinda Conners j,k, Francis Daunt f, Karine Delord l, Kyle Elliott m, Aymeric Fromant e, Jos´e Pedro Gran­ adeiro n, Jonathan A. Green o, Lewis G. Halsey p, Keith C. Hamer q, Motohiro Ito r, Ruth Jeavons s, Jeong-Hoon Kim t, Nobuo Kokubun u, Shiho Koyama v, Jude V. Lane s,w,1, Won Young Lee t, Sakiko Matsumoto v, Rachael A. Orben x, Ellie Owen y, Vitor H. Paiva z, Allison Patterson m, Christopher J. Pollock s, Jaime A. Ramos z, Paul Sagar aa, Katsufumi Sato ab, Scott A. Shaffer h, Louise Soanes ac, Akinori Takahashi ad, David R. Thompson ae, Lesley Thorne k, Leigh Torres af, Yutaka Watanuki ag, Susan M. Waugh ah,b,1, Henri Weimerskirch l, Shannon Whelan m, Ken Yoda v, Jos´e C. Xavier z, Maria P. Dias b,ai,1. Martin Beal a,b,*, Paulo Catry a, Richard A. Phillips c, Steffen Oppel d, John P.Y. Arnould e, Maria I. Bogdanova f, Mark Bolton g, Ana P.B. Carneiro b, Corey Clatterbuck h,i, Melinda Conners j,k, Francis Daunt f, Karine Delord l, Kyle Elliott m, Aymeric Fromant e, Jos´e Pedro Gran­ adeiro n, Jonathan A. Green o, Lewis G. Halsey p, Keith C. Hamer q, Motohiro Ito r, Ruth Jeavons s, Jeong-Hoon Kim t, Nobuo Kokubun u, Shiho Koyama v, Jude V. Lane s,w,1, Won Young Lee t, Sakiko Matsumoto v, Rachael A. Orben x, Ellie Owen y, Vitor H. Paiva z, Allison Patterson m, Christopher J. Pollock s, Jaime A. Ramos z, Paul Sagar aa, Katsufumi Sato ab, Scott A. Shaffer h, Louise Soanes ac, Akinori Takahashi ad, David R. Thompson ae, Lesley Thorne k, Leigh Torres af, Yutaka Watanuki ag, Susan M. Waugh ah,b,1, Henri Weimerskirch l, Shannon Whelan m, Ken Yoda v, Jos´e C. Xavier z, Maria P. Dias b,ai,1. Corrigendum to “Quantifying annual spatial consistency in chick-rearing seabirds to inform important site identification” [Biol. Conserv. 281 (2023) 109994] 95060, USA k ag Faculty of Fisheries Sciences, Hokkaido University, Minato-cho 3- 1-1, Hakodate 041-8611, Japan k School of Marine and Atmospheric Sciences, Stony Brook Univer­ sity, Stony Brook, NY USA 11794-5000 k School of Marine and Atmospheric Sciences, Stony Brook Univer­ sity, Stony Brook, NY USA 11794-5000 l Centre d’Etudes Biologiques de Chiz´e, CNRS UMR 7372 - La Rochelle Universit´e, 79360 Villiers en Bois, France y y l Centre d’Etudes Biologiques de Chiz´e, CNRS UMR 7372 - La Rochelle Universit´e, 79360 Villiers en Bois, France ah Ligue de la Protection des Oiseaux, Fonderies Royales, CS90263, 17305 Rochefort, France m Department of Natural Resource Sciences, McGill University, 21111 Lakeshore Rd, Ste Anne-de-Bellevue, QC H9X3V9, Canada ai cE3c Center for Ecology, Evolution and Environmental Changes & CHANGE - Global Change and Sustainability Institute, Department of Animal Biology, Faculdade de Ciˆencias, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal n CESAM, Departamento de Biologia Animal, Faculdade de Ciˆencias, Universidade de Lisboa, Rua Ernesto Vasconcelos, 1749-016 Lisboa, Portugal
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Transurethral needle electrode resection and transurethral holmium laser resection of bladder cancer
World journal of surgical oncology
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© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. RESEARCH Open Access Zhou et al. World Journal of Surgical Oncology (2020) 18:166 https://doi.org/10.1186/s12957-020-01943-3 Zhou et al. World Journal of Surgical Oncology (2020) 18:166 https://doi.org/10.1186/s12957-020-01943-3 Abstract Purpose: The aim of the present study was to explore the efficacy and safety of transurethral needle electrode resection and transurethral holmium laser resection of non-muscular invasive bladder cancer (NMIBC). Patients and methods: In this prospective, case-control study, patients from the Urinary Surgery or Oncology Department who met the inclusion and exclusion criteria received transurethral needle electrode resection (n = 52) or transurethral holmium laser resection (n = 51). Results: A total of 103 patients with NMIBC were included in the present study, with 68 males and 35 females. Their mean age was 57.3 years. Sixty-two patients had Ta, 15 patients had T1, and 26 patients had Tis. Operative time, intraoperative blood loss, postoperative gross hematuria time, bladder irrigation time, and postoperative hospitalization time were all significantly lower in the transurethral holmium laser resection group than the transurethral needle electrode resection group. After resection, transurethral holmium laser resection significantly decreased the value of HGF, TSH, and TNF-α versus the transurethral needle electrode resection group. The incidence of obturator reflex was significantly lower in the transurethral holmium laser resection group than the transurethral needle electrode resection group. There was no significant difference in disease-free survival rate and progression-free survival rate between the two groups. Keywords: Non-muscular invasive bladder cancer, Transurethral needle electrode resection, Transurethral holmium laser resection muscular invasive bladder cancer, Transurethral needle electrode resection, Transurethral holmium laser incidence of urinary reproductive system tumors in China. Pathologically, bladder cancer is divided into muscle- and non-muscle-invasive bladder cancer (NMIBC). NMIBC, which can further develop into muscle-infiltrating bladder cancer, accounts for 70 ~ 80% bladder cancer cases, and 20 ~ 25% of patients with NMIBC have poor prognosis [1–3]. At present, the main problems in clinical practice are insuf- ficient understanding of the risk of NMIBC, which is gener- ally considered to be a low-grade malignancy with a low diagnosis rate of carcinoma in situ (CIS), irregular treat- ment of special histological types of NMIBC, inadequate treatment of high-risk NMIBC, irregular bladder perfusion treatment, and other problems worthy of attention. The Transurethral needle electrode resection and transurethral holmium laser resection of bladder cancer Yu Zhou†, Zheng-Long Zhang†, Mao-Hua Luo and Hua Yang* Yu Zhou†, Zheng-Long Zhang†, Mao-Hua Luo and Hua Yang* Introduction Bladder cancer is a common malignancy of the urinary sys- tem, and its incidence ranks 9th in the world, 6th in men, and 10th in women. The ratio of male to female incidence is 3:1. According to a 2018 epidemiological report, there were about 78,100 new cases of bladder cancer in China (including about 60,600 cases of male, 17,500 of female), and the incidence is 5.71/10 (8.65/10 of male, 2.62/10 of fe- male). Currently, bladder cancer still carries the highest * Correspondence: chufeng323@126.com †Yu Zhou and Zheng-Long Zhang contributed equally to this work. Department of Urology, Renmin Hospital, Hubei University of Medicine, No. 39 Chaoyang Middle Road, Maojian District, Shiyan 442000, Hubei, China * Correspondence: chufeng323@126.com †Yu Zhou and Zheng-Long Zhang contributed equally to this work. Department of Urology, Renmin Hospital, Hubei University of Medicine, No. 39 Chaoyang Middle Road, Maojian District, Shiyan 442000, Hubei, China Zhou et al. World Journal of Surgical Oncology (2020) 18:166 Page 2 of 9 Page 2 of 9 hemoglobin < 80 g/L, neutrophils < 1.5 × 109/L, platelets < 75 × 109/L, or other hematological diseases; (9) known im- munodeficiency, acquired or congenital immunodeficiency, or a history of organ transplantation; (10) metastasis or inva- sion of the tumor center or various mental disorders; (11) patients had severe cardiovascular and cerebrovascular dis- eases, such as myocardial infarction, severe/unstable angina pectoris, coronary/peripheral artery bypass graft, symptom- atic congestive heart failure, cerebrovascular accident or transient ischemic attack, or pulmonary embolism, who can- not tolerate transurethral resection of bladder tumor (TURBT) surgery; (12) severe hypertension (systolic blood pressure ≥180 mmHg, diastolic blood pressure ≥110 mmHg), or serious complications of hypertension, or serious complications of diabetes; (13) subjects with fever exceeding 38 °C and significant active infections that may affect the clinical trial, such as acute pneumonia, active tuberculosis, and severe urinary tract infection; (14) those who partici- pated in other clinical trials within 3 months before screen- ing; (15) patients who had received chemotherapeutic drugs or bacille Calmette-Guérin (BCG) perfusion therapy within the last 3 months; (16) severe side effects (symptoms of blad- der irritation, etc.) occurred during the treatment of bladder perfusion and the patient was unable to tolerate them; (17) pregnant women, critically ill subjects, and other cases with contraindications, in case of serious cardiovascular disease, significant abnormal coagulation function, non-transitional epithelial tumor (such as adenocarcinoma, squamous cell carcinoma), acute cystitis, spinal deformity and patients can- not lie flat, urethral stricture, etc.; and 18 those who were considered by the researchers to compromise surgical effi- cacy and safety evaluation or have poor compliance. surgical methods of NMIBC include transurethral resection of bladder tumor, photodynamic therapy, transurethral laser surgery, partial cystectomy, and radical cystectomy (RC). RC is divided into three types: open, laparoscopic, and robot-assisted laparoscopic RC. There is substantial evidence that RC is still the most commonly used treatment for high-risk NMIBC or patients who develop muscle-layer infiltration [4, 5]. In this study, we aimed to explore the efficacy of trans- urethral needle electrode resection and transurethral holmium laser resection for NMIBC based on a pro- spective case-control study and to provide a basis for the selection of clinical treatment. Patients and methods Patient selection This prospective case-control study was conducted from January 2015 to November 2016 at the Department of Ur- ology, Renmin Hospital, Shiyan, China. The study was ap- proved and monitored by the Ethics Committee of Renmin Hospital, Hubei Medical University (approval no. 2017- 017). Patients, who were admitted to the Urinary Surgery or Oncology Department, were recruited if they met the following criteria: (1) age ≥18 years; (2) pathologically or histologically confirmed primary non-muscular invasive urothelial carcinoma of the bladder (Ta, Tis, T1); (3) East- ern Cooperative Oncology Group (ECOG) performance status (PS) score 0–2; (4) imaging examination showed that bladder cancer did not involve the bladder muscle layer and there was no lymph node metastasis or distant metas- tasis; (5) subjects who agreed to receive transurethral needle electrode resection or transurethral holmium laser resec- tion and who can be followed up according to postopera- tive routine perfusion treatment; (6) the function of the main organs (heart, liver, lung, and kidney) was basically normal and ECOG performance status (PS) score 0–2; (7) bladder capacity ≥200 mL; and (8) patients provided writ- ten informed consent. Fig. 1 Comparison of tumor recurrence in the follow-up time between the two groups (log-rank test: χ2 = 0.569, P = 0.451) Exclusion criteria were as follows: (1) patients diagnosed with muscle-infiltrating urothelial carcinoma of the bladder (stage T2 or above); (2) patients had a history of bladder con- tracture or bladder functional volume less than 200 mL; (3) severe urethral stricture did not allow entry of a cystoscope; (4) pelvic computed tomography (CT) scan or systemic physical examination revealed tumor metastasis; (5) patients had other concurrent uropoiesis reproductive system tumors (above upper tract urothelial carcinoma, etc.) or tumor in other organs; (6) patients had hereditary or acquired coagula- tion disorders or were using anticoagulant drugs; (7) signifi- cant dysfunction of the main internal organs, such as 1.5 times greater than the upper normal limit of creatinine, as- partate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin; (8) decreased hematopoietic func- tion of the bone marrow: leucocytes < 3.0 × 109/L, Fig. 1 Comparison of tumor recurrence in the follow-up time between the two groups (log-rank test: χ2 = 0.569, P = 0.451) Zhou et al. Transurethral needle electrode resection Transurethral needle electrode resection The location, number, diameter, and base of the tumor in the bladder were observed by transurethral electrotomy after continuous epidural anesthesia and disinfection. Then, 5% (v/v) mannitol rinse solution was applied to adjust the output power of the electric cutting current to 120 W and the output power of the electric coagulation current to 80 W. The bladder was kept in a filling state, about 1 cm away from the base of the tumor, and the mucosa, submucosa, and deep muscle layer were excised cystoscopically with a needle electrode close to the bladder mucosa. A blunt nee- dle electrode was used to push and peel the deep surface of the deep muscle layer, and the tumor tissues were lifted up- ward by the impact of water flow, and the tumor base was well exposed. Under the endoscope, tumor tissues and nor- mal tissues could be clearly distinguished. Clockwise or counterclockwise electric cutting was performed along the tumor base until the whole tumor pedicle was completely removed. During the operation, coagulation and cutting were performed to effectively control bleeding and keep the visual field clear. If necessary, the fat in the outer layer of the bladder can be cut. Small tumors were sucked out dir- ectly by Elick, while larger tumors were hooked out by a ring-like electrotomy from the operating channel. One tri- Fig. 3 Imaging examination lumen indwelling catheter was placed postoperatively, and the bladder was irrigated with normal saline. lumen indwelling catheter was placed postoperatively, and the bladder was irrigated with normal saline. Patients and methods Patient selection World Journal of Surgical Oncology (2020) 18:166 This study aimed to explore the efficacy of transureth- ral needle electrode resection and transurethral holmium laser resection for Non-muscular invasive bladder cancer (NMIBC). The patients were prospectively included in this study and divided into the two groups by the types of surgery. lumen indwelling catheter was placed postoperatively, and the bladder was irrigated with normal saline. Fig. 3 Imaging examination Fig. 3 Imaging examination Patients and methods Patient selection World Journal of Surgical Oncology (2020) 18:166 Page 3 of 9 Table 1 Patients’ basic characteristics of the two surgical groups Index Transurethral needle electrode resection (n = 52) Transurethral holmium = 51) Age (year) (mean ± sd) 55.8 ± 12.8 58.8 ± 12.9 Gender [n (%)] Male 34 (65.4) 3 4(66.7) Female 18 (34.6) 17 (33.3) Height (cm) (mean ± sd) 166.6 ± 11.2 163.3 ± 10.0 Weight (kg) (mean ± sd) 64.5 ± 14.9 60.4 ± 12.6 BMI (mean ± sd) 23.0 ± 3.8 22.5 ± 3.5 Tumor size (mm) (mean ± sd) 25.7 ± 12.6 25.7 ± 13.9 Tumor size grouping [n (%)] < 3 cm 37 (71.2) 33 (64.7) ≥3 cm 15 (28.8) 18 (35.3) Tumor focality [n (%)] Unifocal 43 (82.7) 43 (84.3) Multifocal 9 (17.3) 8 (15.7) Tumor location [n (%)] Lateral wall 31 (59.6) 31 (60.8) Trigone 6 (11.5) 5 (9.8) Dome 7 (13.5) 7 (13.7) Posterior wall 3 (5.8) 3 (5.9) Anterior wall 3 (5.8) 2 (3.9) Less than 1.5 cm from the ureter mouth 2 (3.8) 3 (5.9) Pathological classification [n (%)] Ta 33 (63.5) 29 (56.9) T1 7 (13.5) 8 (15.7) Tis 12 (23.1) 14 (27.5) Preoperative tumor grade [n (%)] G1 44 (84.6) 43 (84.3) G2 8 (15.4) 7 (13.7) G3 0 (0.0) 1 (2.0) Postoperative tumor grade [n (%)] G1 44 (84.6) 43 (84.3) G2 7 (13.5) 7 (13.7) G3 1 (1.9) 1 (2.0) Comorbid disease [n (%)] None 30 (57.7) 29 (56.9) Diabetes 6 (11.5) 6 (11.8) High blood pressure 8 (15.4) 7 (13.7) Coronary heart disease (CHD) 4 (7.7) 5 (9.8) Prostatic hyperplasia 4 (7.7) 4 (7.8) Table 1 Patients’ basic characteristics of the two surgical groups Index Transurethral needle electrode resection (n = 52) Transurethral holmium laser resection (n = 51) Total (n = 103) P Age (year) (mean ± sd) 55.8 ± 12.8 58.8 ± 12.9 57.3 ± 12.9 0.236 Gender [n (%)] Male 34 (65.4) 3 4(66.7) 68 (66.0) 0.891 Female 18 (34.6) 17 (33.3) 35 (34.0) Height (cm) (mean ± sd) 166.6 ± 11.2 163.3 ± 10.0 165.0 ± 10.7 0.120 Weight (kg) (mean ± sd) 64.5 ± 14.9 60.4 ± 12.6 62.4 ± 13.9 0.133 BMI (mean ± sd) 23.0 ± 3.8 22.5 ± 3.5 22.8 ± 3.6 0.487 Tumor size (mm) (mean ± sd) 25.7 ± 12.6 25.7 ± 13.9 25.7 ± 13.2 0.998 Tumor size grouping [n (%)] < 3 cm 37 (71.2) 33 (64.7) 70 (68.0) 0.483 ≥3 cm 15 (28.8) 18 (35.3) 33 (32.0) Tumor focality [n (%)] Unifocal 43 (82.7) 43 (84.3) 86 (83.5) 0.825 Multifocal 9 (17.3) 8 (15.7) 17 (16.5) Tumor location [n (%)] Lateral wall 31 (59.6) 31 (60.8) 62 (60.2) 0.993 Trigone 6 (11.5) 5 (9.8) 11 (10.7) Dome 7 (13.5) 7 (13.7) 14 (13.6) Posterior wall 3 (5.8) 3 (5.9) 6 (5.8) Anterior wall 3 (5.8) 2 (3.9) 5 (4.9) Less than 1.5 cm from the ureter mouth 2 (3.8) 3 (5.9) 5 (4.9) Pathological classification [n (%)] Ta 33 (63.5) 29 (56.9) 62 (60.2) 0.791 T1 7 (13.5) 8 (15.7) 15 (14.6) Tis 12 (23.1) 14 (27.5) 26 (25.2) Preoperative tumor grade [n (%)] G1 44 (84.6) 43 (84.3) 87 (84.5) 1.000 G2 8 (15.4) 7 (13.7) 15 (14.6) G3 0 (0.0) 1 (2.0) 1 (1.0) Postoperative tumor grade [n (%)] G1 44 (84.6) 43 (84.3) 87 (84.5) 1.000 G2 7 (13.5) 7 (13.7) 14 (13.6) G3 1 (1.9) 1 (2.0) 2 (1.9) Comorbid disease [n (%)] None 30 (57.7) 29 (56.9) 59 (57.3) 1.000 Diabetes 6 (11.5) 6 (11.8) 12 (11.7) High blood pressure 8 (15.4) 7 (13.7) 15 (14.6) Coronary heart disease (CHD) 4 (7.7) 5 (9.8) 9 (8.7) Prostatic hyperplasia 4 (7.7) 4 (7.8) 8 (7.8) Table 1 Patients’ basic characteristics of the tw Index Transurethra = 52) Age (year) (mean ± sd) 55.8 ± 12.8 Gender [n (%)] Male 34 (65.4) Female 18 (34.6) Height (cm) (mean ± sd) 166.6 ± 11.2 Weight (kg) (mean ± sd) 64.5 ± 14.9 BMI (mean ± sd) 23.0 ± 3.8 Tumor size (mm) (mean ± sd) 25.7 ± 12.6 Tumor size grouping [n (%)] < 3 cm 37 (71.2) ≥3 cm 15 (28.8) Tumor focality [n (%)] Unifocal 43 (82.7) Multifocal 9 (17.3) Tumor location [n (%)] Lateral wall 31 (59.6) Trigone 6 (11.5) Dome 7 (13.5) Posterior wall 3 (5.8) Anterior wall 3 (5.8) Less than 1.5 cm from the ureter mouth 2 (3.8) Pathological classification [n (%)] Ta 33 (63.5) T1 7 (13.5) Tis 12 (23.1) Preoperative tumor grade [n (%)] G1 44 (84.6) G2 8 (15.4) G3 0 (0.0) Postoperative tumor grade [n (%)] G1 44 (84.6) G2 7 (13.5) G3 1 (1.9) Comorbid disease [n (%)] None 30 (57.7) Diabetes 6 (11.5) High blood pressure 8 (15.4) Coronary heart disease (CHD) 4 (7.7) Prostatic hyperplasia 4 (7.7) 62 (60.2) 0.791 Page 4 of 9 Zhou et al. Transurethral holmium laser resection After continuous epidural anesthesia and disinfection, a cystoscope was placed through the urethra to ob- serve the location, number, diameter, and base of the tumor in the bladder. A holmium laser fiber was inserted into the bladder through the cystoscope op- erating hole using normal saline for rinsing. The hol- mium laser fiber was placed close to the tumor body, and the tumor was cut about 1 cm from the tumor Fig. 2 Imaging examination Fig. 4 Imaging examination Fig. 4 Imaging examination Fig. 4 Imaging examination Fig. 2 Imaging examination Fig. Page 5 of 9 Zhou et al. World Journal of Surgical Oncology (2020) 18:166 Fig. 5 Histopathologic examination Fig. 5 Histopathologic examination (%). Outcomes were compared between two groups with two-sided t test for continuous variables and chi-square test for categorical variables. Survival data were analyzed by Kaplan-Meier method. All tests were two-sided, and P < 0.05 was considered to be statistically significant. base. When the muscle layer was cut, the tumor tis- sue was lifted up with water flow. The small tumors were sucked out by Elick directly, while larger tumors were hooked out by a ring-like electrotomy from the operating channel. One tri-lumen indwelling catheter was placed postoperatively, and the bladder was irri- gated with normal saline. Data collection Patient baseline information and clinical efficacy data were collected. The baseline information included age, gender, height, weight, body mass index (BMI), tumor size, tumor focality, tumor location, pathological classification, pre- operative/postoperative tumor grade, and comorbid dis- ease. The clinical efficacy data included operative time, intraoperative time, postoperative gross hematuria time, bladder irrigation time, postoperative hospitalization time, indwelling time, complications, secondary operation, tumor residual rate at the second operation, disease-free survival rate (1-year, 2-year, 3-year), progression-free sur- vival rate (1-year, 2-year, 3-year), hepatocyte growth factor (HGF), tumor-specific growth factor (TSGF), and tumor necrosis factor alpha (TNF-α). In the follow-up period, B ultrasound and cystoscopy were performed to evaluate re- currence or progress. Figures 2, 3, and 4 presented the imaging examin- ation of the bladder tumor on the right posterior wall; Fig. 5 is the histopathologic examination about papillary urothelial carcinoma; Fig. 6 is the histopath- ologic examination about infiltrating papillary urothe- lial carcinoma; Fig. 7 is the surgical process about Comparison of baseline characteristics in patients Comparison of baseline characteristics in patients A total of 103 patients with NMIBC were included in the present study, 52 patients in the transurethral needle electrode resection group, and 51 patients in the trans- urethral holmium laser resection group (Fig. 1). The pa- tients included 68 males and 35 females, with a mean age of 57.3 years. The pathological classification of NMIBC was as follows: 62 patients in Ta, 15 patients in T1, and 26 patients in Tis. The tumor size was 25.7 (12.6) mm in the transurethral needle electrode resec- tion group, 25.7 (13.9) mm in the transurethral holmium laser resection group. Table 1 shows a comparison of baseline characteristics in patients between the two groups. No statistically significant differences were ob- served in baseline characteristics of the patients between the two groups (all P > 0.05). Comparison of clinical outcome variables in patients Comparison of clinical outcome variables in patients A comparison was made of the perioperative indicators, complications, and secondary operation after resection be- tween the groups (Table 2). In the transurethral holmium laser resection group, the operative time (28.8 ± 3.6 min vs. 31.5 ± 4.4 min), estimated intraoperative blood loss (27.0 ± 3.9 mL vs. 34.2 ± 6.2 mL), postoperative gross hematuria time (2.8 ± 1.5 days vs. 3.5 ± 1.1 days), bladder irrigation time (1.9 ± 0.5 days vs. 2.2 ± 0.4 days), and postoperative hospitalization time (6.2 ± 1.3 days vs. 6.7 ± 1.3 days) were all significantly decreased after resection when compared with the transurethral needle electrode resection group (P < 0.05 or P < 0.01). The incidence of obturator reflex was significantly lower in the transurethral holmium laser resec- tion group (2.0%) than the transurethral needle electrode resection group (15.4%) (P < 0.05). There was no significant difference in indwelling time, urethral stricture, secondary operation, and tumor residual rate at the second operation between the two groups (all P > 0.05). In the subgroup analysis by tumor size, there was a significant difference in operative time, estimated intra- operative blood loss, and bladder irrigation time between the two groups with tumor size < 3 cm; there was a sig- nificant difference in postoperative gross hematuria time between the two groups with tumor size ≥3 cm. In the subgroup analysis by tumor focality, there was a signifi- cant difference in operative time, estimated intraopera- tive blood loss, postoperative gross hematuria time, and bladder irrigation time between the two groups with uni- focal tumor. In the subgroup analysis by tumor location, there was a significant difference in operative time, esti- mated intraoperative blood loss, postoperative gross hematuria time, bladder irrigation time, and postopera- tive hospitalization time between the two groups with lateral wall tumor; there was a significant difference in operative time, postoperative gross hematuria time, and bladder irrigation time between the two groups with posterior wall tumor. Statistical analysis Statistical analyses were completed using SPSS17.0. Data were reported as mean (standard deviation) or number Fig. 6 Histopathologic examination Fig. 6 Histopathologic examination Page 6 of 9 Zhou et al. World Journal of Surgical Oncology (2020) 18:166 Table 2 Comparison of perioperative indicators between the two surgical groups Index Groups P Transurethral needle electrode resection (n = 52) Transurethral holmium laser resection (n = 51) Perioperative indicators (mean ± sd) Operative time (min) 31.5 ± 4.4 28.8 ± 3.6 0.001 Intraoperative blood loss (ml) 34.2 ± 6.2 27.0 ± 3.9 0.000 Postoperative gross hematuria time (days) 3.5 ± 1.1 2.8 ± 1.5 0.009 Bladder irrigation time (days) 2.2 ± 0.4 1.9 ± 0.5 0.000 Postoperative hospitalization time (days) 6.7 ± 1.3 6.2 ± 1.3 0.039 Indwelling time (days) 3.4 ± 1.4 3.2 ± 1.0 0.428 Complications [n (%)] Urethral stricture 3 (5.8) 2 (3.9) 1.000 Obturator reflex 8 (15.4) 1 (2.0) 0.031 Secondary operation [n (%)] 14 (26.9) 17 (33.3) 0.478 Tumor residual rate at the second operation [n (%)] 3 (21.4) 4 (23.5) 0.889 Table 2 Comparison of perioperative indicators between the two surgical groups Fig. 7 Surgical process Fig. 7 Surgical process transurethral holmium laser resection; and Fig. 8 is the surgical process about transurethral needle elec- trode resection. Comparison of clinical outcome variables in patients In the subgroup analysis by tumor stage, there was a significant difference in operative time, estimated intraoperative blood loss, and bladder irriga- tion time between the two groups with Ta tumor; there was a significant difference in estimated intraoperative blood loss between the two groups in T1 tumor; and there was a significant difference in estimated intraoper- ative blood loss and bladder irrigation time between the two groups in Tis tumor. Fig. 8 Surgical process Fig. 8 Surgical process Zhou et al. World Journal of Surgical Oncology (2020) 18:166 Page 7 of 9 Table 3 Comparison of tumor disease-free survival rate and progression-free survival rate of the two surgical groups [n (%)] Group Disease-free survival rate Progression-free survival rate 1 year 2 years 3 years 1 year 2 years 3 years Transurethral needle electrode resection 41 (78.8) 35 (71.4) 31 (64.6) 38 (73.1) 30 (61.2) 21 (43.8) Transurethral holmium laser resection 44 (86.3) 41 (82.0) 34 (73.9) 41 (80.4) 36 (72.0) 25 (54.3) P 0.321 0.213 0.328 0.38 0.255 0.304 In the transurethral needle electrode resection group, the 1-, 2-, and 3-year disease-free survival rate was 78.8%, 71.4%, and 64.6%, respectively; the 1-, 2-, and 3-year progression-free survival rate was 73.1%, 61.2%, and 43.8%, respectively. In the transurethral holmium laser resection group, the 1-, 2-, and 3-year disease-free survival rate was 73.1%, 61.2%, and 43.8%, respectively; the 1-, 2-, and 3-year progression-free survival rate was 80.4%, 72.0%, and 54.3%, respectively. There was no significant difference in disease- free survival rate and progression-free survival rate between the two groups (Table 3). time is within 3 months after the surgery), etc., while pathological grade and stage of tumors are associated with tumor progression [6–10]. Traditional TURBT has been widely used in clinical practice, but this method requires the removal of the tumor in sections and layers. During the operation, the tumor is morselized and removed. This method violates the principle of no tumor in traditional surgery and may lead to tumor cell dispersion and implantation, increas- ing the recurrence rate of bladder tumor after surgery. Secondly, this kind of operation inevitably destroys the tumor layer, and the specimen may miss the muscle layer, resulting in the inaccuracy of postoperative patho- logical staging, thus affecting subsequent treatment. Comparison of clinical outcome variables in patients In addition, some specific sites, such as tumors located near the lateral posterior wall of the bladder, are prone to ob- turator nerve reflex during surgery because the pelvic segment of the obturator nerve travels along the lateral wall of the pelvis and is close to the lateral posterior wall of the bladder [11–16]. There was no significant difference in the periopera- tive value of HGF, TSH, and TNF-α between the two groups (all P > 0.05, Table 4). After the resection, the transurethral holmium laser resection group had a sig- nificant decrease in the levels of HGF (10.89 ± 2.47 vs. 16.80 ± 1.45), TSH (2.4 ± 0.4 vs. 3.6 ± 0.3), and TNF-α (76.01 ± 4.82 vs. 99.14 ± 6.01 days) as compared to the transurethral needle electrode resection group (all P > 0.05, Table 4). Currently, the treatment options of NMIBC include surgical treatment, postoperative cystic-perfusion chemotherapy, and cystic-perfusion immunotherapy. The surgical treatment includes transurethral resection of bladder tumor, transurethral laser surgery, and photo- dynamic therapy. TURBT is the current “gold standard” for the treatment of NMIBC and also the main treat- ment [17–19]. The objectives of TURBT are as follows: first, complete removal of all tumors within the field of vision to achieve the effect of radical surgical resection; second, resection of tissues for evaluation of pathological grade and stage, determination of the next postoperative treatment plan of the patient, and assessment of progno- sis and risk of recurrence. In 1966, lasers began to be used in urology. This technique has been widely used in clinical practice for its advantages of safety, simplicity, *Three days after the operation Discussion Bladder tumors can be divided into two categories: stage Tis, Ta, and T1 non-muscle-infiltrating bladder tumors, and above T2 muscle-infiltrating bladder tumors. NMIBC, also known as superficial bladder tumors, is confined to the mucosa (Ta-Tis) and submucosa (T1) and accounts for about 80 ± 5%. Muscular invasive blad- der tumors account for 20 ± 5%. The proportion of Ta, T1, and Tis lesions in NMIBC is about 70%, 20%, and 10%, respectively. According to different recurrence probabilities and prognosis risks, NMIBC can be divided into three levels: low risk, medium risk, and high risk. Risk factors for recurrence are the number of primary tumors, the university, the degree of infiltration and the recurrence probability (especially whether the recurrence Table 4 Comparison of relevant perioperative and postoperative serum indexes of the two surgical groups Group HGF(μmol/L) TSGF(U/mL) TNF-α(μg/mL) Preoperative Postoperative* Preoperative Postoperative* Preoperative Postoperative* Transurethral needle electrode resection (n = 52) 34.88 ± 4.10 16.80 ± 1.45 5.4 ± 0.8 3.6 ± 0.3 220.33 ± 12.51 99.14 ± 6.01 Transurethral holmium laser resection (n = 51) 34.51 ± 4.12 10.89 ± 2.47 5.6 ± 0.7 2.4 ± 0.4 216.75 ± 12.68 76.01 ± 4.82 P 0.647 < 0.001 0.153 < 0.001 0.245 < 0.001 *Three days after the operation Page 8 of 9 Zhou et al. World Journal of Surgical Oncology (2020) 18:166 Page 8 of 9 Zhou et al. World Journal of Surgical Oncology (2020) 18:166 less damage, and definite curative effect. In recent years, holmium laser, green laser, and thulium laser have been developed and put into clinical use, showing a good clin- ical application prospect. Holmium laser was used to treat bladder tumors in the mid-1990s and has been widely used because of its good cutting and electro- coagulation, shallow tissue penetration, and low heat loss [20–23]. less damage, and definite curative effect. In recent years, holmium laser, green laser, and thulium laser have been developed and put into clinical use, showing a good clin- ical application prospect. Holmium laser was used to treat bladder tumors in the mid-1990s and has been widely used because of its good cutting and electro- coagulation, shallow tissue penetration, and low heat loss [20–23]. postoperative recovery, and lower recurrence rate versus traditional transurethral needle electrode resection. Ethics approval and consent to participate This was a prospective study involving human subjects treated according to the standard clinical practice. All permissions granted by competent Ethics Committees and other authorities were obtained, and all informed consents required by the existing law and regulations were collected. Availability of data and materials The datasets during and/or analyzed during the current study are available from the corresponding author on reasonable request. Received: 31 March 2020 Accepted: 3 July 2020 Received: 31 March 2020 Accepted: 3 July 2020 Received: 31 March 2020 Accepted: 3 July 2020 In our study, the operative time, estimated intraopera- tive blood loss, postoperative gross hematuria time, blad- der irrigation time, and postoperative hospitalization time were all significantly lower in the transurethral hol- mium laser resection group as compared to the trans- urethral needle electrode resection group. After resection, transurethral holmium laser resection signifi- cantly decreased the value HGF, TSH, and TNF-α as compared to transurethral needle electrode resection. The incidence of obturator reflex was significantly lower in the transurethral holmium laser resection group than the transurethral needle electrode resection group. References 1. Antoni S, Ferlay J, Soerjomataram I, Znaor A, Jemal A, Bray F. Bladder cancer incidence and mortality: a global overview and recent trends. Eur Urol. 2017;71(1):96–108. 2. Chavan S, Bray F, Lortet-Tieulent J, Goodman M, Jemal A. International variations in bladder cancer incidence and mortality. Eur Urol. 2014;66(1): 59–73. 3. Dy GW, Gore JL, Forouzanfar MH, Naghavi M, Fitzmaurice C. Global burden of urologic cancers, 1990-2013. Eur Urol. 2017;71(3):437–46. 4. Aron M. Variant histology in bladder cancer-current understanding of pathologic subtypes. Current urology reports. 2019;20(12):80. 5. Gurram S, Muthigi A, Egan J, Stamatakis L. Imaging in localized bladder cancer: can current diagnostic modalities provide accurate local tumor staging? Current urology reports. 2019;20(12):82. 6. Bladder Cancer Treatment (PDQ(R)): Patient Version. In: PDQ Cancer Information Summaries. edn. Bethesda (MD); 2002. 7. Richters A, Aben KKH, Kiemeney L. The global burden of urinary bladder cancer: an update. World J Urol. 2019. 8. Veskimae E, Espinos EL, Bruins HM, Yuan Y, Sylvester R, Kamat AM, Shariat 3. Dy GW, Gore JL, Forouzanfar MH, Naghavi M, Fitzmaurice C. Global burden of urologic cancers, 1990-2013. Eur Urol. 2017;71(3):437–46. 4. Aron M. Variant histology in bladder cancer-current understanding of pathologic subtypes. Current urology reports. 2019;20(12):80. 5. Gurram S, Muthigi A, Egan J, Stamatakis L. Imaging in localized bladder cancer: can current diagnostic modalities provide accurate local tumor staging? Current urology reports. 2019;20(12):82. adder Cancer Treatment (PDQ(R)): Patient Version. In: PDQ Cancer 6. Bladder Cancer Treatment (PDQ(R)): Patient Version Information Summaries. edn. Bethesda (MD); 2002. 7. Richters A, Aben KKH, Kiemeney L. The global burden of urinary bladder cancer: an update. World J Urol. 2019. Funding g This study was performed without funding. g This study was performed without funding. Holmium laser is a pulsed solid-state laser, which gener- ates strong heat in an instant and can be absorbed by super- ficial tissues efficiently. Its tissue penetration depth is 4 mm, and it has good vaporization cutting and coagulation hemostatic ability, which can precisely cut tumor tissues to effectively avoid bladder perforation. In addition, the hol- mium laser does not generate an electric field during surgery and can completely eliminate the obturator nerve reflex. It is suitable for patients with cardiac pacemaker and arrhythmia. It is worth noting that the temperature of the local tissue can be as high as 100 ~ 300 °C [28]. In contrast, the local tissue temperature during laser treatment of NMIBC is only 40 ~ 75 °C, and the thermal damage of the surrounding tissues is relatively small. Therefore, postoperative wound healing is fast and the indwelling catheter time is short. Authors’ contributions YZ, ZLZ, and HYM contributed to the study conception and design. YZ, ZLZ, and MHL contributed to data collection and analysis. YZ and ZLZ contributed to the literature review and drafting of the manuscript. HYM contributed to the critical review and approval of the final manuscript. The authors read and approved the final manuscript. Discussion The limitations of this study include the small number of cases and lack of randomized controlled study with TURBT, so it is still necessary to carry out a large-scale randomized con- trolled clinical study with long-term follow-up to verify its long-term therapeutic effect. Acknowledgements Acknowledgements None. Abbreviations TURBT is the preferred surgical method for the treatment of NMIBC at present. It mainly relies on electrothermal effect to remove tumors. Due to the physical characteristics of electrotomy, this surgical method has some unavoidable defects. The TURBT circuit current can easily induce obturator nerve re- flex during tumor resection of the lateral wall of the bladder. Strong obturator nerve reflex can cause com- plications such as bladder perforation. If the tumor location is unique, risks such as ureteral orifice stric- ture, urethral stricture, tumor residue, and dissemi- nated implantation may occur after TURBT, as well as disadvantages such as easy burning of tumor speci- mens to form eschar and difficulty in accurate patho- logical staging [24–27]. CIS: Carcinoma in situ; HGF: Hepatocyte growth factor; NMIBC: Non-muscular invasive bladder cancer; RC: Radical cystectomy; TNF-α: Tumor necrosis factor alpha; TSGF: Tumor-specific growth factor; RC: Radical cystectomy; ECOG: Eastern Cooperative Oncology Group; PS: Performance status; CT: Computed tomography; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; TURBT: Transurethral resection of bladder tumor; BCG: Bacille Calmette-Guérin; BMI: Body mass index Competing interests Th h d l h The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 31 March 2020 Accepted: 3 July 2020 Publisher’s Note of urothelial and nonurothelial histological variants of bladder cancer in predicting oncological outcomes in patients with muscle-invasive and metastatic bladder cancer? A European association of urology muscle invasive and metastatic bladder cancer guidelines panel systematic review European urology oncology. 2019;2(6):625–42. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 9. Taylor J, Becher E, Steinberg GD. Update on the guideline of guidelines: non-muscle-invasive bladder cancer. BJU Int. 2019. 10. Babjuk M, Burger M, Comperat EM, Gontero P, Mostafid AH, Palou J, van Rhijn BWG, Roupret M, Shariat SF, Sylvester R, et al. European Association of Urology Guidelines on Non-muscle-invasive Bladder Cancer (TaT1 and Carcinoma In Situ) - 2019 Update. Eur Urol. 2019;76(5):639–57. Carcinoma In Situ) - 2019 Update. Eur Urol. 2019;76(5):639–57. 11. Timo K. Nykopp, Jose Batista da Costa, Miles Mannas, Peter C Black: Current clinical trials in non-muscle invasive bladder cancer. Curr Urol Rep. 2018; 19(12):101. 12. Baumeister P, Zamboni S, Mattei A, et al. Histological variants in non-muscle invasive bladder cancer. Transl Androl Urol. 2019;8(1):34–8. 13. Akand M, Muilwijk T, Raskin Y, et al. Quality control indicators for transurethral resection of non-muscle-invasive bladder cancer. Clin Genitourin Cancer. 2019;17(4):e784–92. 14. Onishi T, Sugino Y, Shibahara T, et al. Randomized controlled study of the efficacy and safety of continuous saline bladder irrigation after transurethral resection for the treatment of non-muscle-invasive bladder cancer. BJU Int. 2017;119(2):276–82. 15. Hayashida Y, Miyata Y, Matsuo T, et al. A pilot study to assess the safety and usefulness of combined transurethral endoscopic mucosal resection and en-bloc resection for non-muscle invasive bladder cancer. BMC Urol. 2019; 19(1):56. 16. Onishi T, Sekito S, Shibahara T, et al. The role of continuous saline bladder irrigation after transurethral resection in patients with high-grade non- muscle-invasive bladder cancer. Scand J Urol. 2018;52(5-6):385–8. 17. Ghandour R, Singla N, Lotan Y. Treatment options and outcomes in nonmetastatic muscle invasive bladder cancer. Trends in cancer. 2019;5(7): 426–39. 18. Soria F, Marra G, D'Andrea D, Gontero P, Shariat SF. The rational and benefits of the second look transurethral resection of the bladder for T1 high grade bladder cancer. Translational andrology and urology. 2019;8(1): 46–53. 19. Dobruch J, Borowka A, Herr HW. Clinical value of transurethral second resection of bladder tumor: systematic review. Urology. 2014;84(4):881–5. 20. Gendy R, Delprado W, Brenner P, Brooks A, Coombes G, Cozzi P, Nash P, Patel MI. Conclusion In the treatment of NMIBC, holmium laser resection has the advantages of fewer complications, better safety, faster 8. Veskimae E, Espinos EL, Bruins HM, Yuan Y, Sylvester R, Kamat AM, Shariat SF, Witjes JA, Comperat EM. What is the prognostic and clinical importance Page 9 of 9 Page 9 of 9 Page 9 of 9 Page 9 of 9 Zhou et al. World Journal of Surgical Oncology (2020) 18:166 (2020) 18:166 Zhou et al. World Journal of Surgical Oncology (2020) 18:166 Publisher’s Note Repeat transurethral resection for non-muscle-invasive bladder cancer: a contemporary series. BJU Int. 2016;117(Suppl 4):54–9. 21. Babjuk M, Bohle A, Burger M, Capoun O, Cohen D, Comperat EM, Hernandez V, Kaasinen E, Palou J, Roupret M, et al. EAU guidelines on non- muscle-invasive urothelial carcinoma of the bladder: Update 2016. Eur Urol. 2017;71(3):447–61. 22. Wigmore GJ, Anstey JR, St John A, Greaney J, Morales-Codina M, Presneill JJ, Deane AM, MacIsaac CM, Bailey M, Tatoulis J, et al. 20% human albumin solution fluid bolus administration therapy in patients after cardiac surgery (the HAS FLAIR study). J Cardiothorac Vasc Anesth. 2019;33(11):2920–7. 23. Rolevich AI. Evmenenko AA: [A systematic review and meta-analysis to assess the recurrence-free survival in non-muscle invasive bladder cancer after transurethral resection guided by 5-aminolevulinic acid-induced photodynamic diagnosis compared with white-light transurethral resect]. Urologiia. 2016;4:137–46. 24. Balci M, Tuncel A, Keten T, et al. Comparison of monopolar and bipolar transurethral resection of non-muscle invasive bladder cancer. Urol Int. 2018;100(1):100–4. 24. Balci M, Tuncel A, Keten T, et al. Comparison of monopolar and bipolar transurethral resection of non-muscle invasive bladder cancer. Urol Int. 2018;100(1):100–4. 25. Gordon PC, Thomas F, Noon AP, et al. Long-term outcomes from re- resection for high-risk non-muscle-invasive bladder cancer: a potential to rationalize use. Eur Urol Focus. 2019;5(4):650–7. 25. Gordon PC, Thomas F, Noon AP, et al. Long-term outcomes from re- resection for high-risk non-muscle-invasive bladder cancer: a potential to rationalize use. Eur Urol Focus. 2019;5(4):650–7. 26. Li C, Liang G, Zhang J, et al. The effect of holmium laser resection versus standard transurethral resection on non-muscle-invasive bladder cancer: a systematic review and meta-analysis. Lasers Med Sci. 2020;31. 27. Zapała P, Dybowski B, Poletajew S, et al. Clinical rationale and safety of restaging transurethral resection in indication-stratified patients with high- risk non-muscle-invasive bladder cancer. World J Surg Oncol. 2018;16(1):6. 27. Zapała P, Dybowski B, Poletajew S, et al. Clinical rationale and safety of restaging transurethral resection in indication-stratified patients with high- risk non-muscle-invasive bladder cancer. World J Surg Oncol. 2018;16(1):6. 28. Muta M. Issa: Technological advances in transurethral resection of the prostate: bipolar versus monopolar TURP. J Endourol. 2008;22(8):1587–95. 28. Muta M. Issa: Technological advances in transurethral resection of the prostate: bipolar versus monopolar TURP. J Endourol. 2008;22(8):1587–95.
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Teaching Shared Decision Making to Family Medicine Residents: A Descriptive Study of a Web-Based Tutorial
JMIR medical education
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JMIR MEDICAL EDUCATION JMIR MEDICAL EDUCATION Dion et al Original Paper Abstract Background: DECISION+2, a Web-based tutorial, was designed to train family physicians in shared decision making (SDM) regarding the use of antibiotics for acute respiratory infections (ARIs). It is currently mandatory for second-year family medicine residents at Université Laval, Quebec, Canada. However, little is known about how such tutorials are used, their effect on knowledge scores, or how best to assess resident participation. Objective: The objective of our study was to describe the usage of this Web-based training platform by family medicine residents over time, evaluate its effect on their knowledge scores, and identify what kinds of data are needed for a more comprehensive analysis of usage and knowledge acquisition. Methods: We identified, collected, and analyzed all available data about participation in and current usage of the tutorial and its before-and-after 10-item knowledge test. Residents were separated into 3 log-in periods (2012-2013, 2013-2014, and 2014-2015) depending on the day of their first connection. We compared residents’participation rates between entry periods (Cochran-Armitage test), assessed the mean rank of the difference in total scores and category scores between pre- and posttest (Wilcoxon signed-rank test), and compared frequencies of each. Subsequent to analyses, we identified types of data that would have provided a more complete picture of the usage of the program and its effect on knowledge scores. Results: The tutorial addresses 3 knowledge categories: diagnosing ARIs, treating ARIs, and SDM regarding the use of antibiotics for treating ARIs. From July 2012 to July 2015, all 387 second-year family medicine residents were eligible to take the Web-based tutorial. Out of the 387 eligible residents, 247 (63.8%) logged in at least once. Their participation rates varied between entry periods, most significantly between the 2012-2013 and 2013-2014 cohorts (P=.006). For the 109 out of 387 (28.2%) residents who completed the tutorial and both tests, total and category scores significantly improved between pre- and posttest (all P values <.001). However, the frequencies of those answering correctly on 2 of the 3 SDM questions did not increase significantly (P>.99, P=.25). Distribution of pre- or posttest total and category scores did not increase between entry periods (all P values >.1). Available data were inadequate for evaluating the associations between the tutorial and its impact on the residents’ scores and therefore could tell us little about its effect on increasing their knowledge. Teaching Shared Decision Making to Family Medicine Residents: A Descriptive Study of a Web-Based Tutorial Maxime Dion1,2, BSc; Ndeye Thiab Diouf1,3, MSc; Hubert Robitaille1, PhD; Stéphane Turcotte1, MSc; Rhéda Adekpedjou1,4, MPHM, MD; Michel Labrecque1,5, MSc, CCFP, FCFP, MD, PhD; Michel Cauchon1,5, CCFP, FCFP, MD; France Légaré1,5, MSc, CCFP, FCFP, MD, PhD 1Population Health and Practice-Changing Research Group, CHU de Québec Research Centre, Saint-François-d'Assise Hospital, Quebec, QC, Canada 2Department of Mathematics and Statistics, Université Laval, Quebec, QC, Canada 3Department of Community Health, Université Laval, Quebec, QC, Canada 4Department of Social and Preventive Medicine, Université Laval, Quebec, QC, Canada 5Department of Family Medicine and Emergency Medicine, Université Laval, Quebec, QC, Canada Corresponding Author: France Légaré, MSc, CCFP, FCFP, MD, PhD Population Health and Practice-Changing Research Gro CHU de Québec Research Centre Saint-François-d'Assise Hospital 10, rue de l'Espinay Quebec, QC, G1L 3L5 Canada Phone: 1 418 525 4437 Fax: 1 418 525 4194 Email: France.Legare@mfa.ulaval.ca Email: France.Legare@mfa.ulaval.ca JMIR Med Educ 2016 | vol. 2 | iss. 2 | e17 | p. 1 (page number not for citation purposes) http://mededu.jmir.org/2016/2/e17/ Introduction Acute respiratory tract infections (ARIs) are the main cause of consultation in family medicine units in North America [1]. Despite numerous evidence-based guidelines [2-8] demonstrating that antibiotics are ineffective for treating most ARIs [9-12], primary care physicians seem unable to break the habit [13,14]. Widespread overuse of antibiotics for treating ARIs ultimately creates antibiotic resistance [15,16]. In a shared decision making (SDM) approach, health professionals explain the risks and benefits of the available treatment options to patients based on the best available scientific data and take into account patients’ values and preferences before making the treatment decision together [17,18]. Over the years, SDM has been recognized as an effective strategy for reducing the overuse of treatment options not clearly associated with benefits for patients [19]. Despite the willingness of policy makers in many industrialized countries to implement SDM in their health care systems, implementation has not been widespread in clinical practice [20], and few medical curricula include SDM training [21,22]. For this situation to change, SDM should be taught as early as possible in medical training and also as part of continuing education programs [23-25]. The potential of Web-based learning as an instructional tool for medical education has been recognized for many years [34,35]. However, little is known about how effective Web-based learning is for increasing SDM knowledge among physicians [36], how to evaluate usage and participation, and what kind of data are needed for these purposes. The objectives of this study were, therefore, to (1) describe the use of this Web-based training platform by the family medicine residents over time and its effect on knowledge scores (primary outcomes) and (2) note any gaps in data available for these purposes (secondary outcome). Web-based learning has become an increasingly popular approach to medical education [26,27] and is now ubiquitous in university education [28]. Although some have raised concerns about its effectiveness [27], Web-based learning modules have proved to be efficient in targeting many types of health professionals [26,29,30] for various purposes, including reducing the overuse of antibiotics. Little et al [30] recently conducted a study to assess the impact of a Web-based training intervention that aims to optimize the prescription of antibiotics for ARIs among general practitioners in 6 European countries. Their training program showed that Web-based training to enhance communication skills significantly contributed to a decrease in the prescription of antibiotics for treating ARIs. KEYWORDS decision making; patient participation; education, medical, graduate; educational measurement; program evaluation; computer-assisted instruction Laval and for all health care professionals in the university’s family practice training units (FPTUs) [25,32]. Its final version contained a Web-based tutorial entitled “Shared decision making to treat ARI,” a 2-hour workshop in the form of a classroom course, and a decision aid. The impact of the full DECISION+2 was assessed as part of a cluster randomized trial that included 9 FPTUs and first- and second-year residents [33]. The results of this study showed that DECISION+2 contributed to reducing the number of patients deciding to use antibiotics for ARIs by facilitating their involvement in the treatment decision (to take antibiotics or not). The Web-based tutorial is currently mandatory for second-year family medicine residents at Université Laval, Quebec, Canada. Introduction Although they did not focus specifically on SDM, there is widespread consensus that risk communication skills are one of its most important components [31]. Web-based programs have also been shown to be popular among residents. In 2005, Cook et al led a randomized controlled trial to find out whether there was a difference in internal medicine residents’preferences between a Web-based module and printed materials and the level of knowledge achieved [26]. They found that the participants preferred the Web-based module because it saved time and concluded that Web-based learning was effective, well-accepted, and efficient [26]. (JMIR Med Educ 2016;2(2):e17) doi: 10.2196/mededu.6442 (JMIR Med Educ 2016;2(2):e17) doi: 10.2196/mededu.6442 Abstract Conclusion: Residents’ use of this Web-based tutorial appeared to increase between entry periods following the changes to the SDM program, and the tutorial seemed less effective for increasing SDM knowledge scores than for diagnosis or treatment scores. http://mededu.jmir.org/2016/2/e17/ JMIR Med Educ 2016 | vol. 2 | iss. 2 | e17 | p. 1 (page number not for citation purposes) JMIR Med Educ 2016 | vol. 2 | iss. 2 | e17 | p. 1 (page number not for citation purposes) XSL•FO RenderX XSL•FO RenderX JMIR MEDICAL EDUCATION Dion et al However, our results also highlight the need to improve data availability before participation in Web-based SDM tutorials can be properly evaluated or knowledge scores improved. JMIR Med Educ 2016 | vol. 2 | iss. 2 | e17 | p. 2 (page number not for citation purposes) http://mededu.jmir.org/2016/2/e17/ The Web-Based Tutorial The SDM training program has changed twice over 3 years. It was introduced in the family medicine residency program in 2011 as a multicomponent program: a Web-based tutorial entitled “Shared decision making to treat ARI,” a 2-hour workshop in the form of a classroom course, and a decision aid. However, the 2-hour workshop was withdrawn after the 2012-2013 residency period, leaving only the Web-based tutorial and the decision aid. The rationale for this removal was the time constraints in the residents’ schedule. Before July 2014, the Web-based tutorial contained 5 modules including information about diagnosis, treatment, and key components of the SDM process in the treatment of ARIs in primary care. After the 2013-2014 residency period, a sixth module on integrating the knowledge acquired in the first 5 modules was added. This addition also integrated parts of the workshop that had been removed 1 year earlier. Figure 1 shows a timeline of the major modifications made over the 3 residency periods. The SDM tutorial was offered to all second-year residents in the entire network of 12 FPTUs of the Department of Family Medicine and Emergency Medicine at Université Laval, Quebec, Canada. This tutorial is one of the several tutorials available In 2010, our team developed a multicomponent intervention (DECISION+2) for family medicine residents at Université http://mededu.jmir.org/2016/2/e17/ JMIR MEDICAL EDUCATION Dion et al [33]. The tutorial included videos, exercises, a link to access the decision aid, and a pre-post knowledge test. This tutorial was designed to be completed in 2-3 hours. It was required as part of the family medicine curriculum but not specifically evaluated. However, at the end of their residency, residents were examined on all the subjects they learned via the intranet. via the intranet on the department’s Web platform. At the beginning of their second year of residency, residents were given the link and invited to complete the tutorial and the pre-posttests over the course of the year. On the Department home page, residents entered their student identification number and password to access the tutorial. Then, they encountered a brief description of the tutorial before starting it (Textbox 1) Textbox 1. DECISION+2, a Web-based self-tutorial in shared decision making. Participants We included all second-year family medicine residents in the Department of Family Medicine and Emergency Medicine at Université Laval from July 2012 to July 2015 who logged in on the department’s Web platform to register for the tutorial, whether they completed it or not. Residency begins on July 15 of each year. The knowledge test used for the pretest and the posttest contained 10 multiple-choice questions and was only available in French (Université Laval is a French-language university). It was based on information in the tutorial and contained key elements considered by the authors as essential knowledge for the practice of SDM regarding the use of antibiotics for treating ARIs. Some questions were multiple response while others were single response, and participants did not know which kind they were answering. Four questions were concerned with diagnosis (all single response), 3 were concerned with treatment (2 single response and 1 multiple response), and 3 were concerned with SDM (1 single response and 2 multiple response; Multimedia Appendix 1). The knowledge scores were displayed as follows: for single response items, 1 point for a correct answer and 0 points for any incorrect answer; for multiple response items, 1 point if all answers were correct and 0 if any answer was missing or incorrect. The maximum score was 10 points. Residents could only see their scores at the end of the posttest questions. The Web-Based Tutorial For example, a resident who logged in to the tutorial on March 23, 2013, was classified in the 2012-2013 cohort because he or she began between July 15, 2012, and July 14, 2013. We considered the tutorial as completed if the residents answered all the pretest and posttest questions. of their first connection to the tutorial. For example, a resident who logged in to the tutorial on March 23, 2013, was classified in the 2012-2013 cohort because he or she began between July 15, 2012, and July 14, 2013. We considered the tutorial as completed if the residents answered all the pretest and posttest questions. JMIR Med Educ 2016 | vol. 2 | iss. 2 | e17 | p. 4 (page number not for citation purposes) The Web-Based Tutorial Module 1: Introduction • Introduce the shared decision-making process and acute respiratory infections Module 2: Diagnostic probabilities • Know the most useful signs and symptoms for the diagnosis of acute respiratory infections • Integrate notions of diagnostic probabilities • Know how to use diagnostic tools Module 3: Treatment • Know evidence on the effects of antibiotics in treating acute respiratory infections • Integrate the concepts of probability associated with the effects of antibiotics in treating acute respiratory infections • If the option for antibiotics is selected, choose which one Module 4: Effective communication of risk and benefits • Understand the essential elements of effective communication of treatment options and their benefits and risks • Use the communication tool on the benefits and risks associated with using antibiotics or not to treat acute respiratory infections Module 5: Promoting active patient participation • Ask questions related to patient preferences and values, such as questions regarding their concerns about the benefits and risks associated with taking antibiotics or not • Use a visual tool to help patients clarify their values and preferences about the benefits and risks associated with taking an antibiotic or not • Verify patient comfort with the decision made Module 6 (added after July 2014): Integrate all acquired knowledge • Estimate diagnostic probabilities • Effectively communicate the benefits and risks • Identify the values and the preferences of the patient • Promote an informed choice based on the best evidence available and that reflects what is important for the patient Textbox 1. DECISION+2, a Web-based self-tutorial in shared decision making. Textbox 1. DECISION+2, a Web-based self-tutorial in shared decision making. Module 2: Diagnostic probabilities JMIR Med Educ 2016 | vol. 2 | iss. 2 | e17 | p. 3 (page number not for citation purposes) http://mededu.jmir.org/2016/2/e17/ http://mededu.jmir.org/2016/2/e17/ XSL•FO RenderX Dion et al JMIR MEDICAL EDUCATION Figure 1. History of Université Laval SDM training program between July 2012 and July 2015. SDM: shared decision making; ARIs: acute respiratory tract infections. niversité Laval SDM training program between July 2012 and July 2015. SDM: shared decision making; ARIs: acute respirato Figure 1. History of Université Laval SDM training program between July 2012 and July 2015. SDM: shared decision making; ARIs: acute respiratory tract infections. of their first connection to the tutorial. Statistical Analysis We performed simple descriptive statistics including frequencies, median, and interquartile range (minimum and maximum) to summarize characteristics and modalities of use among all family medicine residents who logged in to the Web-based tutorial, and to understand how residents used the tutorial. Also, we estimated proportions of family medicine residents, per period, who logged in to the tutorial, did the pretest only, did the posttest only, or did both. The Cochran-Armitage test for trend was performed to test the change in proportions of family medicine residents who logged in over time. Because the knowledge score was an ordinal variable and did not respect the normality assumption, we used nonparametric tests. To describe the change in the level of knowledge among those who completed the tutorial, we used Wilcoxon signed-rank test to identify significant pre- or posttest differences between the total scores and scores on each of the 3 knowledge categories (diagnosis, treatment, SDM). This kind of test is used especially for paired samples. We used McNemar test to measure if residents answered the questions correctly after doing the tutorial. Finally, the distribution of knowledge total scores and category scores between periods were compared using Mann-Whitney U test. We considered a difference statistically significant when the P value was <.05. We performed statistical analysis using the SAS version 9.4 (SAS Institute Inc). Data Collection To describe the participation in and usage of the DECISION+2 Web-based tutorial used to teach SDM at Université Laval and its effect on knowledge scores, we collected all available data about (1) its content and scoring system; (2) its history and the incentives offered for undertaking it; (3) residents’participation in and usage of the tutorial; and (4) changes in their pre-post test scores. In observing the history of the SDM program, we noted any changes made to it and the reasons for change. Data for tutorial registrations between July 2012 and July 2015 were extracted from department’s Web platform. Data included identification number of each registrant, date of the first and last connection, frequency of connection, total time spent on the tutorial, and answers to each of the pretest and posttest questions. With the connection frequencies we were able to determine how many registered residents started the tutorial. Residents who logged in were separated into 3 entry periods (2012-2013, 2013-2014, and 2014-2015) depending on the day http://mededu.jmir.org/2016/2/e17/ XSL•FO RenderX XSL•FO RenderX JMIR MEDICAL EDUCATION Dion et al Web-based tutorial was part of an academic program and data were provided anonymously. Participants’ Use of the Tutorial All 387 second-year family medicine residents were eligible to take the Web-based tutorial. Out of the 387 residents, 247 (63.8%) logged in to the Web-based tutorial. Among the 247 who logged in, 109 (44.1%) completed both the pre- and posttest, 95 (38.5%) completed the pretest only, 2 (0.8%) completed the posttest only, and 41 (16.6%) logged in but did not complete either test. In total, only 28.2% (109/387) of all eligible family medicine residents completed the tutorial (Figure 2). Table 1 shows simple descriptive statistics of participants for the different entry periods. Proportions of women and the median number of connections were similar between cohorts. However, a relative increase in the median of time spent in the tutorial was observed between the 2013-2014 and 2014-2015 cohorts. Also, the proportion of registered residents who logged in to the tutorial per entry period was 53.7% (65/121) in 2012-2013, 67.7% (90/133) in 2013-2014, and 69.2% (92/133) in 2014-2015 (Cochran-Armitage test; P=.006). Participation rates increased between each succeeding entry period and seemed more pronounced between the 2012-2013 and 2013-2014 cohorts. JMIR Med Educ 2016 | vol. 2 | iss. 2 | e17 | p. 5 (page number not for citation purposes) JMIR Med Educ 2016 | vol. 2 | iss. 2 | e17 | p. 5 http://mededu.jmir.org/2016/2/e17/ Ethics P valueb Posttest, median (IQR) Pretest, median (IQRa) Category <.001 7 (6-8) 4 (3-5) All (out of 10) <.001 3 (2-4) 2 (1-2) Diagnosis (out of 4) <.001 3 (2-3) 2 (1-2) Treatment (out of 3) <.001 1 (1-1) 1 (0-1) Shared decision making (out of 3) aIQR: interquartile range. bDifference assessed with Wilcoxon signed-rank test. P values do not represent the median difference but represent improvement in the mean rank of the difference in scores between the pre- and posttests. Dion et al JMIR MEDICAL EDUCATION Figure 2. Flowchart of the participants. Figure 2. Flowchart of the participants. improved significantly between the pre- and posttest (Wilcoxon signed-rank test; all P values <.001). Table 3 shows the frequencies of those who answered each question correctly pre- and posttest. More participants answered the posttest questions correctly (McNemar test; all P values <.01) except for questions 7, 8, and 9 (the SDM category). In addition, low frequencies of those who answered correctly were observed for both pre- and posttest on questions 8 and 9. Family Medicine Residents’ Knowledge Scores Twelve out of 109 residents who completed both tests had a posttest score equal to or lower than their pretest score, that is, the percentage of participants whose total knowledge score did not improve was 11%. Table 2 shows the medians and the interquartile ranges in the total knowledge scores and in each category among those who completed the tutorial. The median total knowledge score and each of the 3 category scores Table 2. Medians and interquartile ranges in the knowledge total scores and for each category of those who completed the tutorial. P valueb Posttest, median (IQR) Pretest, median (IQRa) Category <.001 7 (6-8) 4 (3-5) All (out of 10) <.001 3 (2-4) 2 (1-2) Diagnosis (out of 4) <.001 3 (2-3) 2 (1-2) Treatment (out of 3) <.001 1 (1-1) 1 (0-1) Shared decision making (out of 3) aIQR: interquartile range. bDifference assessed with Wilcoxon signed-rank test. P values do not represent the median difference but represent improvement in the mean rank of the difference in scores between the pre- and posttests. ans and interquartile ranges in the knowledge total scores and for each category of those who completed the tutorial. aIQR: interquartile range. Q q g bDifference assessed with Wilcoxon signed-rank test. IQR: interquartile range. bDifference assessed with Wilcoxon signed-rank test. P values do not represent the median difference but represent improvement in the mean rank of the difference in scores between the pre- and posttests. Ethics As the study was supported by the institution where it was performed, no ethical approval was requested because the Table 1. Description of the characteristics and modalities of use of family medicine residents who entered the tutorial. Total 2014-2015 2013-2014 2012-2013 Characteristics N=247 n=92 n=90 n=65 Gender, n (%) 193 (78) 70 (76) 69 (77) 54 (83) Female 54 (22) 22 (24) 21 (23) 11 (17) Male Number of connections 2 3 3 2 Median (1, 4) (2, 4) (1, 5) (1, 4) IQRa 1-14 1-10 1-14 1-7 Range Total time passed (hours) 2.22 2.77 1.87 1.54 Median (0.38, 3.32) (1.24, 3.87) (0.49, 3.10) (0.09, 2.71) IQRa 0.00-19.98 0.00-19.98 0.00-9.29 0.00-10.13 Range Tests done, n (%) 41 (17) 8 (9) 15 (17) 18 (28) None 95 (38) 17 (18) 34 (38) 44 (68) Pretest only 2 (1) 0 (0) 2 (2) 0 (0) Posttest only 109 (44) 67 (73) 39 (43) 3 (4) Pre- and posttest a il Table 1. Description of the characteristics and modalities of use of family medicine residents who entered the tutorial. http://mededu.jmir.org/2016/2/e17/ XSL•FO RenderX XSL•FO RenderX JMIR MEDICAL EDUCATION Dion et al Figure 2. Flowchart of the participants. Family Medicine Residents’ Knowledge Scores Twelve out of 109 residents who completed both tests had a posttest score equal to or lower than their pretest score, that is, the percentage of participants whose total knowledge score did not improve was 11%. Table 2 shows the medians and the interquartile ranges in the total knowledge scores and in each category among those who completed the tutorial. The median total knowledge score and each of the 3 category scores improved significantly between the pre- and posttest (Wilcoxon signed-rank test; all P values <.001). Table 3 shows the frequencies of those who answered each question correctly pre- and posttest. More participants answered the posttest questions correctly (McNemar test; all P values <.01) except for questions 7, 8, and 9 (the SDM category). In addition, low frequencies of those who answered correctly were observed for both pre- and posttest on questions 8 and 9. Table 2. Medians and interquartile ranges in the knowledge total scores and for each category of those who completed the tutorial. Ethics Medians and interquartile ranges in the knowledge total scores and for each category between the 2013-2014 and essed with Mann-Whitney U test. P values do not represent the median difference but represent a distribution difference bDistribution difference assessed with Mann-Whitney U test. P values do not represent the median difference but represent a distribution difference between the 2 entry periods. Table 4 shows the medians and the interquartile ranges, and the P values of the 2-sided Wilcoxon rank-sum test performed to verify whether the distribution of total scores and the category scores pre- and posttest were the same between entry periods. The 2012-2013 period was not assessed because only 3 family medicine residents completed the tutorial during that period. All P values were greater than 5%, that is, there was no significant difference in the distribution of the total score or in any of the category scores for the pre- and posttests between the 2013-2014 and 2014-2015 cohorts. on knowledge scores were limited. For example, sex was the only demographic data available, and data on time spent on the tutorial by residents per connection period, which pages they visited per connection period, and their participation in the classroom workshop were not available. JMIR Med Educ 2016 | vol. 2 | iss. 2 | e17 | p. 7 (page number not for citation purposes) Ethics P values do not represent the median difference but represent improvement in the mean rank of the difference in scores between the pre- and posttests. q g bDifference assessed with Wilcoxon signed-rank test. P values do not represent the median difference but represent improvement in the mean rank of the difference in scores between the pre- and posttests. JMIR Med Educ 2016 | vol. 2 | iss. 2 | e17 | p. 6 (page number not for citation purposes) JMIR Med Educ 2016 | vol. 2 | iss. 2 | e17 | p. 6 (page number not for citation purposes) http://mededu.jmir.org/2016/2/e17/ http://mededu.jmir.org/2016/2/e17/ XSL•FO RenderX JMIR MEDICAL EDUCATION Table 3. Frequencies of those who correctly answered each question. P valuea Posttest, n (%) Pretest, n (%) Category Diagnosis .008 106 (97.2) 96 (88.1) Question 1 <.001 64 (58.7) 14 (12.8) Question 2 <.001 68 (62.4) 27 (24.8) Question 3 <.001 90 (82.6) 41 (37.6) Question 4 Treatment <.001 101 (92.7) 40 (36.7) Question 5 <.001 90 (82.6) 36 (33.0) Question 6 .41 103 (94.5) 100 (91.7) Question 7 Shared decision making >.99 6 (5.5) 6 (5.5) Question 8 .25 13 (11.9) 9 (8.3) Question 9 <.001 92 (84.4) 66 (60.6) Question 10 aTutorial effect assessed with McNemar test. Dion et al JMIR MEDICAL EDUCATION Table 3. Frequencies of those who correctly answered each question. aTutorial effect assessed with McNemar test. aTutorial effect assessed with McNemar test. Table 4. Medians and interquartile ranges in the knowledge total scores and for each category between the 2013-2014 and 2014-2015 cohorts. P valueb 2014-2015, median (IQR) 2013-2014, median (IQRa) Knowledge test Pretest .17 4 (3, 5) 4 (3, 5) All categories .96 1 (1, 2) 2 (1, 2) Diagnosis .11 2 (1, 2) 1 (1, 2) Treatment .69 1 (0, 1) 1 (0, 1) SDMc Posttest .95 7 (6, 8) 7 (6, 8) All categories .45 3 (3, 4) 3 (2, 4) Diagnosis .36 3 (2, 3) 3 (3, 3) Treatment .98 1 (1, 1) 1 (1, 1) SDM aIQR: interquartile range. bDistribution difference assessed with Mann-Whitney U test. P values do not represent the median difference but represent a distribution difference between the 2 entry periods. cSDM: shared decision making and interquartile ranges in the knowledge total scores and for each category between the 2013-2014 and 2014-2015 cohorts. the knowledge total scores and for each category between the 2013-2014 and 2014-2015 cohorts. Table 4. http://mededu.jmir.org/2016/2/e17/ Principal Findings This study described the residents’use of a Web-based training platform over time and attempted to assess whether the residents’ knowledge scores about the diagnosis and treatment of ARIs and SDM regarding the use of antibiotics for ARIs Overall, we observed that the data available regarding residents’ participation in, use of, and effects of the Web-based tutorial XSL•FO RenderX XSL•FO RenderX JMIR MEDICAL EDUCATION Dion et al Dion et al evaluation of knowledge acquired. In this era of rapidly growing numbers of SDM training programs [40] and national efforts to offer them on the Web platform, the most effective methods of delivery and evaluation urgently need to be standardized. were improved by this Web-based training. It also provided an opportunity to identify what kinds of data are appropriate for evaluating the usage of the training platform and its impact on knowledge. The main results were that residents’ use of the Web-based tutorial increased over time, but not their knowledge scores; residents appeared to perform better on knowledge scores about diagnosing ARIs and treatment options than on SDM; just over a quarter completed the tutorial and one-third did not even start it; and little data appropriate for evaluating the course’s effectiveness were available. Our results led us to make 4 main observations. were improved by this Web-based training. It also provided an opportunity to identify what kinds of data are appropriate for evaluating the usage of the training platform and its impact on knowledge. The main results were that residents’ use of the Web-based tutorial increased over time, but not their knowledge scores; residents appeared to perform better on knowledge scores about diagnosing ARIs and treatment options than on SDM; just over a quarter completed the tutorial and one-third did not even start it; and little data appropriate for evaluating the course’s effectiveness were available. Our results led us to make 4 main observations. Third, a difficulty we encountered was inadequacy of data organization or availability at the university level, which made it hard to evaluate the usage of the SDM training and its effects on knowledge. Indeed, the data available were not adequate for evaluating associations between participation in the tutorial and its impact on the residents’ knowledge scores. Moreover, no data were available that could inform us about whether the loss of the workshop component of the SDM program caused the increase in residents’ use of the tutorial. Limitations The limitations of this study included contamination by residents who spent more than 1 year completing their second-year residency, and potential confounding variables. Lack of available demographic data compounded these problems. In terms of the tutorial’s effectiveness, participants were not classified by FPTU. Therefore, we cannot be sure that belonging to the same FPTU did not influence their answers. Also, participants might have logged in to the tutorial and then, rather than doing the tutorial, left the connection open and done something else for several hours (offline) before disconnecting. This might have distorted the time shown as spent on the tutorial. Finally, the psychometric properties of the pre- and post-knowledge tests had not been validated, and therefore scores might not have been valid, consistent, or reliable. Second, our results suggested that the Web-based tutorial had a significantly positive effect on knowledge scores about the diagnosis and treatment of ARIs. However, the questions that the most residents failed were in the SDM category. This could be because the questions were poorly written, or it could be due to the nature of SDM. Unlike diagnosis or treatment, SDM is a subject that is inherently antithetical to unidirectional learning—it is about person-to-person communication and sharing information [17,18]. Perhaps Web-based tutorials performed in solitude are not an appropriate platform for teaching some of the essentials components of SDM to family medicine residents, namely presenting options, communicating risks and benefits, and clarifying values of patients. Moreover, knowledge scores may be an inadequate form of evaluation for SDM. However, in a 2013 randomized controlled trial on physician communication regarding prostate cancer screening, Feng et al assessed a 30-minute Web-based module and found, at 3-month follow-up, that the family physicians who used the Web-based tutorial had more shared decision-making behaviors and were more likely to encourage patients to consider different screening options compared with usual education [38]. Together, these results suggested that Web-based learning about SDM needs to be reassessed in further studies. They might also reflect the significant heterogeneity among SDM training programs [39], not only in their content but in their modes of delivery and Principal Findings In addition, although all participants were second-year family medicine residents, we were unable to collect any demographic data on participants except for gender. If we had been able to analyze data extracted from log-in dates, visited pages, and time passed on each page per connection, our interpretation would have been more meaningful. To improve a Web-based training such as this, more information is needed about how the residents use the tutorial, and data collection needs to be adapted to reflect modifications in the program when they take place [41]. With the increase in Web-based interventions, the potential for data extraction is growing exponentially [42]. Moreover, sophisticated data analysis methods already exist that take account of the structure of more complex data such as this [43]. Our findings highlight the need to strengthen partnerships with residency programs so that data are made available in an appropriate form to be useful for evaluation purposes, both by faculties and by researchers. First, we reported an increase in the participation rate between the 2012-2013 and 2013-2014 cohorts. This improvement could be explained by the removal of the workshop component of the course because 2012-2013 was the last year in which it was offered in addition to the Web-based tutorial. Perhaps family medicine residents saw the Web-based tutorial as an unnecessary addition to the classroom workshop on SDM in their curriculum and saw it as too much time spent on the topic. Some studies suggest that such Web-based tutorials should be brief and not too complex or intensive for medical students [24,37]. Furthermore, the average increase of about 1 hour in the median of the time spent in the tutorial observed between the 2013-2014 and 2014-2015 cohorts could have been caused by the addition of the sixth module. However, the added module was supposed to integrate all the knowledge acquired in the first 5 modules and compensate for the removal of the classroom workshop. We expected to see an increase in the distribution of the knowledge scores (total and per category) between these 2 periods, but this was not the case. Perhaps the inclusion of an additional review module was not relevant and may even have unnecessarily extended the duration of the training. More data would be needed to confirm this. JMIR Med Educ 2016 | vol. 2 | iss. 2 | e17 | p. 8 (page number not for citation purposes) http://mededu.jmir.org/2016/2/e17/ Authors' Contributions FL, ML, and MC participated in the concept and design of the study. MD, NTD, HR, and RA participated in data acquisition. MD and ST performed the statistical analysis. All authors were involved in interpretation of the data and critical revision for important intellectual content of the manuscript. MD and FL drafted the manuscript. MD and HR coordinated the study. All authors read and approved the final manuscript. Multimedia Appendix 1 Knowledge test. [PDF File (Adobe PDF File), 33KB-Multimedia Appendix 1] Acknowledgments The authors wish to thank Louisa Blair for editing this manuscript. Conclusions Residents’ use of this Web-based tutorial appeared to increase between entry periods following the changes to the SDM program, and the tutorial seemed less effective in the SDM categories than in the diagnosis and treatment categories. However, to evaluate the use of a Web-based tutorial properly and its impact on knowledge, data collection needs to include the different log-in dates, visited pages, time passed on each page per connection, and more complete sociodemographic characteristics. There is still work to be done to improve data sharing, quality, and availability for evaluation purposes, so XSL•FO RenderX XSL•FO RenderX JMIR MEDICAL EDUCATION Dion et al that implementation of SDM in the context of antibiotics use for treating ARIs becomes a feature of everyday family practice. References 1. Schappert SM, Rechtsteiner EA. 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JMIR Public Health Surveill 2016;2(1):e10 [FREE Full text] [doi: 10.2196/publichealth.5018] [Medline: 27227145] Edited by CL Parra-Calderón, G Eysenbach; submitted 03.08.16; peer-reviewed by T Irizarry, K Yakubu; comments to author 22.11.16; revised version received 05.12.16; accepted 07.12.16; published 19.12.16 Please cite as: Dion M, Diouf NT, Robitaille H, Turcotte S, Adekpedjou R, Labrecque M, Cauchon M, Légaré F Teaching Shared Decision Making to Family Medicine Residents: A Descriptive Study of a Web-Based Tutorial JMIR Med Educ 2016;2(2):e17 URL: http://mededu.jmir.org/2016/2/e17/ doi: 10.2196/mededu.6442 Abbreviations Edited by CL Parra-Calderón, G Eysenbach; submitted 03.08.16; peer-reviewed by T Irizarry, K Yakubu; comments to author 22.11.16; revised version received 05.12.16; accepted 07.12.16; published 19.12.16 Please cite as: Dion M, Diouf NT, Robitaille H, Turcotte S, Adekpedjou R, Labrecque M, Cauchon M, Légaré F Teaching Shared Decision Making to Family Medicine Residents: A Descriptive Study of a Web-Based Tutorial JMIR Med Educ 2016;2(2):e17 URL: http://mededu.jmir.org/2016/2/e17/ doi: 10.2196/mededu.6442 PMID: 27993760 ©Maxime Dion, Ndeye Thiab Diouf, Hubert Robitaille, Stéphane Turcotte, Rhéda Adekpedjou, Michel Labrecque, Michel Cauchon, France Légaré. Originally published in JMIR Medical Education (http://mededu.jmir.org), 19.12.2016. 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, first published in JMIR Medical Education, is properly cited. The complete bibliographic information, a link to the original publication on http://mededu.jmir.org/, as well as this copyright and license information must be included. JMIR Med Educ 2016 | vol. 2 | iss. 2 | e17 | p. 11 (page number not for citation purposes) JMIR Med Educ 2016 | vol. 2 | iss. 2 | e17 | p. 11 (page number not for citation purposes) http://mededu.jmir.org/2016/2/e17/ http://mededu.jmir.org/2016/2/e17/
https://openalex.org/W2339819427
https://iris.unimore.it/bitstream/11380/1150614/2/Single-Phase%20series%20active%20filter.pdf
English
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Single‐phase series active power filter with transformer‐coupled matrix converter
IET power electronics
2,016
cc-by
8,400
1 Introduction Many solutions are based on proportional–integral (PI) or PI–derivative controllers, with different tuning approaches. In [14], artificial neural networks (ANNs) are used. Linear control theory is used in [15] through Bode diagrams to tune the controller of a single-phase shunt active filter, and in [16] through the Routh criterion to determine the stability bounds. Active power filters (APFs) are used to eliminate unwanted frequency components from power lines or sensitive loads. They were proposed in the 1970s, but they rose to mainstream utilisation only recently, thanks to advances in power electronics (with the emergence of fast and efficient power switches such as metal-oxide-semiconductor field effect transistors (MOSFETs) and insulated gate bipolar transistors (IGBTs)) and microelectronics (with the advent of cheap and abundant embedded computing power in microcontrollers, digital signal processors (DSPs), field-programmable gate arrays (FPGAs)). Furthermore, some researchers are applying predictive controllers (PCs) to APFs. PCs promise the fastest dynamic response but are very sensitive to parameter drift [13, 20]. A safer alternative with fast dynamic response is hysteresis controllers. In addition to being less sensitive to parameter variations, hysteresis controllers have intrinsic peak current limiting capabilities, a feature that is generally lacking in PCs [21]. In [2], the authors propose a solution in which a hysteresis controller drives a UPQC with series capacitors that enable operation with reduced DC-link voltage. APFs can be divided into three main categories: shunt filters, series filters and unified power quality conditioners (UPQCs), that are the combination of a series and a shunt filter [1–3]. Shunt filters operate in parallel with the load and are usually employed to compensate the harmonics introduced by non-linear loads connected to the grid. They are generally very efficient due to the fact that they need to draw only the unwanted current components. On the other hand, series filters carry the whole load current but are more versatile, being able to operate on the fundamental as well as on the unwanted harmonic components [4]. They are employed to protect sensitive loads in polluted grid scenarios [5]. The possibility to reduce, or even to eliminate, DC energy storage components is very appealing, since the DC-link capacitor is among the weakest and most reliability-critical components in an electronic power converter. Among the converter architectures operating without DC-link capacitors, some researchers are studying the use of matrix converters (MCs) in APFs for both three-phase [5, 22] and single-phase applications [23]. 1 Introduction Series filters need galvanic isolation between the converter and the power line in order to avoid short-circuit paths. Isolation is usually achieved using a transformer, either inside the converter (high-frequency transformer) or at the output of the converter (line frequency transformer) [6–9]. MCs were first proposed in the 1980s, but viable real-world applications took long to take off since, even to this day, some technological challenges remain, such as non-ideal commutations of the power devices and the consequent voltage and current distortions [24]. MCs can connect any input line to any output line through bidirectional switching devices, and their main advantage is the elimination of the reactive components used for energy storage (line inductors in CSIs, bus capacitors in VSIs) [25]. The goal of eliminating unwanted harmonic components can be logically divided into two tasks: detecting the unwanted components and filtering them out. Some authors stress the importance of the detection stage to the overall performance of the filter [9–11] while, in most other cases, a closed-loop architecture makes it difficult to separate detection from filtering [12–16]. This paper employs a series active filter, based on a single-phase MC (SPMC), to protect sensitive loads from the effects of voltage disturbances at the point of common coupling. The SPMC feeds a step-down transformer connected in series with the load. The voltage waveform inserted by the transformer is added to the distorted grid voltage, obtaining a pure sinusoidal voltage waveform across the load. The use of a step-down transformer allows to employ power switches with lower current ratings, while the series connection enables a limited control of the fundamental voltage amplitude. The key advantage of this solution is that, with respect to a back-to-back converter (Fig. 1a), it requires less passive components (the grid side inductor and DC-link capacitor) Several solutions can be found in the existing literature for the control of APFs. Many authors use variations of the synchronous reference frame or d–q transform control with the attractive feature of controlling constant quantities in steady state. In [17], a d–q controller is enhanced with the use of an adaptive pole placement technique for optimal control. In [18], the d–q control is applied to a current-source inverter (CSI) that ensures rapid control response and intrinsic short-circuit protection, but can result in lower efficiency with respect to voltage-source inverters (VSIs). IET Power Electron., 2016, Vol. 9, Iss. 6, pp. 1279–1289 & The Institution of Engineering and Technology 2016 Single-phase series active power filter with transformer-coupled matrix converter ISSN 1755-4535 Received on 13th June 2015 Revised on 27th November 2015 Accepted on 16th January 2016 doi: 10.1049/iet-pel.2015.0443 www.ietdl.org ISSN 1755-4535 Received on 13th June 2015 Revised on 27th November 2015 Accepted on 16th January 2016 doi: 10.1049/iet-pel.2015.0443 www.ietdl.org Giampaolo Buticchi1 ✉, Davide Barater2, Carlo Concari2, 1Chair of Power Electronics, University of Kiel, Kiel, Germany 2Department of Information Engineering, University of Parma, Parma, Italy ✉E-mail: gibu@tf.uni-kiel.de Giampaolo Buticchi1 ✉, Davide Barater2, Carlo Concari2, Giovanni Franceschini2 1Chair of Power Electronics, University of Kiel, Kiel, Germany 2Department of Information Engineering, University of Parma, Parma, Italy ✉E-mail: gibu@tf.uni-kiel.de Abstract: This study presents a series active power filter based on a single-phase matrix converter. Back-to-back voltage- source converters with nested control loops and passive filters are normally used for this application. A matrix converter, as proposed in this study, allows a simpler implementation both in terms of hardware (no grid filter and no DC link) and of control. Moreover, a novel zero-voltage compensation technique is introduced in order to reduce the distortion around the input voltage zero crossing. Simulations and experiments are used to validate the considered control methods, confirming the feasibility of the proposed architecture. [19]. Also, in this case the controlled variables are constant in ideal steady-state conditions. IET Power Electronics IET Power Electronics Research Article 1 Introduction A similar approach considers the use of the instantaneous active and reactive power theory (p–q control) to control active filters 1279 Fig. 1 Active filter topologies a Back-to-back single-phase series active filter b Topology of the proposed active filter Fig. 1 Active filter topologies a Back-to-back single-phase series active filter b Topology of the proposed active filter Fig. 1 Active filter topologies 2 Active filter control strategy and a simple control is sufficient. Moreover, with the proposed modulation strategy, no current sensors are needed. Instead, a back-to-back converter requires a fast grid current control and DC-link voltage control [26]. The drawback of the MC solution is the reduced compensation capabilities due to the absence of a stable DC voltage. Moreover, the input current quality of the MC is inferior, since the back-to-back converter can absorb sinusoidal current [27]. The proposed circuit is shown in Fig. 1b. Every bidirectional switch consists of two IGBTs in series, with the emitters in common, driven by the signals SxA and SxB. Lf, Cf constitute the output filter for rejecting the high-frequency harmonics introduced by the modulation, whereas Cin is the input filter capacitor. The input filter of a MC represents a non-trivial design topic, since it must be ensured that the high-frequency current harmonics generated by the converter do not pollute the grid voltage or current. For this reason, in a three-phase MC, an inductor–capacitor (LC) filter is normally adopted, and particular care is taken to avoid resonance (by adding a resistor in parallel with the inductor) [30]. Considering a single-phase application, and that normally the transformer of the APF is designed with a turn ratio smaller than one, effectively reducing the input current, for this application a single capacitor at the input is adopted. This solution proved to be satisfying even considering a wide range of line impedance values. The topology adopted in this work was already proposed in literature [23] for the protection of sensitive loads from fast grid voltage variations (i.e. voltage sags and swells) and harmonic pollution. Nevertheless, the strategy in [23] did not involve a closed-loop control: the voltage waveform to compensate the distorted grid voltage was calculated by subtracting the desired load voltage vload from the sampled grid voltage vgrid. This method is very fast to compensate voltage sags or swells, but the harmonics rejection accuracy can be poor, since all the control variables are extracted from the input grid voltage and no feedback is provided from the load. Moreover, the control is sensitive to variations of the system parameters, since they cannot be compensated as with closed-loop controls. y g g g p The block scheme of the APF control is shown in Fig. 2. 2 Active filter control strategy The load voltage is processed by an array of harmonic compensators, and the result is the MC voltage set point, v∗ mx. To generate the correct output voltage, a duty cycle m for the MC is computed dividing the voltage In [28], the same control strategy proposed in [23], but with a different pulse-width modulation (PWM), is implemented to compensate only the harmonic distortion of the grid voltage. In this case, a low-pass filter (LPF) in the d–q reference frame is employed to detect the fundamental component vgrid of the grid voltage, which is then subtracted from the polluted grid voltage to generate the compensating waveform. Fig. 2 Block scheme of the control g p g In this paper, the active filter’s performance is enhanced by a harmonic closed-loop compensator based on the fast Fourier transform. Furthermore, a zero-voltage compensation strategy is proposed to reduce the output voltage distortion that occurs in SPMCs in correspondence with the input voltage zero crossing. A version of this paper appeared in the proceedings of the IEEE IECON 2013 conference [29]. This paper is better organised, describes the control strategy in detail, presents a thorough review of the state of the art, reports further experimental results and proposes a novel PWM technique that allows removing the output current sensor. The paper is organised as follows. Section 2 describes the proposed converter and the control strategy. Section 3 describes the PWM technique and the zero-voltage compensation strategy, comparing the adopted control techniques with the previous works proposed in literature. The following sections describe and discuss the simulations and experiments used to validate the proposed system; the last section reports the concluding remarks. In this paper, the active filter’s performance is enhanced by a harmonic closed-loop compensator based on the fast Fourier transform. Furthermore, a zero-voltage compensation strategy is proposed to reduce the output voltage distortion that occurs in SPMCs in correspondence with the input voltage zero crossing. A version of this paper appeared in the proceedings of the IEEE IECON 2013 conference [29]. This paper is better organised, describes the control strategy in detail, presents a thorough review of the state of the art, reports further experimental results and proposes a novel PWM technique that allows removing the output current sensor. The paper is organised as follows. Section 2 describes the proposed converter and the control strategy. 2 Active filter control strategy Section 3 describes the PWM technique and the zero-voltage compensation strategy, comparing the adopted control techniques with the previous works proposed in literature. The following sections describe and discuss the simulations and experiments used to validate the proposed system; the last section reports the concluding remarks. Fig. 2 Block scheme of the control Fig. 2 Block scheme of the control Fig. 2 IET Power Electron., 2016, Vol. 9, Iss. 6, pp. 1279–1289 & The Institution of Engineering and Technology 2016 1280 Fig. 3 Simplified system model and Thevenin equivalent circuit of the system a Simplified system model b Thevenin equivalent of the system at the low-voltage side of the transformer set point by the input voltage, m = v∗ mx/vgrid. The PWM block generates the correct switching sequence for the converter, as will be thoroughly explained in the following section. Regarding the harmonic detection algorithms, several solutions are present in literature. In the following, the most relevant will be briefly described, and then a choice will be made. In [31], the instantaneous reactive power (IRP) theory is used; in a three-phase system transformed in αβ coordinates, the average of the instantaneous power is related to the fundamental frequency, while the oscillating part corresponds to the harmonics. In [11, 32], the harmonics are separated in the d–q current frame, so that the fundamental components assume DC values and can be easily isolated with low-pass filtering. A modified approach that uses the decomposition of multi-phase voltage vectors to represent different power components is presented in [9]. A further possibility is to use an array of adaptive second-order generalised integrators [33] and isolate the harmonics of the output voltage. Unfortunately, given the limited frequency separation between the grid harmonics, very narrow bandwidth must be ensured for this solution, triggering potential numerical stability problems. A straightforward solution is to perform the discrete Fourier transform (DFT) of the output voltage. As a matter of fact, this method allows to obtain the coefficients of the Fourier series with an extremely high rejection of the other harmonics. In [34], the Fryze–Buchholz–Depenbrock power theory was proposed. This theory states that a utility-connected load that absorbs non-sinusoidal currents can be decomposed into conductance and susceptance at the different harmonics. Once these values are calculated, it is possible to isolate the harmonics via low-pass filtering. ANNs [35] were also employed to detect harmonics in power systems. 2 Active filter control strategy Kalman filters (KF) can also be adopted for this kind of operation. Fig. 3 Simplified system model and Thevenin equivalent circuit of the system a Simplified system model b Thevenin equivalent of the system at the low-voltage side of the transformer b Thevenin equivalent of the system at the low-voltage side of the transformer and Z2 mainly represents the transformer leakage inductance at the low-voltage side (grid side). For the analysis, the output filter capacitor and the transformer capacitance were not taken into account, since only the low-frequency behaviour of the circuit was considered. The equivalent Thevenin circuit of the system, at the low-voltage side of the transformer, is depicted in Fig. 3b and the system equations are given in the following equation The aforementioned compensation strategies were evaluated in [36]. Each solution presented good harmonic rejection capabilities, in particular the KF and the DFT methods presented similar accuracy, with the KF being more sensitive to measurement noise. The IRP and the d–q decomposition are suitable only for single-phase systems. While the DFT is apparently the most demanding methods in terms of computation and memory requirements, the recursive DFT (RDFT) can replace the DFT in real-time applications and obtain the same accuracy. Considering the extensive work present in literature, and limiting the choice to single-phase and low-cost systems, it is the opinion of the authors that the RDFT represents the best solution, being more robust than the KF with high noise and demanding less computational power than the ANN approach. The only drawback is the increased working memory needed to store the old samples. vm−eq = vmxZm n(Z1 + Zm) Zeq = Z1//Zm n2 Z1 = Rf + s(Lf + L1) Z2 = R2 + sL2 Zm = sLm Ztot = Z2 + Zeq Iload = Vload Rl Vload = (Vgrid −vm−eq) Rl (Rl + Ztot) ⎧ ⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩ (1) (1) Considering the circuit inFig. 1b, it is possibleto obtain the simplified model of the system as shown in Fig. 3a. The voltage generator vmx simulates the MC output voltage, and impedance Z1 represents the matrix output filter inductor plus the transformer leakage inductance. Zm takes into account the magnetising inductance of the transformer In the RDFT scheme, the harmonics of vload are isolated by DFTs tuned at different frequencies. PI controllers on the in-phase and quadrature components of the DFT allow infinite gain at the harmonic frequencies (Fig. 4). IET Power Electron., 2016, Vol. 9, Iss. 6, pp. 1279–1289 & The Institution of Engineering and Technology 2016 2 Active filter control strategy It is important to note that in Fig. 4 the Fig. 4 Block scheme of the RDFT harmonic compensator Fig. 4 Block scheme of the RDFT harmonic compensator IET Power Electron., 2016, Vol. 9, Iss. 6, pp. 1279–1289 & The Institution of Engineering and Technology 2016 IET Power Electron., 2016, Vol. 9, Iss. 6, pp. 1279–1289 & The Institution of Engineering and Technology 2016 1281 switches is used to turn off the transistor whose antiparallel diode is conducting, thus preventing input short circuits [25]. implementation of the RDFT window can be realised in a computational-efficient way with circular buffers. For completeness, it should be said that the scheme is very similar to a PI in a synchronous reference frame (with poles on the imaginary axis). The use of the moving average allows to completely decouple the different harmonics. In this case, Hf can be expressed as in (2), where a is the ratiobetween the proportional andintegralgains of the controller In [39], an evolution of this technique is adopted for a SPMC operating as an AC/DC converter. Instead of the current through each bidirectional switch, the output current of the converter is used to choose the adequate switching sequence. This method can still lead to open load conditions in proximity of the current zero crossing, since errors in the sampled signals can always be present. This issue is normally addressed by a specific clamp circuit offering a free-wheeling path for the output current. Hf =  n=3,5,7 K 2as s2 + (nv0)2 + 2(s2 −(nv0)2) (s2 + (nv0)2) 2   (1/Ts(1 −e−sTs)) (2) (2) A similar four-step switching sequence can be realised with the information of the voltage across the bidirectional switches. Differently from the previous case, three devices are gated in specific time intervals. The short circuit is avoided by gating devices with a series diode that cannot be turned on. This is the method used in [28], where the sign of the input voltage (i.e. the grid voltage) is taken as reference for the commutation sequences. It is worth to be noted that in [28] all the driving signals are disabled during a narrow window around the input voltage zero crossings, in order to avoid possible short circuits due to measurement errors or to a change of the input polarity. 2 Active filter control strategy the maximum variation of the input voltage that can happen over a switching cycle). 2 Active filter control strategy Considering that the active filter must have satisfactory performance with arbitrary load, it must be made sure that particular load conditions do not cause resonance problems. While the non-linear and resistive/ inductive load does not present particular challenges for the harmonic compensator, as will be shown in the simulations, a capacitive load can resonate with the converter’s output filter. In this case, considering a purely capacitive load Cload, the transfer function of the output filter (from the output of the converter to vmx) can be written as A hybrid approach was pursued in [29]. If the information of the grid voltage is considered reliable, i.e. above a certain threshold, voltage-based commutation is used. Otherwise, the bidirectional switches commutate with the current sequence. Due to the fact that the SPMC feeds a transformer, the magnetising current should create a sufficient phase displacement between voltage and current to allow proper commutation. If both the current and the voltage are below the threshold that allows a safe commutation, current commutation is preferred. Open load situations are then addressed by the use of small RC snubbers. The presence of the snubbers is justified also by the fact that they prevent overvoltages due to the stray inductance of the tracks. The principal drawback with respect to [28, 39] is the need for two sensors: one for the input voltage and one for the SPMC output current. Hout = Lm s2LmLfCload/n2 + Lf + Lm (3) (3) Obviously, considering that the transformer magnetising inductance is much larger than the output filter one, resonance between the filter inductors and the load can happen. A simple method to damp this resonance (and obtain a flatter filter output response) is simply to add a virtual resistor in series with the filter inductance (active damping), as in Fig. 2. In this work, the PWM strategy is further simplified by using a different approach when the voltage measurement is unreliable. When the input voltage is very low, the output voltage capability of the MC is obviously limited; for this reason, as a simplification, the MC can be driven in order to generate a null voltage output by turning on S1A-S1B-S3A-S3B (or S2A-S2B-S4A-S4B), as in Fig. 1b, thus short circuiting the output. The MC remains in this state as long as the modulus of the measured input voltage stays below a certain threshold (e.g. 3.2 Input voltage zero-crossing compensation strategy Differently from a three-phase MC, in a SPMC during input voltage zero crossings the output voltage is inherently limited. This limitation results in load voltage distortion if the SPMC output voltage needed for harmonic compensation is different from zero during the grid voltage zero crossing. The converter simply cannot synthesise the desired output voltage around vgrid zero crossings. IET Power Electron., 2016, Vol. 9, Iss. 6, pp. 1279–1289 & The Institution of Engineering and Technology 2016 3 PWM and zero-crossing compensation strategies They often occur simultaneously, but they are addressed separately in the following for clearer presentation. 3 PWM and zero-crossing compensation strategies As mentioned previously, single-phase MCs are particularly appealing for the possibility to downsize power electronics in AC/AC applications, due to the absence of DC bus capacitors. Nevertheless, they inherit some issues from their three-phase counterparts. In fact, the proper commutation of the bidirectional switches requires particular care, since no free-wheeling paths exist in a MC for the load currents. This means that the commutation from one bidirectional switch to another must happen avoiding both short circuiting the input voltages and opening the loads. Since this is a notorious problem also for three-phase MCs, many works exist in literature proposing different approaches to achieve safe commutations. The above strategy is shown in Fig. 5a that reports the gate signals of one leg during a transition of the input voltage with negative derivative. The other leg behaves in a similar way. The four-step commutation restarts when the input voltage reaches the threshold again. To evenly distribute the commutation stress among the devices, during the positive derivative intervals of the input voltage zero crossing the low-side devices can be used instead of the high-side ones. In this way, the PMW control sequence technique requires only one sensor, as in [28, 39]. It is worth to be noted that this method appears similar to that proposed in [23], but differs from it in a key point. In [23], a bidirectional switch was driven by only one signal, and no information was reported about how to avoid short circuits. Furthermore, SPMCs are affected by another major issue: during the input voltage zero crossings the output voltage is inherently limited. This limitation leads to voltage distortion if the output voltage desired by the control is different from zero during the grid voltage crossover. The zero-crossing limitation affects only single-phase MCs; multiphase MCs are intrinsically immune [37, 38]. These two problems represent key issues for the control of single-phase MCs. They often occur simultaneously, but they are addressed separately in the following for clearer presentation. Furthermore, SPMCs are affected by another major issue: during the input voltage zero crossings the output voltage is inherently limited. This limitation leads to voltage distortion if the output voltage desired by the control is different from zero during the grid voltage crossover. The zero-crossing limitation affects only single-phase MCs; multiphase MCs are intrinsically immune [37, 38]. These two problems represent key issues for the control of single-phase MCs. 4 Stability analysis The active filter and the proposed control strategies were simulated in the MATLAB/Simulink environment with the PLECS toolbox. The circuit in Fig. 1b was implemented with the simulation parameters of Table 1. Third, fifth and seventh harmonics were added to the 50 Hz fundamental component of the grid voltage, obtaining the expression (5). The resulting input THD was 5.3%. The load was represented by a resistor Rl Stability and robustness analyses were performed in order to prove the correct operation of the proposed control strategy. The virtual resistor effect is taken into account in the stability analysis. To this aim, it is important to note that the virtual resistor contribution is affected by a delay due to the sampling. This actually modifies the equation of Z1. The effect of the ZVC is more complex because it is heavily non-linear. To simplify the analysis it can be observed that the procedure of sampling the regulator’s output and adding a sinusoidal signal can be modelled as a low-pass filtering discretised at the grid frequency (ZVC(s)). As a consequence, the closed-loop transfer function of the SPMC acts as a high-pass filter. The equations are reported in (4). vgrid = 230 2 √ sin (vt) + 10 sin (3vt + 3p/10) + 10 sin (5vt) −10 sin (7vt) (5 vgrid = 230 2 √ sin (vt) + 10 sin (3vt + 3p/10) + 10 sin (5vt) −10 sin (7vt) ( (5) Several simulations were performed in order to study the performance of the MC and the control strategy. In particular, the harmonic rejection capabilities of the RDFT controller were examined with and without the zero-voltage compensation strategy. The following simulation result figures show the grid voltage vgrid, the output voltage and current vload and iload, and the MC output voltage vmx. For a = Kp/KI = 1/6, K = 15 the system is stable and presents an infinite gain at the selected harmonics Z1 = Rf + s(Lf + L1) + Rv 1.5s/ fsw + 1 ZVC(s) = 1 1.5s/( fgrid) + 1 1 s/(2p fcut) + 1 v′ mx(s) = vmx 1 1 + ZVC(s) ⎧ ⎪⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎪⎩ (4) From Fig. 7 it is obvious that, without the ZVC, vmx cannot follow the reference in proximity of the grid voltage zero crossing, deteriorating the harmonic rejection as a consequence. 3.1 Bidirectional switches commutation strategy Different approaches are viable in order to achieve safe commutations of the bidirectional switches. With the knowledge of the voltage across the bidirectional switch or the direction of the current, specific switches can be appropriately gated in sequence to avoid dangerous situations. A possible solution is proposed in this paper. Under the hypothesis of odd harmonics compensation, a suitable signal can be added to v∗ mx, in order to make its zero crossings coincide with the input voltage zero crossings. If the operating voltage of the load can be moved around a sufficiently wide window of rms value, a first harmonic can be added to v∗ mx, slightly modifying the amplitude of the first harmonic of vload. The four-step current commutation is one of the most widely adopted solutions for three-phase MCs. With this scheme, information about the current flowing through the bidirectional IET Power Electron., 2016, Vol. 9, Iss. 6, pp. 1279–1289 & The Institution of Engineering and Technology 2016 1282 Fig. 5 Zero-crossing issue a Modified PWM strategy during the zero crossing of the input voltage b Waveforms of the zero-voltage compensation Fig. 5 Zero-crossing issue a Modified PWM strategy during the zero crossing of the input voltage b Waveforms of the zero-voltage compensation Fig. 5 Zero-crossing issue a Modified PWM strategy during the zero crossing of the input voltage b Waveforms of the zero-voltage compensation a Modified PWM strategy during the zero crossing of the input voltage b Waveforms of the zero-voltage compensation Fig. 6 shows the Nyquist diagram results for the RDFT harmonic compensator in the different conditions. In Fig. 6a, the diagram is shown for the nominal conditions without the ZVC. The converter is considered ideal, i.e. the voltage limitations are neglected. In Fig. 6b, the ZVC and virtual resistor are added. As a matter of fact, for a resistive load, the addition of the virtual resistor has no effect. Even the introduction of the ZVC, despite its slow dynamic behaviour, has no effect on system stability. A serious drawback of this solution is that the load voltage amplitude and phase are modified, limiting its applicability to phase sensitive loads. The quadrature signal is the one that allows to partially compensate the zero-crossing distortion with the minimum change to the rms value of the fundamental. 3.1 Bidirectional switches commutation strategy Considering that the SPMC’s output is constrained to the input voltage, it must be considered if the benefits of a better total harmonic distortion (THD) overcome the phase displacement issues. If it is known that the most important operating point coincides with a condition where the SPMC must output a voltage different from zero at the input voltage zero crossing, a specific tuning of the LC filter can be used to prevent the intervention of the zero- voltage compensation (ZVC). The robustness analysis was conducted by changing the output inductance (half the nominal value). As can be seen from Fig. 6c, with the chosen parameters the effect on the stability is negligible. Other parameters were also changed with very little effect. It should be said that a big difference of the SPMC with respect to the back-to-back inverter is that no current loops are present, because the SPMC acts intrinsically as a voltage generator. For this reason, parameters like the filter inductance and the load have little effect on the system stability. The block scheme of Fig. 2 shows that the output of the harmonic compensator is sampled at one of the zero crossings of the grid voltage. This value is then fed through a LPF with a very slow time constant. The output of the filter is the amplitude of the first harmonic in quadrature with the grid voltage which is then added to the harmonic compensator’s output v∗ mx. If only the odd harmonics need to be compensated, the resulting v∗ mx is zero at both zero crossings of the grid voltage. This is illustrated in Fig. 5b, where the system behaviour is shown with and without the zero-crossing compensation strategy. To show the limits of the control, the value of K was increased in Fig. 6d. This modification heavily affects the phase margin and brings the system very close to instability. 4 Stability analysis The simulated THD reaches the almost ideal value of 0.3% with the ZVC, while without the compensation the THD is 0.6%. (4) IET Power Electron., 2016, Vol. 9, Iss. 6, pp. 1279–1289 & The Institution of Engineering and Technology 2016 IET Power Electron., 2016, Vol. 9, Iss. 6, pp. 1279–1289 & The Institution of Engineering and Technology 2016 1283 Fig. 6 Stability analysis with RDFT harmonic compensator a HOL without ZVC and virtual R, phase margin (PM) = 75° b HOL with ZVC and virtual R c HOL with ZVC, virtual R and low load Fig. 6 Stability analysis with RDFT harmonic compensator Table 1 Simulation parameters Name Description Value vgrid grid voltage 230 V rms fgrid grid frequency 50 Hz fsw switching frequency 10 kHz Lf AC inductor filter 0.75 mH Lm Transformer magnetising inductance 120 mH Cf AC output capacitor filter 2.2 μF Cin AC input capacitor filter 2.2 μF n transformer turn ratio 2.8 Rl load resistor 17 Ω Table 1 Simulation parameters oscillations. From the simulations, values in the order of 1–2 Ω are enough to damp the resonance without implying excessive power losses (<1 W). As explained in Section 2, a virtual resistor is added to damp the resonance with capacitive loads. The bode diagram of the output transfer function with the simulation parameters is shown in Fig. 8a, where a 3 kVAR load capacitor and a 2 Ω virtual resistor were employed. To implement this solution, however, a current sensor is needed at the output of the MC, increasing cost and complexity. The simulation results with this purely capacitive load are reported in Fig. 8b, where the output THD equals 0.6%. If the load is not purely capacitive, the intrinsic damping of the circuit should be enough to allow the operation of the active filter even without the active damping. The simulations were repeated with different loads, with the same input harmonic distortion and ZVC enabled. Fig. 7c shows the simulation results when a diode is inserted in series with the resistor Rl = 17 Ω. The performance still remains good, with a THD of 0.4%. It is worth noting that resonance problems of the LCL output filter can happen due to the current harmonics of the non-linear load. 4 Stability analysis In this case, a simple solution, that does not require additional current sensors, is to put a resistor in series with the filter capacitor to passively damp the IET Power Electron., 2016, Vol. 9, Iss. 6, pp. 1279–1289 & The Institution of Engineering and Technology 2016 IET Power Electron., 2016, Vol. 9, Iss. 6, pp. 1279–1289 & The Institution of Engineering and Technology 2016 Fig. 7 Simulation results with RDFT harmonic compensator a No ZVC b ZVC c Simulation results with non-linear load 6 Experimental results A prototype of the active filter was built to test the modulation strategy and the harmonic compensators. It includes the power devices, the gate drivers power supply, the signal conditioners and IET Power Electron., 2016, Vol. 9, Iss. 6, pp. 1279–1289 & The Institution of Engineering and Technology 2016 1284 eescale MPXS2010VLQ120 microcontroller performing signal essing and generating the PWM signals. The test bed was set i d ith th h ti f Fi 1b Th i it with reduced power (Rl = 150 Ω). A picture of the test bed shown in Fig. 9. D t i d ith L HDO 6054 di it l t 7 Simulation results with RDFT harmonic compensator ZVC C mulation results with non-linear load a Freescale MPXS2010VLQ120 microcontroller performing signal processing and generating the PWM signals The test bed was set with reduced power (Rl = 150 Ω). A picture of the test bed shown in Fig 9 Fig. 7 Simulation results with RDFT harmonic compensator a No ZVC b ZVC c Simulation results with non-linear load Fig. 7 Simulation results with RDFT harmonic compensator a No ZVC b ZVC c Simulation results with non-linear load Fig. 7 Simulation results with RDFT harmonic compensator with reduced power (Rl = 150 Ω). A picture of the test bed is shown in Fig. 9. a Freescale MPXS2010VLQ120 microcontroller performing signal processing and generating the PWM signals. The test bed was set up in accordance with the schematic of Fig. 1b. The circuit parameters were the same used for the simulations (Table 1) but Data were acquired with a Lecroy HDO 6054 digital storage oscilloscope through ADP305 (differential voltage) and AP015 IET Power Electron., 2016, Vol. 9, Iss. 6, pp. 1279–1289 & The Institution of Engineering and Technology 2016 1285 (current) probes. The samples were then imported in MATLAB in order to calculate the spectra and plot the figures. The same distorted input waveform was used as in the simulations, with the actual resulting rms value slightly lower due to limitations of the converter used to generate the distorted grid voltage. Fig. 10 shows the results. The same layout of Fig. 7 was kept even for the sampled data in order to facilitate the comparison, and a very good the MC output and voltage oscillations in proximity of crossings are present when the ZVC is not employed. IET Power Electron., 2016, Vol. 9, Iss. 6, pp. 1279–1289 & The Institution of Engineering and Technology 2016 (current) probes. The samples were then imported in MATLAB in order to calculate the spectra and plot the figures. The same distorted input waveform was used as in the simulations, with the actual resulting rms value slightly lower due to limitations of the converter used to generate the distorted grid voltage. Fig. 10 shows the results. The same layout of Fig. 7 was kept even for the sampled data in order to facilitate the comparison, and a very good agreement with the simulations can be seen. Again, distortion of (current) probes. The samples were then imported in MATLAB in order to calculate the spectra and plot the figures. The same distorted input waveform was used as in the simulations, with the actual resulting rms value slightly lower due to limitations of the converter used to generate the distorted grid voltage. Fig. 10 shows the results. The same layout of Fig. 7 was kept even for the sampled data in order to facilitate the comparison, and a very good agreement with the simulations can be seen. Again, distortion of IET Power Electron., 2016, Vol. 9, Iss. 6, pp. 1279–1289 & The Institution of Engineering and Technology 2016 6 Experimental results A version of the MC output and load voltage in proximity o crossing is also reported in Fig. 10c. As can be seen, with the MC output is forced to go to zero, causing a distortio output voltage. With ZVC, this effect is highly mitigated. Fig. 11 shows further details of the experiments. In partic Fig. 8 Operation with capacitive load a Undamped and damped output transfer functions of the MC b Simulation results for pure capacitive load Fig. 9 Picture of the test bed Fig. 8 Operation with capacitive load a Undamped and damped output transfer functions of the MC b Simulation results for pure capacitive load Fig. 8 Operation with capacitive load a Undamped and damped output transfer functions of the MC b Simulation results for pure capacitive load Fig. 9 Picture of the test bed Fig. 9 Picture of the test bed the MC output and voltage oscillations in proximity of the zero crossings are present when the ZVC is not employed. A zoomed version of the MC output and load voltage in proximity of a zero crossing is also reported in Fig. 10c. As can be seen, without ZVC the MC output is forced to go to zero, causing a distortion of the output voltage. With ZVC, this effect is highly mitigated. (current) probes. The samples were then imported in MATLAB in order to calculate the spectra and plot the figures. (current) probes. The samples were then imported in MATLAB in order to calculate the spectra and plot the figures. The same distorted input waveform was used as in the simulations, with the actual resulting rms value slightly lower due to limitations of the converter used to generate the distorted grid voltage. Fig. 10 shows the results. The same layout of Fig. 7 was kept even for the sampled data in order to facilitate the comparison, and a very good agreement with the simulations can be seen. Again, distortion of Fig. 11 shows further details of the experiments. In particular, the input current is reported in Fig. 11a. It is evident that further IET Power Electron., 2016, Vol. 9, Iss. 6, pp. 1279–1289 & The Institution of Engineering and Technology 2016 1286 Experimental results with RDFT harmonic compensator (sampled data acquired by Lecroy HDO6054 DSO) VC d waveform in proximity of the zero crossing Fig. 8 References 1 Khadkikar, V.: ‘Enhancing electric power quality using UPQC – a comprehensive overview’, IEEE Trans. Power Electron., 2012, 27, (5), pp. 2284–2297 A single-phase series APF was developed based on a SPMC. The proposed system can operate bidirectionally and needs no DC capacitor. A four-step voltage commutation technique that employs a single sensor was used, and a particular switching strategy that implies short circuiting the output terminals was proposed to prevent output open-circuit conditions. The control requirements are strongly reduced relative to a standard back-to-back inverter (no need to control the DC link), as well as the number of required sensors (only two voltage sensors) and passive components (no DC-link capacitors nor input inductances). 2 Karanki, K., Geddada, G., Mishra, M., et al.: ‘A modified three-phase four-wire UPQC topology with reduced dc-link voltage rating’, IEEE Trans. Ind. Electron., 2013, 60, (9), pp. 3555–3566 ( ) pp 3 Ketzer, M.B., Jacobina, C.B.: ‘Multivariable load current sensorless controller for universal active power filter’, IET Power Electron., 2014, 7, (7), pp. 1777–1786 4 El-Sadek, M., Ibrahim, E., Kamel, R.: ‘Series active filter for voltage harmonic suppression and 50 hz voltage boosting’. Proc. of MEPCON 2006, El-minia, Egypt, 19–21 December 2006, vol. 1, pp. 59–64 5 Lozano-Garcia, J.M., Ramirez, J.M.: ‘Voltage compensator based on a direct matrix converter without energy storage’, IET Power Electron., 2015, 8, (3), pp. 321–332 A drawback of the MC relative to a back-to-back solution is that the output voltage is not perfectly controllable when the input voltage is close to zero. To mitigate this problem, a zero-voltage compensation technique based on a time shift of the output voltage zero crossing was proposed and tested. This strategy should be used when slight changes in the output rms voltage and phase are allowed. In fact, the first harmonic introduced by the ZVC may limit the remaining voltage available for harmonic compensation. 6 Buticchi, G., Barater, D., Lorenzani, E., et al.: ‘Active common-mode filter for photovoltaic transformerless inverters’. Proc. of IEEE IECON 2012, 2012, pp. 5702–5707 7 Tian, J., Chen, Q., Xie, B.: ‘Series hybrid active power filter based on controllable harmonic impedance’, IET Power Electron., 2012, 5, (1), pp. 142–148 Jianben, L., Shaojun, D., Qiaofu, C., et al.: ‘Modelling and industrial applic 8 Jianben, L., Shaojun, D., Qiaofu, C., et al.: ‘Modelling and industrial application of series hybrid active power filter’, IET Power Electron., 2013, 6, (8), pp. 6 Experimental results 10 Experimental results with RDFT harmonic compensator (sampled data acquired by Lecroy HDO6054 DSO) a No ZVC Fig. 10 Experimental results with RDFT harmonic compensator (sampled data acquired by Lecroy HDO6054 DSO) a No ZVC b ZVC c Zoomed waveform in proximity of the zero crossing c Zoomed waveform in proximity of the zero crossing can be explained with the fact that non-linearities that were not modelled in the simulations, such as the on-state voltage drop of the devices, cannot be compensated by the controllers, and as a consequence a small third harmonic distortion remains. harmonics are produced, but the SPMC is operating as expected. During the peaks of the input voltage the input current decreases (in order to have fundamental current at the output). Fig. 11b shows the start-up transient with the RDFT compensator. The bandwidth of the controller is limited by the RDFT calculation (that introduces a delay of one grid voltage period), but the accuracy is greater than with the other compensator [29]. The fact that a small third harmonic remains does not constitute a stability issue. Theoretically, if a constant third harmonic remained in the output voltage, the infinite gain of the RDFT compensator could lead to numerical instability problem. What actually happens is that the input of the PI regulators (see Fig. 4) oscillates around the zero value. As a matter of fact, the phase of the residual third harmonic is not constant, but changes continually. For completeness, the spectra of the input and output voltages are reported in Fig. 11c with their peak values highlighted. As can be seen, the compensation of the fifth and seventh harmonics is almost perfect, while some third harmonic distortion remains. This IET Power Electron., 2016, Vol. 9, Iss. 6, pp. 1279–1289 & The Institution of Engineering and Technology 2016 1287 Fig. 11 Detail of the experiments with the RDFT compensator a SPMC input current Fig. 11 Detail of the experiments with the RDFT compensator Fig. 11 Detail of the experiments with the RDFT compensator a SPMC input current b Transient response c Spectra of input and output voltages 8 References 1707–1714 9 Mulla, M.A., Rajagopalan, C., Chowdhury, A.: ‘Hardware implementation of series hybrid active power filter using a novel control strategy based on generalised instantaneous power theory’, IET Power Electron., 2013, 6, (3), pp. 592–600 The main drawback is that, differently from a three-phase matrix converter, even if a transformer with unity turn ratio is chosen, the output voltage can never exceed the input voltage. Because of this, particular combinations of harmonics, that need a compensating voltage higher than is available near the input voltage zero crossing, cannot be fully compensated. 10 Asiminoaei, L., Blaabjerg, F., Hansen, S.: ‘Detection is key – harmonic detection methods for active power filter applications’, IEEE Ind. Appl. Mag., 2007, 13, (4), pp. 22–33 11 Biricik, S., Redif, S., Zerdem, Z.C., et al.: ‘Real-time control of shunt active power filter under distorted grid voltage and unbalanced load condition using self-tuning filter’, IET Power Electron., 2014, 7, (7), pp. 1895–1905 IET Power Electron., 2016, Vol. 9, Iss. 6, pp. 1279–1289 & The Institution of Engineering and Technology 2016 1288 12 Buticchi, G., Consolini, L., Lorenzani, E.: ‘Active filter for the removal of the dc current component for single-phase power lines’, IEEE Trans. Ind. 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Design, Preparation and Characterization of a High-Performance Epoxy Adhesive with Poly (Butylacrylate-block-styrene) Block Copolymer and Zirconia Nano Particles in Aluminum- Aluminum Bonded Joints
Journal of inorganic and organometallic polymers and materials
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Design, preparation and characterization of a high- performance epoxy adhesive with Poly (butylacrylate-block-styrene) Block Copolymer and Zirconia nano particles in aluminum- aluminum bonded joints Mojtaba Nikkhah Varkani  University of Isfahan Omid Moini Jazani  (  o.moini@eng.ui.ac.ir ) University of Isfahan Majid Sohrabian  Shahid Beheshti University Amir Torabpour Esfahani  mohsen fallahi  University of Isfahan Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at Journal of Inorganic and Organometallic Polymers and Materials on July 13th, 2023. See the published version at https://doi.org/10.1007/s10904- 023-02790-x. Methods Mechanical properties, thermal stability, and microstructure of the epoxy adhesive was assessed using tensile test, TGA, and FESEM tests, respectively. To investigate the adhesion features, the formulated adhesive was applied in lap joint bonding of an aluminum to aluminum. Background Epoxy adhesives are one of the polymers used as high performance matrix in adhesives. However, the high brittleness and low toughness due to their high-crosslinking degree are critical problems during their service in structural applications. The lack of appropriate thermal stability at high temperature is another drawback of these valuable materials. In this study, the effect of hybrid reinforcement comprising zirconium oxide nanoparticles (NPs), phenolic resin (resol type) and poly (butyl acrylate-block-styrene) copolymer (BCP) on mechanical, adhesion, thermal and morphological properties of the epoxy adhesive was studied. Design, preparation and characterization of a high-performance epoxy adhesive with Poly (butylacrylate-block-styrene) Block Copolymer and Zirconia nano particles in aluminum- aluminum bonded joints Design, preparation and characterization of a high-performance epoxy adhesive with Poly (butylacrylate-block-styrene) Block Copolymer and Zirconia nano particles in aluminum- aluminum bonded joints Mojtaba Nikkhah Varkani1, Omid Moini Jazani1 *, Majid Sohrabian2, Amir Torabpour Esfahani3, Mohsen Fallahi1 1Department of Chemical Engineering, Faculty of Engineering, University of Isfahan, P.O. Box 81746-73441, Isfahan, Iran 2 Department of Mechanical and Energy Engineering, Shahid Beheshti University, Tehran, Iran 3 Department of New Materials and Alloys, Institute of Materials and Energy, Isfahan, Iran * Corresponding Authors: Email address: o.moini@eng.ui.ac.ir (O. Moini Jazani) Highlights: Highlights: 1- A new approach was developed for designing epoxy based adhesives with high mechanical, adhesion and thermal properties by adding butyl acrylate block styrene copolymer, phenolic resin, and zirconia nanoparticles. 1- A new approach was developed for designing epoxy based adhesives with high mechanical, adhesion and thermal properties by adding butyl acrylate block styrene copolymer, phenolic resin, and zirconia nanoparticles. 2- The hybrid of butyl acrylate block styrene copolymer, phenolic resin, and zirconia nanoparticles showed synergistic effects on the aluminium - aluminium single lap joint, butt joint tensile strength, and T-peel strength. 2- The hybrid of butyl acrylate block styrene copolymer, phenolic resin, and zirconia nanoparticles showed synergistic effects on the aluminium - aluminium single lap joint, butt joint tensile strength, and T-peel strength. 3- Temperature enhancement and decrement declined the adhesion strength of the aluminium - aluminium single lap joint strength and butt joint tensile strength. 3- Temperature enhancement and decrement declined the adhesion strength of the aluminium - aluminium single lap joint strength and butt joint tensile strength. 4- Proposed toughening mechanisms for finding structure-property association in epoxy adhesives. 4- Proposed toughening mechanisms for finding structure-property association in epoxy adhesives. Graphical Abstract Significant findings A new approach was developed for designing advanced adhesives with high mechanical, adhesion and thermal properties by adding hybrid additives. Based on the tensile test results, adding 5 phr of zirconium oxide nanoparticles to the epoxy adhesive increased the tensile strength, modulus, and the toughness of the dumbbell-shaped samples by 69%, 33% and 175% as compared with the neat epoxy adhesive, respectively. Furthermore, the highest improvement of shear strength in the single lap joint was observed in the sample containing 10 phr phenolic resin, 5 phr zirconia NPs, and 2.5 phr block copolymer, showing a 420% increment compared to the pure epoxy, reflecting the synergistic impact of these compounds at the mentioned percentage. The TGA results indicated the highest initial degradation temperature in the sample containing 5 phr zirconia NPs which was 54.4℃ higher than that of the pure epoxy. The images of the fracture surface of the optimal samples in the tensile test showed that the cavitation, shear band formation, crack deviation, and crack tip blunting are among the major mechanisms in the increase of the toughness of the samples. Keywords: epoxy adhesive, zirconia nanoparticles, tensile strength, adhesion strength, thermal stability 1. Introduction Epoxy resins have found extensive applications in the production of composites, especially in the engineering fields due to their high load-bearing properties. They are also widely used in coatings due to their excellent chemical resistance and electrical resistivity. As a high- performance polymeric matrix, epoxy resin has found unique applications in the construction, automotive, and aerospace fields [1]. The structure of the epoxy resin, however, suffers from poor resistance against the crack initiation and growth and low toughness due to its high crosslink degree which can limit its applications in the fields requiring high fracture strength. The Tensile strength and toughness of the epoxy resin depend on various factors such as the type of hardener, diluent, and filler [2]. Numerous studies have recently addressed the enhancement of the fracture strength and toughness of the epoxy resin, among which, block copolymers can be mentioned. Block copolymers (BCP) refer to a type of polymers that includes epoxy-phillic and epoxy-phobic blocks within an epoxy matrix which spontaneously form intertwined spherical and cylindrical micelles depending on the block ratios and the content. The fracture toughness can be remarkably enhanced by adding a small amount of these BCPs (less than 5 phr) while showing no significant negative impact on the Tg and modulus [3]. It has been also proven that the incorporation of a small content of nanoparticles into the epoxy adhesive can elevate its toughness, glass transition temperature, and single lap shear strength at room temperature [4]. Studies have revealed that inorganic NPs such as SiO2 [5,6], Nano clay [7], Al2O3 [8], Fe2O3 [9], and GO [10] can improve the mechanical and thermal features of the epoxy resin. Among these NPs, zirconia nanoparticles (ZrO2) have shown high strength, stiffness, corrosion resistance, hardness, and fracture toughness, introducing them as a proper candidate for enhancing the mechanical properties of the polymeric composites [11, 12]. However, a limited number of studies have addressed this material. Brochier et al. assessed the effect of zirconia NPs on the single lap joint of the epoxy adhesives and showed that the incorporation of 1% zirconia NPs can increment the single lap shear strength by 60% [13]. Khosravi et al. reported 27, 62, and 110% enhancement in the tensile strength, stiffness, and fracture toughness of the epoxy/glass fiber upon adding zirconia NPs, respectively [14]. Azizi and Eslami examined the influence of silane-modified zirconia NPs on epoxy/basalt composite. 1. Introduction Their results indicated that the incorporation of 3% zirconia NPs managed to improve the bending strength, bending modulus, bending fracture elongation, tensile strength, tensile modulus, and tensile fracture elongation by 90, 74, 84, 76, 85, and 14%, respectively as compared with samples free of zirconia NPs [15]. Rosa Medina et al. evaluated the effect of zirconia NPs on the epoxy resin. They found that the addition of 10% zirconia NPs can improve the modulus and fracture toughness by 37 and 100%, respectively. Moreover, the incorporation of 8% zirconia NPs enhanced the glass transition temperature by 8% when compared with the pure epoxy samples [16]. Jialiang Li et al. assessed the influence of silane-modified zirconia NPs on the mechanical properties of the epoxy resin, their results indicated that the incorporation of 3% NPs to the epoxy incremented the toughness by 53.3 and 39.4% at room temperature and 90 K, respectively (compared to pure epoxy sample). The reason could be the proper dispersion of zirconia NPs in the epoxy matrix and their good interactions and adhesion to the epoxy matrix. Further incorporation of NPs led to their agglomeration, which declined the mechanical properties [17]. Xiaoqian Ma et al. examined the effect of zirconia NPs on the epoxy resin and indicated that the best mechanical properties could be achieved at 3 wt.% NPs content showing 44, 29, and 63% increment in the tensile strength, tensile modulus, and fracture toughness, respectively (compared to the pure epoxy sample) [18]. Bascom and Cottington reported a 35% decline in the tensile strength of the epoxy resin at 50℃. They also obtained the glass transition temperature of 68℃ for the developed adhesive [19]. Adams et al. assessed the shear strength at room temperature as well as low temperatures and indicated that the stresses due to the adhesive contractions had a lower influence on the single lap strength compared to the thermal stresses [20]. Adams et al. assessed the shear strength at room temperature as well as low temperatures and indicated that the stresses due to the adhesive contractions had a lower influence on the single lap strength compared to the thermal stresses [20]. Bai et al. studied the effect of temperature on the single lap metal joint of carbon-epoxy adhesive composite and showed an 80% reduction in the single lap strength and stiffness at temperatures beyond the glass transition temperature [21]. Lu Ke et al. 1. Introduction addressed the effect of temperature on the single lap joint of carbon-epoxy composite and revealed a 50% reduction in the adhesion strength at 70 ℃ as compared with the room temperature [22]. Banea and da Silva studied the influence of temperature on the epoxy single lap strength at three temperatures of -40, room temperature, and 80℃. They observed a 30 and 10% reduction in the shear strength of the composite at 80℃ compared to the room temperature and -40 ℃, respectively [23]. Na et al. also evaluated the effect of temperature on the mechanical features of the basalt fiber- reinforced composite. They found a decrease in Young’s modulus and tensile strength upon temperature elevation, while the elongation showed an increment [24]. Baneaa et al. also investigated the effect of temperature on single lap metal-to-metal joints at 100, 125, 175, and 200 ℃. At temperatures below the glass transition temperature, the shear strength increased by approximately 9% at 100 ℃ and 30% at 125 ℃. However, at temperatures above the glass transition temperature, a noticeable decrease can be seen in shear strength. Single lap shear strength decreased by 54% and 75% at 175 and 200℃, respectively [25]. In 2015, T. Gomez-del Río et al. explored the effect of styrene-butadiene methyl methacrylate (SBM) block copolymer and CNT on epoxy. DSC results indicated that the addition of SBM reduced Tg up to 4 ℃ compared to pure epoxy. The addition of CNT did not affect Tg. A hybrid of these two compounds slightly reduced Tg. The incorporation of 5 phr SBM increased the fracture toughness and fracture energy by 115% and 410%, respectively. Moreover, adding 0.25 phr CNT increased the fracture toughness by 14.5%; while the hybrid of these two compounds exhibited no effect on fracture toughness [26]. In 2018, Dong Quan et al. examined the effect of MWCNT and core rubber shell (CSR) of butadiene-acrylic copolymer on the mechanical properties of epoxy adhesives. Based on SEM images, the best dispersion was observed in the composites containing 0.1 and 0.2% MWCNT in the presence of 10% CSR. The fracture toughness of pure epoxy increased from 1.22 to 2.41 in the composite containing 0.3% MWCNT and 10% CSR. The highest shear strength (30.1 MPa) was observed in the sample containing 0.5% MWCNT and 10% CSR [27]. In 2019, Jojibabu Pantaa et al. 1. Introduction studied the effect of the block copolymer and surface-modified carbon nanotubes on the lap shear strength. The results revealed that the addition of surface- modified CNT to pure epoxy increased its shear strength by 26%. A hybrid of surface-modified CNT and SBM tri-block copolymer (styrene-butadiene methyl methacrylate) elevated shear strength by 137% compared to pure epoxy, reflecting their synergistic effect at this percentage [28]. In the present study, we propose a systematic approach to fabricate high performance epoxy adhesive using block copolymer (as toughening agent) and zirconia nanoparticles to prepare adhesive for aluminum- aluminum lap joints(At room, high and low temperature). The aim of this research is a comprehensive study on the effect of zirconia nanoparticles and block copolymer (butyl acrylate-block-styrene) on mechanical properties, adhesion strength, and thermal stability of epoxy-based nanocomposite adhesives by studying the microstructure and toughening mechanisms. Materials In this research, DGEBA epoxy with the brand name of EPONATE 500, a viscosity of 14000- 11000 cp, and room temperature density of 1.16 g/cm3 and cycloaliphatic hardener with the brand name of EPIKURE F205 made by Bitex composite Turkey were prepared at the resin/hardener ratio of 100:55 and density of 0.98 gr/cm3. Furthermore, liquid phenolic resin (Resol) with the brand name of IL-800/1, a viscosity of 3000-2500 cp, and density of 1.2 g/cm3 made by Rezitan Iran, xylene (Merck; Germany) with a density of 0.86 gr/cm3 as the epoxy diluent, butyl acrylate-block-styrene block copolymer (ResinFam Company; Iran) with a viscosity of 2000-5000 cp, and zirconia nanoparticles with a diameter of 20-30 nm, 99% purity, and density of 5.89 g/cm3 (Sahand Petro plastic Company; Iran) were prepared. Preparation method To prepare the samples, epoxy resin (20 g) was first poured into a suitable container followed by adding 2 ml of xylene. The mixture was then mechanically stirred for 3 minutes at 200 rpm. Zirconia nanoparticles were then added to the mixture in specified percentages (before addition, the zirconia nanoparticles were weighed and placed in the oven at 110 ℃ for 2 hours for dehumidification). To better mix the nanoparticles in the epoxy matrix, they were also stirred by a mechanical stirrer at a certain speed. In the continue, (2, 4, and 6 g) liquid phenolic resin was added and mechanically stirred for 15 min at 200 rpm. Afterward, (0.25, 0.5, and 0.75 g) toughening agent (butyl-acrylate-block- styrene) was added and stirred for 20 min at 200 rpm. The mixture was then placed in an oven to remove bubbles that may enter the mixture during the stirring process. Subsequently, the curing agent (10 g) was added and mechanically stirred for 10 min at 200 rpm. The prepared adhesive was then poured into dumbbell-shaped molds to prepare specimens for mechanical evaluation tests, some parts of the adhesive were also used for metal-metal lap joint, butt joint, and T-peel tests. The samples were removed from the molds after 24 h and placed at ambient temperature for 7 days for final curing. Formulation The following formulations were designed to assess the effect of the contents of phenolic resin, BCP, and zirconia NPs on the mechanical properties of the epoxy adhesive. The formulation of epoxy adhesives with different component were designed as presented in Table.1. Table 1: Composition of the epoxy adhesive formulations used in this research Test number Sample code Phenolic resin (phr) Block Copolymer (phr) Zirconia (phr) 1 E 0 0 0 2 10P 10 0 0 3 20P 20 0 0 4 30P 30 0 0 5 1.25C 0 1.25 0 6 2.5C 0 2.5 0 7 3.75C 0 3.75 0 8 1Zr 0 0 1 9 2Zr 0 0 2 10 5Zr 0 0 5 11 2.5C - 5Zr 0 2.5 5 12 10P – 5Zr 10 0 5 13 10P– 2.5C 10 2.5 0 14 2.5C-10P-5Zr 10 2.5 5 Table 1: Composition of the epoxy adhesive formulations used in this research Note: for simplicity, abbreviations are used if needed. In sample coding, E, P, C, and Zr respectively denote epoxy, phenolic resin, copolymer, and zirconia NPs. The digits also refer to the weight percentage of the corresponding material. Dumbbell-shaped samples are shown by D; while L, B, and Tp respectively stand for metal-metal lap joint, metal butt joint, and T-peel strength. Tensile test on dumbbell-shaped specimens The tensile test of the dumbbell-shaped samples was carried out according to ISO 527-1,2,3 to determine the tensile properties of the polymers. The length, width, and thickness of the dumbbell-shaped samples were 75, 25, and 5 mm, respectively. Each sample was tested in 5 replicates. According to the mentioned standard, the loading rate of the sample for the tensile test was 2 mm/min. This test was performed by a SANTAM STM-20 tensile device in the central lab of Isfahan University. Effect of temperature on the mechanical properties of the optimal pure epoxy sample In this test, the optimal composition containing 10 phr phenolic resin, 2.5 phr toughening agent, and 5 phr nanoparticles was added to the epoxy. Then, dumbbell-shaped samples and Al-Al single lap joint were prepared at room temperature as well as low temperature (-60 ℃). Al-Al butt joint samples were also fabricated at room, low (-60℃), and high (+120 and 180 ℃) temperatures. T-peel samples were also prepared at room temperature to evaluate the mechanical properties. The following table (Table2) lists the factors and their corresponding levels in the samples. Table 2: Effect of temperature on the tensile, T-peel, single lap shear, and tensile butt joint strength Test number Sample code Phenolic resin (phr) Block Copolymer (phr) Zirconia (phr) Test temperature (℃) 15 D-Epoxy 0 0 0 -60 16 L-Epoxy 0 0 0 -60 17 D-10P-2.5C-5Zr 10 2.5 5 -60 18 D-10P-2.5C-5Zr 10 2.5 5 120 19 L-10P-2.5C-5Zr 10 2.5 5 -60 20 L-10P-2.5C-5Zr 10 2.5 5 120 21 B-10P-2.5C-5Zr 10 2.5 5 25 Effect of temperature on the tensile, T-peel, single lap shear, and tensile butt joint strength 22 B-10P-2.5C-5Zr 10 2.5 5 -60 23 B-10P-2.5C-5Zr 10 2.5 5 120 24 B-10P-2.5C-5Zr 10 2.5 5 180 25 Tp- Epoxy 0 0 0 25 26 Tp- 10P-2.5C-5Zr 10 2.5 5 25 Surface treatment Prior to applying adhesive on the Al surface, anodizing treatment was carried out as follows: Al foil (as the anode) was connected to the positive pole of the source while the auxiliary electrode (Pt) was connected to the negative pole to serve as a cathode. They were placed in sulfuric acid (15%) solution by a clamp to conduct electrochemical anodizing at 9 V for 5 min. Lap shear strength test This test can measure the strength of metal-metal joints based on the ASTM D1002 standard. Al pieces were cut into 100*25*2 mm and the lap domain was 12.5 mm. Each sample was tested in 5 replicates. According to the mentioned standard, the thickness of the adhesive was 0.8 mm while the tensile rate was set to 1.3 mm/min. tested in 5 replicates. According to the mentioned standard, the thickness of the adhesive was 0.8 mm while the tensile rate was set to 1.3 mm/min. Butt joint strength test Al-Al joint specimens were placed in the tensile test apparatus according to the ASTMD2094- 00 standard designed to characterize adhesives in metal-metal joints. A definite length of the samples (12.7 mm) was placed in the pins while the loading rate was set to 0.5 mm/min. According to the mentioned standard, the thickness of the applied adhesive was 0.1 mm while the lap domain was 3.24 mm2. This test was carried out at room temperature as well as -60, 120 and 180℃. The two latter temperatures were prepared using dry ice and a furnace, respectively. The results of this test were presented as the findings of butt joint strength test. The butt joint test samples with different conditions are shown in Figure 1. a b c a b c d d Fig.1 a. Butt joint sample; b. butt joint tensile strength at room temperature; c. butt joint tensile strength at -60 ℃ supplied by dry ice, and d. butt joint tensile strength at 120 and 180 ℃ using a furnace furnace T-peel strength test This test is indeed a groove test; a T-shape is the major type of peeling according to the ASTM D1876. Two Al layers with similar thickness are attached by an adhesive; then the ends of the test sample are detached by a tensile test device at the rate of 127 mm/min. The T-Peel test device and the dimensions of the sample used in it are shown in Figure 2. a b Fig. 2 a. T-peel test specimens and b. T-peel test b Fig. 2 a. T-peel test specimens and b. T-peel test Fig. 2 a. T-peel test specimens and b. T-peel test Fourier-transforms infrared (FTIR) spectroscopy To evaluate the reaction and the bond formation during the curing process, the dumbbell- shaped tensile test samples with the best response were milled into fine powder and assessed by an FTIR spectrometer (6300 Japan). Field-emission scanning electron microscopy (FESEM) To achieve a clearer image, the samples must be coated with a thin layer of gold before the imaging. After the preparation process, the samples were placed in the imaging device. The toughening mechanism and distribution of zirconia NPs in the epoxy matrix were explored by the FESEM technique (MIRA 3, TESCAN). The imaging was carried out on the dumbbell- shaped samples of the tensile test which revealed the best response. Thermogravimetric analysis (TGA) TGA was carried out to check the thermal stability of the samples on the tensile test samples with the best response. The samples were heated from the ambient temperature to 500 ℃ under the nitrogen atmosphere at a rate of 10 ℃/𝑚𝑖𝑛. The sample weight was 10 g which was cut into small plates and placed on Al planes to be heated up to 500 ℃. The initial degradation temperature (TIDT), maximum degradation temperature (Tmax), and residual char or non- volatile fraction were determined up to the temperature of 500 ℃. A TGA device model DSC1 METTLER TOLEDO, Switzerland was employed for this purpose. Differential scanning calorimetry (DSC) The cure kinetics and the effect of the optimal formulation components on the cure kinetics were examined by DSC analysis. Three samples (pure epoxy, epoxy containing 5 phr zirconia NPs, and epoxy containing 2.5 phr BCP along with the curing agent) were prepared. The tests were conducted in the central lab of Isfahan University using a DSC device (DSC 131, Setarm, France). Results and discussions Fig 3 a. The results of the tensile test for dumbbell-shaped samples Fig 3 a. The results of the tensile test for dumbbell-shaped samples Fig. 3a depicts the findings of the tensile test conducted on the dumbbell-shaped samples. According to Fig. 3a, tensile strength and modulus declined upon adding phenolic resin to the epoxy which can be assigned to the lower strength and modulus of the phenolic resin compared to the epoxy. Moreover, phenolic resin shows high shrinkage during the curing process, giving rise to pores and cracks in the adhesive structure; which can consequently decrement the strength and modulus compared to the pure epoxy [8, 29]. The highest decline in the modulus and tensile strength (1672 and 7.3 MPa) was observed in the sample containing 30 phr liquid phenolic resin. The toughness of the system also decreased at all phenolic resin levels. As suggested in Fig. 3a, the modulus continuously decremented upon adding BCP to the epoxy at all levels. Such a decrement can be attributed to the presence of soft blocks of butyl acrylate in the copolymer with a lower modulus relative to the epoxy. Tensile strength also increased up to the BCP level of 2.5 phr followed by a decrease which can be due to the hydrogen bonding between the carbonyl groups of the butyl acrylate and the hydroxyl group of the epoxy [30]. The highest tensile strength (39.4 MPa) was related to the BCP level of 2.5 phr, showing a 40% rise compared to pure epoxy. Also, the highest toughness was observed at this BCP level, exhibiting a 78% increment compared to the pure epoxy due to the proper interaction of the BCP with the epoxy and the formation of nanostructured spherical micelles. The increase in toughness can be also assigned to the enhancement of the elastic energy stored in butyl acrylate rubber particles during the elongation of the sample [31]. Beyond the BCP level of phr 2.5, the modulus and tensile strength decreased to 2009.25 and 35.5 MPa, respectively; due to the enlargement of the BCP structure in the epoxy matrix, reducing its interface with epoxy chains and declining the mechanical properties [32]. According to Fig. Results and discussions 3a, the modulus and strength rose by adding zirconia NPs to epoxy such that the highest modulus and strength respectively reached 2867 and 47.61 MPa at the NPs level of 5%, showing respective increments of 33% and 69% compared to the pure epoxy. The reason can be the higher modulus and strength of nanoparticles compared to the epoxy. It can also be said that the addition of zirconia NPs restricted the movement of epoxy chains [12,15]. Zirconia nanoparticles prevent the opening of a growing crack due to their spherical geometry. Therefore, more energy is required for crack growth and as a result, the energy absorption increases. On the other hand, the hydroxyl groups on zirconia NPs facilitate the curing reaction, leading to higher ring-opening and improved mechanical properties. The highest toughness was also observed in 5wt.%, exhibiting 175% enhancement compared to pure epoxy [15-17]. Fig. 3 b. Results of ternary and quaternary dumbbell-shaped samples Fig. 3 b. Results of ternary and quaternary dumbbell-shaped samples Fig. 3b depicts the findings of the tensile test ternary and quaternary on the dumbbell-shaped samples. According to Fig. 3b, the modulus and strength increased by elevating the amount of zirconia NPs in the epoxy/phenolic matrix, such that the highest modulus, strength, and toughness were According to Fig. 3b, the modulus and strength increased by elevating the amount of zirconia NPs in the epoxy/phenolic matrix, such that the highest modulus, strength, and toughness were recorded in the 10P-5Zr sample. The rise in NPs content compensated for the structural defect due to the presence of resin and increased the mechanical properties. Overall, the mechanical properties decremented relative to pure epoxy at these levels of materials, mainly due to the presence of phenolic resin in the structure of the adhesive, which releases formaldehyde during the curing process, causing cavitation and defects in the adhesive structure and reducing the modulus, strength, and toughness [7,8,26]. According to Figure 3b, the addition of zirconia NPs to the epoxy/BCP matrix increased the modulus, strength, and toughness in an ascending manner; such that the greatest improvement in mechanical properties was observed in the 2.5C-5Zr sample whose modulus and strength were 1566 and 31 MPa, respectively while it's the toughness reached 1239 kJ/m3. The toughness increased by 117% compared to pure epoxy. Such an enhancement can be due to the good adhesion and strong interface of the nanoparticles with the matrix [27,28]. Results and discussions Based on Fig. 3b, the tensile strength and toughness of the adhesive in the 10P-C2.5 dumbbell- shaped sample increased by the incorporation of BCP. The tensile strength and toughness respectively reached 32.9 MPa and 672 kJ/m3 showing 17% and 18% enhancement compared to pure epoxy. According to Fig. 3b, in the optimal 2.5C-10P-5Zr sample, the modulus and strength respectively decreased by 69% and 54% compared to pure epoxy, reflecting a negative interaction of materials at this level. However, the toughness increased by 51%. Instead of reacting with epoxy groups, the hardener was surrounded by acrylate groups of BCP, hydroxyl groups of phenolic, and nanoparticles, thus failing to open the epoxy oxirane rings. This reduced the crosslinking degree and enhanced the toughness while decreasing the modulus and strength. It can be also said that at this level of the materials, the viscosity of the adhesive increased, hindering the exit of the bubbles and thus, reducing the mechanical properties [8,37,38]. Predictive modeling of mechanical properties of nanocomposites The properties of the polymer and the reinforcement, the volume fraction, the distance between the reinforcement particles, and their distribution and interaction with the matrix are among the important and influential factors in the mechanical properties of polymer nanocomposites. However, the nanocomposites are mainly developed through experimental approaches where the control of properties is not an easy task. In this regard, modeling the mechanical properties of nanocomposites is highly important. Among the modeling methods of mechanical properties, continuum media mechanical methods have gained increasing popularity due to their feasibility and the ability to predict properties regardless of the type of microstructure in atomic dimensions [68]. The properties of the polymer and the reinforcement, the volume fraction, the distance between the reinforcement particles, and their distribution and interaction with the matrix are among the important and influential factors in the mechanical properties of polymer nanocomposites. For this purpose, theoretical predictive models of the mechanical properties of nanocomposites such as Halpin–Tsai, series, parallel, and Kerner models were used and compared with the experimental data in terms of Young's modulus of epoxy nanocomposites and zirconia nanoparticles which is shown in Figure 3c. Ec=Em( 1+𝜉𝜂V𝑓 1−𝜂V𝑓) (Halpin-Tsai) (1) 𝜂= 𝐸𝑓 𝐸𝑚−1 𝐸𝑓 𝐸𝑚+𝜉 (2) Ec =EmVm +EfVf (parallel) (3) 1 𝐸c = 𝑉m 𝐸m + 𝑉f 𝐸f (series) (4) 𝐸c 𝐸m = 1 + 𝜈𝑓15(1−𝑉m) 𝜈𝑚(8−10𝑉m) (Kerner) (5) In the above equations, Ec, Em, and Ef respectively denote the modulus of the composite, matrix, and reinforcement. Moreover, Vf and Vm show the volume fraction of the reinforcement and matrix, respectively; while νm in the Kerner equation (Equation number 5) represents the Poisson coefficient of the epoxy resin which is equal to 0.33. In the Halpin–Tsai equation (Equation number 1), ξ =2 for spherical particles. The modulus of the pure epoxy was calculated as 2.149 GPa while the modulus and density of zirconia NPs were 200 GPa and 5.89 g/cm3, respectively. The volume fraction of zirconia NPs was determined 0.1, 0.2, and 0.5 [35, 36]. Fig. 3 c. Predictive model for Young modulus of epoxy/zirconia NPs Fig. 3 c. Predictive model for Young modulus of epoxy/zirconia NPs The general rule of mixtures is one of the basic theories of composites with extensive applications. This theory has upper (parallel) and lower (series) bounds. Predictive modeling of mechanical properties of nanocomposites The upper bound can be obtained by assuming equal strains for filler and matrix; while the series model can be achieved by assuming equal stress for the filler and matrix. Halpin–Tsai equation is also a semi empirical equation capable of evaluating the mechanical properties of nanocomposites based on the matrix and geometry of filler particles. According to Figure 3c Experimental results indicated a 33% increment in the modulus at 5 vol.% of zirconia NPs. As mentioned before, the higher modulus of nanoparticles, their proper dispersion, and interaction with the epoxy matrix are due to their hydroxyl groups. The Young moduli predicted by Kerner, series, and Halpin–Tsai models showed a proper consistency with the empirical results at NP content of 0.1 vol.%. However, with increasing the volume fraction of NPs, predictive models showed no significant enhancement in the modulus. In the parallel model, the modulus showed a 10% increase compared to the experimental results at NPs volume fractions of 0.5% and 0.2%. It can be said that theoretical models do not have good accuracy in predicting the elastic modulus as they consider an ideal bond between nanoparticles while ignoring defects such as bubbles and aggregation as well as the defects of the nanoparticles themselves [33,34,35]. Results of single lap shear strength test for metal-metal joints Fig. 4 a. Results of single lap strength of epoxy samples at phenolic resin levels of 10, 20, and 30 phr. Fig. 4 a. Results of single lap strength of epoxy samples at phenolic resin levels of 10, 20, and 30 phr. As shown in Fig. 4a, the shear strength continuously increased with enhancing phenolic resin levels. Due to the increase in hydroxyl groups in phenolic resin, a proper interaction is formed with the substrate surface, incrementing the adhesion of the adhesive to the Al substrate. It can be also said that phenolic hydroxyl groups increased the ring-opening and crosslinking degree, thus, augmenting the adhesion strength of the adhesive to the substrate. The highest shear strength (1.48 MPa) was detected in the sample containing 30 phr phenolic resin, exhibiting a 41% increment compared to pure epoxy [6,7,40]. Fig 4 b. Results of single lap strength of pure epoxy and copolymer at levels of 1.25, 2.5, and 3.75 h Fig 4 b. Results of single lap strength of pure epoxy and copolymer at levels of 1.25, 2.5, and 3.75 phr According to Fig. 4b, the incorporation of BCP continuously enhanced the shear strength; such that the highest shear strength (1.72 MP) was observed at the BCP level of 3.75 phr level, showing a 63% increment compared to the pure epoxy. Such an enhancement can be attributed to the hydrogen bonds between the hydroxyl groups of epoxy and the ester groups of butyl acrylate in the BCP. The presence of spherical BCP micelles in the adhesive increased energy dissipation, improving the stress transfer from the epoxy matrix in the presence of BCP, thus, increasing the shear strength [41,42]. Fig. 4c. Results of single lap shear strength test in the presence of zirconia NPs (1, 2, and 5 phr) Fig. 4c. Results of single lap shear strength test in the presence of zirconia NPs (1, 2, and 5 phr) As presented in Fig. 4c, the shear strength increased ascendingly with raising NPs content, such that the highest shear strength (3.2 MPa) was observed in the 5Zr sample, exhibiting a 212% increment compared to the pure epoxy. Such an enhancement can be ascribed to the chemical interactions between zirconia NPs and epoxy chains as the hydroxyl groups on the surface of zirconia NPs can also form hydrogen bonds with epoxy. Results of single lap shear strength test for metal-metal joints 5 depicts the proposed mechanism for the interaction between the materials in the adhesive with the anodized aluminum substrate which indicates the hydrogen bonding between the adhesive formulation components together and the anodized substrate with the hydroxyl groups of the phenolic resin, BCP, and zirconia NPs. Fig. 5. Proposed mechanism describing the interaction of materials in a single lap metal-metal joint Fig. 5. Proposed mechanism describing the interaction of materials in a single lap metal-metal joint Fig. 5. Proposed mechanism describing the interaction of materials in a single lap metal-metal joint Results of single lap shear strength test for metal-metal joints Higher percentages of zirconia NPs enhanced the surface roughness, hence, improving their adhesion to the Al substrate. Moreover, the increased adhesion between zirconia nanoparticles and epoxy can augment the adhesion of nanocomposite adhesive to the metal substrate as ZrO2 NPs bridge between the epoxy adhesive and the substrate, further incrementing the shear strength [43- 45,65]. between the epoxy adhesive and the substrate, further incrementing the shear strength [43- 45,65]. 45,65]. Fig. 4d. Results of single lap strength test in the presence of phenolic resin (10, 20, and 30) and zirconia NPs (1, 2, and 5 phr) Fig. 4d. Results of single lap strength test in the presence of phenolic resin (10, 20, and 30) and zirconia NPs (1, 2, and 5 phr) zirconia NPs (1, 2, and 5 phr) Based on Fig. 4d, the shear strength increased ascendingly with increasing the content of zirconia NPs in the epoxy/phenolic matrix. The highest shear strength (4.01 MPa) was observed in the 30P-5Zr sample, showing a 382% enhancement compared to the pure epoxy. The presence of hydroxyl groups on zirconia NPs and phenolic resin led to strong bonding between the adhesive and the surface [40,43,65]. Fig. 4e. Results of the single lap strength test for the optimal triplet and quaternary samples Fig. 4e. Results of the single lap strength test for the optimal triplet and quaternary samples Fig. 4e. Results of the single lap strength test for the optimal triplet and quaternary samples According to Fig. 4e, the shear strength reached 4.34 MPa in the optimal sample of 2.5C-5Zr, showing a 313% increment compared to pure epoxy. In the case of the optimal 2.5C-10P-5Zr sample, the shear strength further grew to 5.47 MPa, offering a 420% rise relative to the pure epoxy. The stress transfer from the matrix was improved due to the proper interaction and hydrogen bonding between the hydroxyl groups of phenolic resin, carbonyl groups of BCP, and hydroxyl groups on the surface of zirconia NPs with epoxy rings and the anodized aluminum substrate, which significantly increased the shear strength in the adhesive formulation. At these levels, materials can be said to have a synergistic effect [42,43,44]. Fig. T-peel test analysis T-peel strength depends on the angle of peeling, test speed, nature of the adhesive, physical and mechanical properties of the substrate, temperature, and humidity of the environment. Fig. 6 shows the results of the T-peel test for pure epoxy and the optimal sample, which indicates the highest records in the lap shear strength test. In the case of pure epoxy, the T-peel strength was 27.8 N/mm which rose to 83.4 N/mm in the 10p-2.5C-5Zr quaternary sample, showing a three-fold enhancement. It can be said that the presence of zirconia nanoparticles and BCP transferred the stress to the substrate. Moreover, the energy dissipation during the crack growth in the T-peel test enhanced the T-peel strength in the quaternary specimen [66,67]. Fig. 6. T-peel test results for pure epoxy and optimal quaternary sample Analysis of the dumbbell-shaped samples at -60 and 120℃ Fig. 6. T-peel test results for pure epoxy and optimal quaternary sample Analysis of the dumbbell-shaped samples at -60 and 120℃ Analysis of the dumbbell-shaped samples at -60 and 120℃ Temperature is an effective factor in the strength of the adhesive joints with a decisive role in the design of these joints. The important factor is the applicability of the adhesive in a wide range of temperatures. Temperature also affects the structure of the adhesive, especially at temperatures near the glass transition temperature (Tg). At temperatures above Tg, the properties of the adhesive such as elasticity, fracture strength, and elastic modulus are declined. At temperatures far below Tg, however, these features may show reverse behavior [25]. Temperature is an effective factor in the strength of the adhesive joints with a decisive role in the design of these joints. The important factor is the applicability of the adhesive in a wide range of temperatures. Temperature also affects the structure of the adhesive, especially at temperatures near the glass transition temperature (Tg). At temperatures above Tg, the properties of the adhesive such as elasticity, fracture strength, and elastic modulus are declined. At temperatures far below Tg, however, these features may show reverse behavior [25]. High temperatures decrement the strength of the adhesive; whereas low temperatures may result in its brittleness. The strength of the adhesive generally declines with the rise of temperature; while its ductility shows an increment. High-temperature adhesives are often highly brittle at lower temperatures; while those designed for low-temperature applications usually exhibit poor strength at high temperatures [24, 25]. T-peel test analysis Fig. 7 presents the results of the tensile test for pure epoxy and optimal quaternary dumbbell- shaped specimens at room temperature as well as -60 and 120 ℃. The modulus of the pure epoxy and 10P-2.5C-5Zr showed a slight increase at -60 ℃ compared to the room temperature which can be assigned to the harder movement of the polymeric chains and a reduction in the free space between the chains. For the case of pure epoxy and optimal quaternary samples, toughness showed a 32 and 12% decline due to the decrease in the toughening mechanisms and easier initiation of the crack growth. The toughening mechanisms used by zirconia NPs and BCP to increase plastic deformation and energy loss got more difficult at low temperatures, lowering the toughening effect of zirconia NPs and BCP. At -60 ℃, the tensile strength of pure epoxy and the optimal quaternary samples showed 46% and 23% decrement, respectively. As the temperature decreases, the adhesive fracture occurs earlier, i.e. before the adhesive undergoes massive deformation, which reduces the strength and toughness [46,50]. At 120 ℃, the tensile strength and modulus of optimal quaternary samples showed 41% and 5% decrement, respectively. According to Figure 7, as the temperature increases, the interaction between the materials in the adhesive decreases and reduces the cohesive strength [58]. According to Figure 7, as the temperature increases, the interaction between the materials in the adhesive decreases and reduces the cohesive strength [58]. Fig. 7. Tensile test results of pure epoxy and optimal quaternary dumbbell-shaped specimens at room temperature, -60 and 120 ℃ Fig. 7. Tensile test results of pure epoxy and optimal quaternary dumbbell-shaped specimens at room temperature, -60 and 120 ℃ temperature, -60 and 120 ℃ Analysis of single lap joint test at -60 and 120 ℃ Fig. 8 shows the results of the single lap tensile strength test for pure epoxy and the optimal quaternary samples at room temperature as well as -60 and -120 ℃. In the case of pure epoxy, the shear strength decreased by 26% at -60 ℃ compared to room temperature. The shear strength of the optimal 10P-2.5C-5Zr reached 4.32 MPa at -60 ℃, showing a 78% decrease relative to the optimal sample at room temperature. At low temperatures, the movement of the epoxy chains on top of each other is hindered, which increases external stresses and facilitates crack growth. At low temperatures, the adhesive loses its ductility and energy absorption to some extent and fails to withstand external stresses, which reduces the shear strength. In the optimal sample, a rise in the temperature to 120 ℃ enhanced the shear strength to 1.2 MPa, i.e. approximately 4.5 times lower than that of the room temperature due to the reduction of intermolecular forces and the interactions between the components of the nanocomposite adhesive with each other and the substrate [54,55]. Fig. 8. Results of single lap tensile strength test for pure epoxy and optimal quaternary sample at room temperature as well as -60 and 120 ℃ Fig. 8. Results of single lap tensile strength test for pure epoxy and optimal quaternary sample at room temperature as well as -60 and 120 ℃ Analysis of butt joint tensile strength at different temperatures The butt joint tensile strength of the 10P-2.5C-5Zr sample is depicted in Fig. 9 at room temperatures as well as 120, 180, and -60 ℃. The butt joint tensile strength of the optimal sample was 0.31 MPa at room temperature which declined to 0.22 MPa at 120 ℃, showing a 29% decline compared to the room temperature. At 180 ℃, the butt joint tensile strength reached 0.21 MPa, exhibiting a 32% drop compared to the room temperature. It can be said that at temperatures higher than Tg, the flow ability of the adhesive increased and the adhesive became softer and separated from the substrate. At temperatures above Tg, all the mechanical properties of the adhesive reduced, which is consistent with previous reports [50,51]. [50,51]. According to Figure 9, at -60 ℃, the butt tensile strength reached 0.25 MPa, showing a 19% decrease compared to the room temperature. Similar to the trend of the single lap joint, it can be said that the toughness mechanisms decreased at this temperature, which reduced the tensile strength. Moreover, the glass transition temperature of epoxy adhesive is close to 90 ℃ below which, the adhesive has a brittle state with low fracture energy [52,53,55]. 9. But joint test results for optimal quaternary specimen at room temperature, -60, 120, and Analysis of FTIR spectra of pure epoxy, and optimal binary, ternary, and quaternary samples Fig. 10a depicts the FTIR spectra of the dumbbell-shaped pure epoxy, binary, ternary, and quaternary samples. The peak emerging at 3431 cm-1 can be ascribed to the stretching vibration of the hydroxyl groups of epoxy. The stretching vibration of C-H in the aromatic ring of epoxy also appeared at 3037 cm-1. The peaks at 2973 and 2933 cm-1 can be attributed to the stretching vibrations of CH3 and CH2 groups; while those at 1572 and 1510 cm-1 are related to C-C stretching vibrations in the aromatic ring of epoxy. An absorption peak at 922 cm-1 is attributable to the epoxy ring. The emergence of some peaks at 649 and 559 cm-1 indicate the bending vibration of N-H and C-H. a weak peak at 1731 cm-1 is indicative of the carbonyl groups in BCP. While the one appearing at 765 cm-1 is related to the O-Zr-O bond in zirconia NPs [56, 57, 59]. The intensity of hydroxyl peaks in 5zr, 2.5C, and 10P showed an increase indicating the rise in the ring-opening process and an enhancement in the hydroxyl groups at their level of materials in the mentioned formulation of epoxy resin. FTIR spectrum of pure zirconia NPs was also recorded as presented in Fig. 10b. A broad band at 3500 cm-1 represents the hydroxyl groups in the structure of nanoparticles which can enhance the interactions and H-bonding with the epoxy chain, hence, improving the mechanical and thermal properties as confirmed by the results of mechanical properties [48, 62, 66]. Fig. 10 a. FTIR spectra of dumbbell-shaped pure epoxy and optimal binary, ternary, and quaternary specimens Fig. 10 a. FTIR spectra of dumbbell-shaped pure epoxy and optimal binary, ternary, and quaternary specimens Fig. 10 a. FTIR spectra of dumbbell-shaped pure epoxy and optimal binary, ternary, and quaternary specimens FTIR spectra of dumbbell-shaped pure epoxy and optimal binary, ternary, and quaternary Fig. 10 a. FTIR spectra of dumbbell-shaped pure epoxy and optimal binary, ternary, and quaternary Fig. 10 a. FTIR spectra of dumbbell-shaped pure epoxy and optimal binary, ternary, and quaternary specimens Fig. 10b. FTIR spectrum of pure zirconia NPs Fig. 10b. FTIR spectrum of pure zirconia NPs Thermal stability Table 3. Analysis of FTIR spectra of pure epoxy, and optimal binary, ternary, and quaternary samples Thermal stability indices of the pure epoxy sample as well as optimal ones Sample code TIDT (oC) Tmax (oC) Residual char at 500 oC (Wt%) Epoxy 175.8 375 9.63 10P 188 381 15.56 20P 194.2 384.2 19.27 30P 201.7 385 23.09 1.25C 195.8 384.2 10.54 2.5C 215 407.5 11.12 3.75C 214.2 385 11.99 10P-2.5C 179.2 373.3 15.65 1Zr 222.7 376.66 10.39 Table 3. Thermal stability indices of the pure epoxy sample as well as optimal ones 2Zr 223 378.33 10.49 5Zr 230.2 390 10.81 10P-5Zr 189.16 382.5 15.96 2.5C-5Zr 193.84 386.66 12.01 10P-2.5C-5Zr 215 394.16 16.38 Table 3 and Fig. 11 present the thermal stability of pure epoxy and optimal samples. Table 3 and Fig. 11 present the thermal stability of pure epoxy and optimal samples. Bisphenol A diglycidyl ether type epoxy resin has good thermal stability with an initial degradation temperature of 175.8 ℃, maximum degradation temperature of 375 ℃, and residual char of 9.63% at 500 ℃ due to the formation of networks with high crosslink density. The thermal degradation of epoxy resin involves three stages. The first stage of degradation occurs at 100-175 ℃ due to the loss of surface moisture and the degradation of the volatile compound with low molecular weight in the epoxy adhesive. Weight loss from 150 ℃ to 350 ℃ 𝑐𝑎𝑛 𝑏𝑒due to the degradation of oxygenated groups such as the hydroxyl groups of epoxy resin. The main weight loss of the second stage at 175-375 ℃ can be assigned to the destruction of bisphenol A aromatic groups, the breakdown of the bonds formed with the curing agents, and the release of flammable gases, amines, and gaseous aromatic compounds. Complete degradation of the epoxy network occurred at 375- 500 ℃ [58,59] According to Table 3, the increase in the amount of residual char can be due to the high thermal stability of the phenolic resin, high breakdown energy of the bonds formed with the phenol rings, and also the increase of the aromatic rings. Residual char acts as an insulating layer against the diffusion of oxygen and heat, preventing further degradation of the sample. The residual char continuously rose by enhancing the phenolic resin content such that the highest residual char (23%) was observed in the sample containing 30 wt.% phenolic resin. Also, the initial degradation temperature reached 201.7 ℃, i.e. 25.8 ℃ higher than pure epoxy. The maximum degradation temperature also reached 385 ℃, which is 10 ℃ higher than pure epoxy [59,60,61]. In 1.25C, 2.5C, and 3.75C samples, the initial and maximum degradation temperatures showed an enhancement. The mobility of the epoxy chains showed a decline due to the proper interactions between the acrylates of the BCP and epoxy rings and their H-bonding. The presence of benzene rings in the styrene structure of the BCP has increased the thermal stability of the system. This increases the energy required for the release of volatiles. Additionally, the residual char continuously increased with elevating the BCP content due to the increment of the benzene rings. The highest residual char was observed in the 3.75C sample, showing a 2.33% increment compared to pure epoxy [8,57]. Table 3 and Fig. 11 present the thermal stability of pure epoxy and optimal samples. According to Table 3, a rise in zirconia content enhanced the initial degradation temperature as well as the maximum degradation temperature compared to the pure epoxy. The enhancement in the initial degradation temperature can be assigned to the proper dispersion of zirconia NPs in the epoxy matrix and their appropriate interaction and adhesion with the epoxy chains due to the presence of hydroxyl groups on the surface of NPs which declines the mobility and slipping of the chains on top of each other, hence higher temperatures and energy will be required to overcome the forces between the epoxy chains and NPs. Due to their inorganic nature, zirconia NPs have intrinsically higher thermal stability, which can elevate the thermal stability of the entire system [53-55]. The initial degradation temperature and maximum degradation temperature of the 10P-5Zr sample showed an increment compared to the pure epoxy. Moreover, the residual char was enhanced due to the presence of phenolic resin in the composition of the adhesive which can augment the thermal stability of the system due to its high bonding energy and proper interaction with epoxy as a consequence of their benzene rings [61, 65]. In the optimal 2.5C-5Zr sample, the initial degradation temperature and maximum degradation temperature also exhibited an increment compared to the pure epoxy. Due to the presence of well- distributed zirconia NPs in the epoxy matrix, the mobility of the polymer chains faces more difficulty. The presence of BCP also promotes interactions of H-bonding with epoxy chains, hindering the release of volatile gases and compounds from the adhesive system [8, 6, 60]. According to Table 3, in the case of optimal 10P-2.5C-5Zr, the initial degradation and maximum degradation temperatures rose compared to the pure epoxy. The residual char was enhanced due to the presence of phenolic resin and BCP and a rise in the content of benzene rings, which incremented the thermal stability. Moreover, it can be said that the mobility and slipping of the epoxy chains decreased due to the proper interactions between zirconia NPs, acrylate blocks, and phenolic in the formulation of the adhesive. Such a decline in mobility is the main obstacle to the release of volatile compounds [8, 57, 60]. Fig. 11. TGA diagram of the pure epoxy and optimal samples Fig. 11. TGA diagram of the pure epoxy and optimal samples Fig. 11. TGA diagram of the pure epoxy and optimal samples DSC analysis Fig. 12 and Table 4 present the results of studying the cure kinetics in pure epoxy as well as 5Zr and 2.5C samples. Fig. 12 and Table 4 present the results of studying the cure kinetics in pure epoxy as well as 5Zr and 2.5C samples. The DSC diagram shows a peak for all three samples. This form of peaks is related to one-step reactions. Considering the shape of the peaks, it can be said that the curing reaction of epoxy adhesives occurred in a single step and the reaction type did not change with the addition of zirconia NPs and BCP. According to Table 4, the initial curing temperature of the optimal epoxy/nano-zirconia and epoxy/copolymer samples decreased compared to the pure epoxy. The incorporation of zirconia NPs and BCP facilitated the reaction of the curing agent with epoxy, thus increasing the crosslinking degree [48]. Furthermore, compared to the pure epoxy, the enthalpy of reaction was higher in the samples containing BCP and NPs, indicating an increase in the released heat, which enhanced the ring-opening and crosslinking degree, thus, augmenting the tensile strength compared to pure epoxy. These results are consistent with the results of dumbbell-shaped samples [62-64]. Table 4 Cure kinetics indices of pure epoxy and optimal epoxy/copolymer and epoxy/zirconium oxide NPs samples sample code Tonset (°C) Tpeak (°C) ΔH (J/gr) Epoxy 54.39 85.02 49.67 5Zr 48.92 83.16 56.52 2.5C 46.22 85.42 154.67 kinetics indices of pure epoxy and optimal epoxy/copolymer and epoxy/zirconium oxide NPs samples Figure. 12. Diagram of heat flow vs. temperature for pure and optimal epoxy samples Figure. 12. Diagram of heat flow vs. temperature for pure and optimal epoxy samples Analysis of FESEM images The fracture cross-section of pure epoxy is shown in Fig. 13 which reveals a perfectly smooth and glossy surface with no plastic deformation, indicating brittle fracture in pure epoxy. The fracture cross-section of epoxy and phenolic resin (at levels of 10, 20, and 30 phr) is depicted in Fig. 13. As seen, with increasing phenolic resin content, parallel lines rose in cross-section, reflecting brittle fracture. The more the number and the deeper the depth of these parallel lines, the higher the brittleness of the facture as also proved by the mechanical features of the dumbbell- shaped samples. A rise in the phenolic resin content reduced all mechanical properties including toughness due to formation of bubbles in the adhesive matrix [7,8]. Fig. 13. Fracture cross-section images of epoxy and epoxy/phenolic samples at a 200-micron scale (a- pure epoxy, b-10P, c-20P, and d-30P) In FESEM images, toughening mechanisms such as crack deviation (red circles), crack tip blunting ( i l ) d h b d f i (bl i l ) k d Fig. 13. Fracture cross-section images of epoxy and epoxy/phenolic samples at a 200-micron scale (a- pure epoxy, b-10P, c-20P, and d-30P) pure epoxy, b-10P, c-20P, and d-30P) In FESEM images, toughening mechanisms such as crack deviation (red circles), crack tip blunting (orange circles), and shear band formation (blue circles) are marked. Figure 14 shows epoxy containing block copolymer at levels of 1.25, 2.5 and 3.75 phr. Fig. 14 shows cavitation and formation of spherical micelles within the BCP-modified epoxy sample. Also, the fracture cross-section changed from a smooth and glossy surface to a rough one, indicating the resistance of the matrix against deformation and increased toughness and plastic deformation of the epoxy matrix. The rougher fracture cross-section also suggests a rise in the fracture toughness energy which can be mainly assigned to the crack deviation due to the presence of spherical micelles in the epoxy matrix. The distribution of spherical micelles within the epoxy matrix led to a plastic deformation zone and a shear band. The cavities created by the BCP are also observable in the images as cavitation is one of the mechanisms of toughness in the studied adhesive. BCP particles can form a shear band in the epoxy matrix, altering the stress rate. The 1.25C sample contains low levels of BCP, leading to a low number of micelles. Analysis of FESEM images The BCP content of 2.5C sample was higher, increasing the number of micelles. By further increase in the BCP content in the 3.75C sample, the size of the micelle structure also rose, which reduced the contact surface of epoxy chains with the copolymer, thus, decreasing the mechanical properties [30-32]. Fig. 14. Fracture cross-section of epoxy/copolymer samples at the 500 micron scale a) 1.25C, b) 2.5C, and c) 3.75C Fig. 14. Fracture cross-section of epoxy/copolymer samples at the 500 micron scale a) 1.25C, b) 2.5C, and c) 3.75C Figure 15 shows epoxy and zirconia nanoparticles in levels 1, 2 and 5 phr. Figure 15 shows epoxy and zirconia nanoparticles in levels 1, 2 and 5 phr. Figure 15 shows epoxy and zirconia nanoparticles in levels 1, 2 and 5 phr. Based on Fig. 15 a-c, dispersion of nanoparticles can be observed in NP level of 1, 2, and 5 phr in the epoxy matrix. The surface also got rougher, suggesting the resistance of the epoxy matrix against fracture and hence, the rise of toughness. This image shows the toughening mechanisms of crack deviation, crack tip blunting, and formation of shear bands [16-18]. a a a b b c c Fig. 15. Fracture cross-section of epoxy/ zirconia NPs samples at the scale of 200-micron a)1Zr, b) 2Zr and c) 5Zr Fig. 16a depicts the fracture cross-section of the 2.5C-5Zr sample which indicates the toughness of epoxy at all additive levels. Toughening mechanisms such as crack deviation and the formation of shear bands can be observed in this image which can be assigned to the tough fracture which increased the fracture energy [27, 28, 65]. Fig. 16 a. Fracture cross-section of 2.5C-5Zr sample at the scale of 200 micron Fig. 16 a. Fracture cross-section of 2.5C-5Zr sample at the scale of 200 micron Fig. 16b illustrates the fracture cross-section of the 10P-5Zr specimen. The toughening mechanisms include crack deviation, crack tip blunting, and formation of the shear band, suggesting a tough fracture in this specimen which coincides with the mechanical properties obtained from the tensile test results. Fig. 16b. Fracture cross-section of 10P-5Zr sample at the scale of 200 micron Fig. 16b. Fracture cross-section of 10P-5Zr sample at the scale of 200 micron Figure 16c also shows the fracture cross-section of the 10P-2.5C-5Zr sample. Mechanisms of toughness in this level of the additives include shear band formation, crack deviation, and crack tip blunting. Analysis of FESEM images Surface roughness also rose compared to the pure epoxy, reflecting plastic deformation and an increase of the fracture energy and toughness [8, 6, 28]. Figure 16c. Fracture cross-section of 10P-2.5C-5Zr sample at 200-micron scale Figure 16c. Fracture cross-section of 10P-2.5C-5Zr sample at 200-micron scale Conflict of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not- for-profit sectors. Conclusion We have successfully developed the epoxy adhesives with improved thermal, mechanical, and adhesion properties by incorporating block copolymer, phenolic resin and nanometer-sized zirconia particles into DGEBA-based epoxy resin. The incorporation of phenolic resin in dumbbell-shaped samples reduced the toughness, modulus, and strength. In the single lap metal-metal joint samples, it increased the adhesion. The highest shear strength was observed in the 10P-2.5C-5Zr sample, showing a 420% increase compared to pure epoxy. Such an enhancement suggests the synergistic effect of materials at this level. The addition of zirconia NPs up to 5 phr incremented the toughness, modulus, and tensile strength compared to pure epoxy due to proper dispersion. The highest toughness was observed in the 5Zr sample, showing a 175% increment compared to the pure epoxy. Concerning the prediction of mechanical properties, the models showed a good agreement with the experimental results at the nanoparticle volume fraction of 0.1%. Further increase of the NPs content led to a deviation in the modeled results relative to the experimental values. In the optimal 10P-2.5C-5Zr sample, the T-peel strength was tripled compared to pure epoxy. Temperature enhancement and decrease altered the butt joint tensile strength in the optimal quaternary sample. The butt joint tensile strength at 180, 120 and -60 ℃ rose by 32%, 29% and declined by 19% compared to the room temperature, respectively. Temperature reduction in the optimal quaternary sample decreased the lap shear strength such that the shear strength at -60 ℃ reached 4.32 MPa, exhibiting a 78% decline compared to the optimal sample at room temperature. By increasing the temperature to 120 ℃, shear strength showed a sharp 4.5-fold decline. sample at room temperature. By increasing the temperature to 120 ℃, shear strength showed a sharp 4.5-fold decline. The highest initial degradation temperature (230.2 ℃) was observed in the 5Zr sample. The highest maximum degradation temperature (407.5 ℃) was also related to the 2.5C specimen; while the 30P sample exhibited the highest residual char (23%). The presence of zirconia nanoparticles and BCP reduced the curing initiation temperature while increasing the curing heat, suggesting a catalytic effect on the curing reaction of epoxy. 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<i>Drosophila</i>poly suggests a novel role for the Elongator complex in insulin receptor–target of rapamycin signalling
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Edinburgh Research Explorer g p Drosophila poly suggests a novel role for the Elongator complex in insulin receptor-target of rapamycin signalling Citation for published version: Bolukbasi, E, Vass, S, Cobbe, N, Nelson, B, Simossis, V, Dunbar, DR & Heck, MMS 2012, 'Drosophila poly suggests a novel role for the Elongator complex in insulin receptor-target of rapamycin signalling', Biology Open, vol. 2, no. 1, pp. 110031. https://doi.org/10.1098/rsob.110031 Citation for published version: Bolukbasi, E, Vass, S, Cobbe, N, Nelson, B, Simossis, V, Dunbar, DR & Heck, MMS 2012, 'Drosophila poly suggests a novel role for the Elongator complex in insulin receptor-target of rapamycin signalling', Biology Open, vol. 2, no. 1, pp. 110031. https://doi.org/10.1098/rsob.110031 Citation for published version: Bolukbasi, E, Vass, S, Cobbe, N, Nelson, B, Simossis, V, Dunbar, DR & Heck, MMS 2012, 'Drosophila poly suggests a novel role for the Elongator complex in insulin receptor-target of rapamycin signalling', Biology Open, vol. 2, no. 1, pp. 110031. https://doi.org/10.1098/rsob.110031 Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 rsob.royalsocietypublishing.org rsob.royalsocietypublishing.org Ekin Bolukbasi†, Sharron Vass, Neville Cobbe‡, Bryce Nelson}, Victor Simossisk, Donald R. Dunbar and Margarete M. S. Heck Author for correspondence: Margarete M. S. Heck e-mail: margarete.heck@ed.ac.uk Research Cite this article: Bolukbasi E, Vass S, Cobbe N, Nelson B, Simossis V, Dunbar DR, Heck MMS. 2011 Drosophila poly suggests a novel role for the Elongator complex in insulin receptor–target of rapamycin signalling. Open Biol 2: 110031. University of Edinburgh, Queen’s Medical Research Institute, University/BHF Centre for Cardiovascular Science, 47 Little France Crescent, Edinburgh EH16 4TJ, UK Drosophila poly suggests a novel role for the Elongator complex in insulin receptor–target of rapamycin signalling rsob.royalsocietypublishing.org Research Cite this article: Bolukbasi E, Vass S, Cobbe N, Nelson B, Simossis V, Dunbar DR, Heck MMS. 2011 Drosophila poly suggests a novel role for the Elongator complex in insulin receptor–target of rapamycin signalling. Open Biol 2: 110031. http://dx.doi.org/10.1098/rsob.110031 Received: 23 November 2011 Accepted: 21 December 2011 Subject Area: cellular biology/developmental biology/ genetics/molecular biology Keywords: cell growth, nucleus, signalling, insulin Author for correspondence: Margarete M. S. Heck e-mail: margarete.heck@ed.ac.uk 1. Summary Multi-cellular organisms need to successfully link cell growth and metabolism to environmental cues during development. Insulin receptor–target of rapamy- cin (InR–TOR) signalling is a highly conserved pathway that mediates this link. Herein, we describe poly, an essential gene in Drosophila that mediates InR–TOR signalling. Loss of poly results in lethality at the third instar larval stage, but only after a stage of extreme larval longevity. Analysis in Drosophila demon- strates that Poly and InR interact and that poly mutants show an overall decrease in InR–TOR signalling, as evidenced by decreased phosphorylation of Akt, S6K and 4E-BP. Metabolism is altered in poly mutants, as revealed by microarray expression analysis and a decreased triglyceride : protein ratio in mutant animals. Intriguingly, the cellular distribution of Poly is dependent on insulin stimulation in both Drosophila and human cells, moving to the nucleus with insulin treatment, consistent with a role in InR–TOR signalling. Together, these data reveal that Poly is a novel, conserved (from flies to humans) mediator of InR signalling that promotes an increase in cell growth and metabolism. Furthermore, homology to small subunits of Elongator demonstrates a novel, unexpected role for this complex in insulin signalling. rsob.royalsocietypublishing.org Open Biol 2: 110031 2 Open Biol 2: 110031 A critical consequence of the activation of InR–TOR sig- nalling is the inhibition of autophagy: a cellular response to starvation in which components of the cytoplasm are engulfed in small double-membrane-enclosed vesicles. The contents of these vesicles are degraded by the autophagic machinery, and breakdown products then serve as a nutrient source for the cell until more nutrients become available in the environment. Alterations to autophagy have been found in cancer and neurodegenerative diseases [17,18]. Herein, we report the identification of Poly as a novel mediator of the InR–TOR signalling pathway. poly is an essential gene in Drosophila that was mutated in a P-element transposon mutagenesis screen [19]. Crucially, the gene pro- duct is conserved in higher eukaryotes, including humans, showing homology to the ELP6 subunit of the Elongator complex. Loss of poly function results in lethality at the late larval stage, but only after extreme larval longevity. Many intriguing phenotypic features are observed in larvae lacking poly, including abnormal nuclear morphology in neuroblasts and the development of large melanotic masses in third instar larvae. We have combined genetic, biochemical and bioinfor- matic approaches to functionally characterize poly. Our data reveal that poly is a novel mediator of InR–TOR signalling and that loss of poly results in downregulation of a number of components of the InR–TOR pathway. We therefore pro- pose that the wild-type Poly protein is a positive regulator of cell growth and metabolism. Expression analysis of poly revealed high levels of mRNA and protein in the first 4 h of embryogenesis, suggestive of maternal loading of the mRNA and possibly protein into oocytes (figure 1h,i). While the level of Poly decreased during the remainder of embryogenesis, the level was still higher than in later stages of development. Overall, poly was expressed throughout development (figure 1h,i). 3.2. Poly is conserved among higher eukaryotes and resembles the Elp6 subunit of the Elongator complex The Drosophila poly gene encodes a 251-amino-acid-long protein, lacking any motifs suggestive of specific function. Sequence similarity searches revealed that Poly is conserved among higher eukaryotes and, importantly, has a human homologue (figure 2a). The apparent orthologue of Poly in Saccharomyces cerevisiae is the Elp6 protein, part of the 6-sub- unit Elongator complex that, in association with the RNA polymerase II holoenzyme, is responsible for transcriptional elongation [21,22]. The Elongator holocomplex is conserved in composition from yeast to humans [23,24], with acetylation activity contributed by the Elp3 catalytic subunit [25]. Intri- guingly, acetylation is directed to different substrates, depending on where in the cell the complex is (e.g. tubulin in the cytoplasm, and histone in the nucleus) [26,27]. Elonga- tor has additionally been linked to translational control through tRNA modification in the cytoplasm. To date, only the Elp3 subunit has been studied in Drosophila, where rsob.royalsocietypublishing.org Open Biol 2: 110031 2 verified that expression of the overlapping CG8790 gene was not affected by the P-element insertion in the poly05137 allele (figure 1b). Two independent experiments additionally corro- borate lesion of the CG9829 gene as being responsible for the mutant phenotype: (i) excision of the P-element following gen- etic exposure to transposase completely reverted the mutant phenotype, and (ii) successful rescue of poly05137 lethality was achieved by using a hs-Gal4 driver to direct expression of a UAS-poly transgene during larval development. 2 rsob.royalsocietypublishing.org TOR is a component of two different complexes: TORC1 and TORC2. Activation of TORC1 has various downstream effects contributing to an increase in cell growth and proliferation. For example, TORC1 directly phosphorylates S6K and 4E-BP, result- ing in an increase in ribosome biogenesis and m7G cap- dependent translation [1]. In addition, TORC1 activation inhibits autophagy [11]. A negative feedback loop signals back to the IRS through S6K, ensuring attenuation of TOR signalling above acer- tain level [12]. The TORC2 complex phosphorylates and activates Akt kinase [13], resulting in the phosphorylation of the forkhead- like transcription factor FoxO. Phosphorylated FoxO is excluded from the nucleus, precluding the transcription of FoxO target genes [14–16]. Mutation of poly results in pleiotropic effects manifesting as a particularly striking phenotype. poly mutants appear to progress normally through embryogenesis, but larval development pro- ceeds much more slowly. While the normal generation time is 10 days at 258C, poly mutant larvae exhibit extreme third instar longevity—up to 21 days—before dying without pupation (figure 1d). When homozygous mutant larvae were examined, the morphology of many tissues appeared abnormal: the brain, ring gland, salivary glands and imaginal discs were reduced in size compared with heterozygous siblings and wild-type ani- mals, suggesting cell growth and/or proliferation defects (figure 1e). Mutant larval neuroblasts were characterized by abnormally shaped nuclei, though mitotic figures, evident even in 20 day old mutant larvae, were normal in appearance. These lobulated nuclei resembled the nuclei of mammalian poly- morphonuclear leucocytes, thus suggesting the name poly (figure 1f ). During the lengthened third instar larval phase, melanotic masses appeared in the haemolymph of poly mutants, increasing in size and number with time (figure 1g). A second hypomorphic allele of poly has recently been reported to result in abnormal nurse-cell chromosome dispersal and oocyte polarity in the Drosophila germline, possibly owing to a proposed interaction with an mRNP complex involved in similar processes [20]. 2. Introduction †Institute of Healthy Ageing, University College London, The Darwin Building, Gower Street, London WCIE 6BT, UK. Multi-cellular organisms have evolved mechanisms to link cellular metabolism and growth to external environmental cues, such as nutrient and growth factor levels, in order to survive and adapt to fluctuations in the availability of these factors. The InR–TOR pathway is one of the key regulators of cellular energy homeostasis and growth [1], and this signalling pathway is evolutionarily con- served among metazoa [1,2]. Thus, studies carried out using Drosophila as a model system have played a major role in expanding our understanding of the mechanisms, as well as downstream consequences, of signalling via this pathway [3–5]. In humans, aberrations of InR–TOR signalling lead to various metabolic syndromes, including diabetes and obesity, as well as to the develop- ment of various types of cancers [6]. ‡School of Biological Sciences, University of Liverpool, The Biosciences Building, Crown Street, Liverpool L69 7ZB, UK. ‡School of Biological Sciences, University of Liverpool, The Biosciences Building, Crown Street, Liverpool L69 7ZB, UK. ‡School of Biological Sciences, University of Liverpool, The Biosciences Building, Crown Street, Liverpool L69 7ZB, UK. }Donnelly Center for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, Ontario, Canada, M5S 3E1. kInstitute of Molecular Oncology, Biomedical Sciences Research Center ‘Alexander Fleming’, 16672 Varkiza, Greece. A cascade of phosphorylation events mediates signalling through the InR– TOR pathway. The binding of insulin to the InR leads to the phosphorylation of insulin receptor substrate (IRS) by the InR. IRS acts as a recruitment site for phosphatidylinositol 3-kinase (PI3K), which catalyses the conversion of phosphatidylinositol (4,5)-bisphosphate (PIP2) into phosphatidylinositol Electronic supplementary material is available at http://dx.doi.org/10.1098/rsob.110031 & 2012 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. (3,4,5)-trisphosphate (PIP3) at the cell membrane. PIP3 in turn recruits PDK1 and Akt to the membrane, where PDK1 phos- phorylates and activates Akt. Phosphorylated Akt signals inhibit the tuberous sclerosis complex (TSC, Tsc1–Tsc2) [7– 9]. When TSC is inhibited, the small GTPase Rheb becomes active [10]. This then activates TOR, integrating TOR into the insulin signalling process. 3.1. Characterization of the poly mutant phenotype emb, embryo (0–4, 4–20 ¼ age in hours); IL, instar larvae (1st, 2nd and 3rd); EP, early pupae; LP, late pupae; adult-m, adult males; adult-f, adult (a) pupation eclosion embryo 1* 2* 3* pupation eclosion embryo 1* 2* 3* embryo 1* 2* 3* wild-type poly/TM6B poly/poly melanotic masses visible 21 days day 1 2 3 4 6 10 (d) (b) poly rp49 wild-type poly/poly (c) poly actin wild-type poly/TM6B poly/poly Dmel2 1 kb poly CG8790 CG9829 P{PZ} in poly05137 CG8790 rsob.royalsocietypublishing.org Open Biol 2: 110031 3 (b) 3 (a) wild-type poly (e) (h) ( f) wild–type ( f) (g) (g) (i) 4 poly poly poly rp49 LP EP adult 1st IL 2nd IL 3rd IL emb (h) (i) Poly actin (i) adult-m adult-f LP EP emb 4-20 emb 0-4 1st IL 2nd IL 3rd IL (h) b 4-20 Figure 1. Characterization of the poly gene and mutant phenotype. (a) Gene map showing the P-element insertion in the first intron of poly. (b) RT-PCR from wild- type and homozygous poly third instar larvae demonstrating the absence of Poly in poly mutant animals. The level of CG8790 is unaffected in poly mutants. RP49 serves as a control. (c) Immunoblotting of extracts from wild-type, heterozygous and homozygous poly third instar larvae demonstrating the absence of Poly in poly mutant animals. Poly is also present in Dmel2-cultured Drosophila cells. (d) Developmental timing of wild-type, heterozygous and homozygous poly animals. (e) Third instar larval brain and imaginal discs dissected from wild-type and poly animals. Note the reduced brain size and absence of imaginal discs in the homozygous poly mutant. (f ) DAPI staining of wild-type and poly larval neuroblasts. Note the abnormally shaped nuclei in the homozygous poly mutant. (g) Homozygous poly larvae develop melanotic masses during the third larval instar, which increase in number with time. (h,i) Expression analysis of poly mRNA and protein throughout development. Northern and immunoblotting of wild-type extracts at specific developmental stages showing Poly mRNA and protein expression levels. emb, embryo (0–4, 4–20 ¼ age in hours); IL, instar larvae (1st, 2nd and 3rd); EP, early pupae; LP, late pupae; adult-m, adult males; adult-f, adult females. the mutant phenotype has recently been reported to be remarkably similar to that described herein [28]. (and can also be identified in flies and humans). 3.1. Characterization of the poly mutant phenotype The poly mutation was isolated in a P-element mutagenesis screen that aimed to generate a large collection of single P-element-induced mutations in Drosophila [19]. The P-element insertion that led to the lethal poly05137 allele was mapped to the single intron of the CG9829 gene, localizing to 87E7-8 on the third chromosome (figure 1a). The poly05137 insertion led to an absence of poly mRNA as assessed by Northern blotting (not shown), reverse transcriptase– polymerase chain reaction (RT-PCR; figure 1b) and Poly protein as revealed by immunoblotting (figure 1c). RT-PCR mutant phenotype has recently been reported to be (and can also be identified in flies and humans) Co Poly actin (i) adult-m adult-f LP EP emb 4-20 emb 0-4 (a) poly rp49 LP EP adult 1st IL 2nd IL 3rd IL 1st IL 2nd IL 3rd IL emb (h) (e) wild-type poly ( f) (g) wild–type poly pupation eclosion embryo 1* 2* 3* pupation eclosion embryo 1* 2* 3* embryo 1* 2* 3* wild-type poly/TM6B poly/poly melanotic masses visible 21 days day 1 2 3 4 6 10 (d) (b) poly rp49 wild-type poly/poly (c) poly actin wild-type poly/TM6B poly/poly Dmel2 1 kb poly CG8790 CG9829 P{PZ} in poly05137 CG8790 e 1. Characterization of the poly gene and mutant phenotype. (a) Gene map showing the P-element insertion in the first intron of poly. (b) RT-PCR f and homozygous poly third instar larvae demonstrating the absence of Poly in poly mutant animals. The level of CG8790 is unaffected in poly muta s as a control. (c) Immunoblotting of extracts from wild-type, heterozygous and homozygous poly third instar larvae demonstrating the absence of Po nt animals. Poly is also present in Dmel2-cultured Drosophila cells. (d) Developmental timing of wild-type, heterozygous and homozygous poly a hird instar larval brain and imaginal discs dissected from wild-type and poly animals. Note the reduced brain size and absence of imaginal discs zygous poly mutant. (f ) DAPI staining of wild-type and poly larval neuroblasts. Note the abnormally shaped nuclei in the homozygous poly mu omozygous poly larvae develop melanotic masses during the third larval instar, which increase in number with time. (h,i) Expression analysis of poly m n throughout development. Northern and immunoblotting of wild-type extracts at specific developmental stages showing Poly mRNA and protein e . 3.1. Characterization of the poly mutant phenotype Consistent with the previous identification of Elp4 and Elp6 proteins as paralogues [29], our resulting phylogeny implies that each of the three different Elongator subunits probably arose from common gene duplication events during eukaryotic evolution Elp6 in budding yeast is part of an Elongator subcomplex that also contains the Elp4 and Elp5 proteins [21,22], which share sufficient homology to be aligned with one another 2b; electronic supplementary material figure S1) interacting physically with Poly immunopre Hs Dm At Sc Hs Dm At Sc Hs Dm At Sc Hs Dm At Sc variable conserved P ol y /E l p 6 E l p 5 E l p 4 Arabidopsis rice Saccharomyces Schizosaccharomyces Caenorhabditis Drosophila human zebrafish Dictyostelium Dictyostelium Arabidopsis rice Saccharomyces Schizosaccharomyces Caenorhabditis Drosophila zebrafish human Drosophila human zebrafish Arabidopsis rice Saccharomyces Schizosaccharomyces Dictyostelium Caenorhabditis 0.5 (a) (b) Alignment and phylogenetic analysis of Poly. (a) Alignment showing Poly homologues in human (Hs; NP_001026873), Drosophila , Arabidopsis thaliana (At; NP_567351) and Saccharomyces cerevisiae (Sc; NP_014043), with shading of identical and similar amino ac HADE program. The coloured band underneath each section of the alignment indicates sequence conservation according to the PAM25 ing window of 10 amino acids. The location of the Walker B motif (ordinarily associated with ATP binding) is indicated by a blue box, a motif required for ATP hydrolysis is similarly conserved. (b) Phylogenetic tree showing the relationship between Poly, identified e of Elp6, and homologous sequences. The scale bar indicates distance according to the WAG substitution matrix. Hs Dm At Sc Hs Dm At Sc Hs Dm At Sc Hs Dm At Sc variable conserved (a) rsob.royalsocietypublishing.org Open Biol 2: 110031 4 Open Biol 2: 110031 Sc variable P ol y /E l p 6 E l p 5 E l p 4 Arabidopsis rice Saccharomyces Schizosaccharomyces Caenorhabditis Drosophila human zebrafish Dictyostelium Dictyostelium Arabidopsis rice Saccharomyces Schizosaccharomyces Caenorhabditis Drosophila zebrafish human Drosophila human zebrafish Arabidopsis rice Saccharomyces Schizosaccharomyces Dictyostelium Caenorhabditis 0.5 (b) Figure 2. Alignment and phylogenetic analysis of Poly. (a) Alignment showing Poly homologues in human (Hs; NP_001026873), Drosophila melanogaster (Dm; AAF40432), Arabidopsis thaliana (At; NP_567351) and Saccharomyces cerevisiae (Sc; NP_014043), with shading of identical and similar amino acids as rendered by the BOXSHADE program. The coloured band underneath each section of the alignment indicates sequence conservation according to the PAM250 matrix, averaged over a sliding window of 10 amino acids. 3.1. Characterization of the poly mutant phenotype The location of the Walker B motif (ordinarily associated with ATP binding) is indicated by a blue box, although no Walker A (P-loop) motif required for ATP hydrolysis is similarly conserved. (b) Phylogenetic tree showing the relationship between Poly, identified as the Drosophila orthologue of Elp6, and homologous sequences. The scale bar indicates distance according to the WAG substitution matrix. P ol y /E l p 6 E l p 5 E l p 4 Arabidopsis rice Saccharomyces Schizosaccharomyces Caenorhabditis Drosophila human zebrafish Dictyostelium Dictyostelium Arabidopsis rice Saccharomyces Schizosaccharomyces Caenorhabditis Drosophila zebrafish human Drosophila human zebrafish Arabidopsis rice Saccharomyces Schizosaccharomyces Dictyostelium Caenorhabditis 0.5 (b) (b) Figure 2. Alignment and phylogenetic analysis of Poly. (a) Alignment showing Poly homologues in human (Hs; NP_001026873), Drosophila melanogaster (Dm; AAF40432), Arabidopsis thaliana (At; NP_567351) and Saccharomyces cerevisiae (Sc; NP_014043), with shading of identical and similar amino acids as rendered by the BOXSHADE program. The coloured band underneath each section of the alignment indicates sequence conservation according to the PAM250 matrix, averaged over a sliding window of 10 amino acids. The location of the Walker B motif (ordinarily associated with ATP binding) is indicated by a blue box, although no Walker A (P-loop) motif required for ATP hydrolysis is similarly conserved. (b) Phylogenetic tree showing the relationship between Poly, identified as the Drosophila orthologue of Elp6, and homologous sequences. The scale bar indicates distance according to the WAG substitution matrix. (figure 2b; electronic supplementary material, figure S1). Thus, Poly is an evolutionarily conserved protein that exhibits homology to the yeast Elp6 Elongator subcomplex component. interacting physically with Poly, immunoprecipitation with an antibody generated against recombinant Poly was per- formed using 0–5 h Drosophila embryo extracts (figure 3a,b). Poly can only be immunoprecipitated with immune serum, and only from wild-type extracts. Samples were analysed using tandem mass spectrometry [30,31]. Immunoprecipi- tates with pre-immune serum served as the control. Among the five Poly-binding proteins identified with significant scores, InR scored highest and was represented by numerous 3.3. Identification of protein interactors of Poly As the poly gene appeared to be a ‘pioneer’, we adopted an unbiased approach to determine the pathways and processes in which Poly might be involved. In order to identify proteins 97 64 51 39 28 19 14 a-poly pre-I 5 4 123 10 9 678 rp49 poly wt InR µ-tub Poly Akt1 wt InR InR poly (a) (c) (e) (d) (b) 97 64 51 39 28 19 poly HC a-poly pre-I Figure 3. Poly and the insulin receptor physically interact. (a) An antibody generated to recombinant Poly immunoprecipitates Poly from wild-type Drosophila third instar larval extracts. HC represents the heavy chain recognized by the secondary antibody during detection. Poly migrates at its predicted size of 28 kDa. (b) Poly was immunoprecipitated from 0–5 h wild-type embryos using Poly antibodies and compared with an identical sample immunoprecipitated with pre-immune serum. Bands numbered 1–5 in the immunoprecipitate lane and their corresponding bands in the pre-immune lane (numbered 6–10) were excised from the Coomassie-stained gel and analysed by mass spectrometry. (c) Immunoblotting using the Poly antibody was carried out on wild-type, InR05545 and Akt104226 larval extracts. (d) RT-PCR was performed using primers specific for poly and rp49 on RNA extracted from wild-type and InR05545 larvae. (e) Melanotic masses (arrows) observed in InR05545 and poly mutant larvae. 97 64 51 39 28 19 14 a-poly pre-I 5 4 123 10 9 678 (b) rsob.royalsocietypublishing.org Open Biol 2: 110031 5 (a) (d) 97 64 51 39 28 19 poly HC a-poly pre-I pre-I (e) (c) (d) InR poly rp49 poly wt InR µ-tub Poly Akt1 wt InR InR Figure 3. Poly and the insulin receptor physically interact. (a) An antibody generated to recombinant Poly immunoprecipitates Poly from wild-type Drosophila third instar larval extracts. HC represents the heavy chain recognized by the secondary antibody during detection. Poly migrates at its predicted size of 28 kDa. (b) Poly was immunoprecipitated from 0–5 h wild-type embryos using Poly antibodies and compared with an identical sample immunoprecipitated with pre-immune serum. Bands numbered 1–5 in the immunoprecipitate lane and their corresponding bands in the pre-immune lane (numbered 6–10) were excised from the Coomassie-stained gel and analysed by mass spectrometry. (c) Immunoblotting using the Poly antibody was carried out on wild-type, InR05545 and Akt104226 larval extracts. (d) RT-PCR was performed using primers specific for poly and rp49 on RNA extracted from wild-type and InR05545 larvae. 3.3. Identification of protein interactors of Poly (e) Melanotic masses (arrows) observed in InR05545 and poly mutant larvae. 3.5. Poly mutation results in decreased signalling activity downstream of the insulin receptor peptides in two distinct bands (table 1). This result suggests that Poly and the InR might coexist within a complex, possibly playing a role in the InR signalling pathway. Based on the results presented so far, we hypothesized that there should be genetic interactions between poly and com- ponents of the InR signalling pathway. Consequences of such interactions could be revealed by the state of down- stream effectors of the InR pathway. 3.4. Poly is decreased in insulin receptor mutants Considering the physical interaction of Poly with InR, we assessed the level of Poly in various InR signalling mutants. Strikingly, the level of Poly was dramatically decreased in InR05545 mutant larvae, whereas the level of Poly in Akt1 mutant larvae was unaffected (Akt1 is situated downstream of InR; figure 3c). The level of poly mRNA was unchanged in the InR mutant, suggesting that the difference in protein level might be due to instability or decreased protein syn- thesis of Poly in the absence of InR (figure 3d). We therefore over-expressed poly in adult fly eyes, using a GMR-Gal4 construct to drive the expression of a UAS-poly transgene [33]. At 278C, this led to a rough eye (disorganized ommatidia) phenotype (figure 4a). Disruption of InR–TOR signalling through mutation of either dAkt1 or dS6K led to a striking suppression of the poly-induced rough eye pheno- type (figure 4b,c), suggesting that an intact InR–TOR signalling cascade was required for Poly to exert its effects. These experiments also critically demonstrate the cell auton- omous importance of Poly to insulin signalling. Further evidence for a connection between Poly and the InR was provided by phenotypic analysis of InR05545 mutant larvae (figure 3e). While this allele was reported previously to be embryonic lethal [32], we noticed that a small number of homozygous InR05545 animals reached the third instar larval stage. Strikingly, the phenotype of these InR mutant larvae resembled that observed in poly larvae with increased larval lifespan and development of melanotic masses (figure 3e). These observations demonstrate that loss of InR leads to a decrease in Poly and that InR05545 mutants exhibit phenotypic features similar to those of poly mutant larvae. We hypothesized that if dAkt and dS6K mutants can act as suppressors of the poly over-expression phenotype, the activity of these kinases might be altered in poly mutant ani- mals. We therefore probed early third instar larval extracts with antibodies that recognize specifically the phos- phorylated (active) forms of dAkt and dS6K. Indeed, phosphorylation of both dAkt and dS6K was decreased upon mutation of poly (figure 4d,e). 3.4. Poly is decreased in insulin receptor mutants These data reveal that the activity of both dAkt and dS6K kinases was decreased poly wt p-dS6K 51 51 64 (e) 51 19 poly wt (g) p-4E-BP 4E-BP expression (f) 0 0.2 0.4 0.6 0.8 wt poly ** 64 51 64 51 poly wt µ-tub µ-tub µ-tub dAkt p-dAkt GMR-Gal4>UAS-poly TM3 GMR-Gal4>UAS-poly Akt1 (b) (a) (d) GMR-Gal4>UAS-pol + GMR-Gal4>UAS-poly TM3 GMR-Gal4>UAS-poly S6K (c) + + Figure 4. Phosphorylation levels of Akt and S6K are decreased in poly larvae. (a–c) Images showing Drosophila compound eyes. (a) A rough eye phenotype is caused by UAS-poly overexpression under the control of GMR-Gal4 driver. (b,c) The rough eye phenotype caused by UAS-poly overexpression under the control of GMR-Gal4 driver is suppressed by Akt104226 and S6K07084 mutations. TM3 represents the balancer chromosome carried by control sibling progeny. (d,e) Immunoblotting of third instar larval extracts using phospho-specific antibodies to dAkt and dS6K reveals decreased phosphorylation of both kinases in poly larval extracts. (f ) d4E-BP transcript levels were assessed by real-time qPCR on RNA isolated from wild-type and poly mutant larvae. d4E-BP levels were normalized to Actin5C levels. The error bars derive from reactions performed on biological triplicate samples. The double asterisk represents significant difference ( p , 0.01) in 4E- BP expression levels between wild-type and poly larvae. (g) Immunoblotting of third instar larval extracts showing decreased phosphorylation of d4E-BP in poly compared with wild-type larvae. poly wt p-dS6K 51 51 64 (e) 4E-BP expression (f) 0 0.2 0.4 0.6 0.8 wt poly ** 64 51 64 51 poly wt µ-tub µ-tub dAkt p-dAkt (d) rsob.royalsocietypublishing.org Open Biol 2: 110031 6 GMR-Gal4>UAS-poly (b) (a) + + 64 51 64 51 poly wt µ-tub dAkt p-dAkt (d) (a) + poly wt p-dS6K 51 51 64 (e) µ-tub (e) GMR-Gal4>UAS-poly Akt1 GMR-Gal4>UAS-poly S6K GMR-Gal4>UAS-poly b) (b) Akt1 TM3 4E-BP expression (f) 0 0.2 0.4 0.6 0.8 wt poly ** GMR-Gal4>UAS-poly (c) GMR Gal4>UAS poly TM3 (c) S6K 51 19 poly wt (g) p-4E-BP µ-tub Figure 4. Phosphorylation levels of Akt and S6K are decreased in poly larvae. (a–c) Images showing Drosophila compound eyes. (a) A rough eye phenotype is caused by UAS-poly overexpression under the control of GMR-Gal4 driver. (b,c) The rough eye phenotype caused by UAS-poly overexpression under the control of GMR-Gal4 driver is suppressed by Akt104226 and S6K07084 mutations. TM3 represents the balancer chromosome carried by control sibling progeny. 3.4. Poly is decreased in insulin receptor mutants (d,e) Immunoblotting of third instar larval extracts using phospho-specific antibodies to dAkt and dS6K reveals decreased phosphorylation of both kinases in poly larval extracts. (f ) d4E-BP transcript levels were assessed by real-time qPCR on RNA isolated from wild-type and poly mutant larvae. d4E-BP levels were normalized to Actin5C levels. The error bars derive from reactions performed on biological triplicate samples. The double asterisk represents significant difference ( p , 0.01) in 4E- BP expression levels between wild-type and poly larvae. (g) Immunoblotting of third instar larval extracts showing decreased phosphorylation of d4E-BP in poly compared with wild-type larvae. Table 1. Mass spectrometry identifies the insulin receptor as a physical interactor of Poly. Analysis of pre-immune and immunoprecipitation samples by tandem mass spectrometry identified interacting proteins specifically found in the immune-precipitate sample. Insulin receptor was identified in bands 2 and 3, with the highest scores. ‘exp score’ quantifies on a log scale (base 10) the expectation that the hit was achieved by chance, calculated using the program X!TANDEM. Table 1. Mass spectrometry identifies the insulin receptor as a physical interactor of Poly. Analysis of pre-immune and immunoprecipitation samples by tandem mass spectrometry identified interacting proteins specifically found in the immune-precipitate sample. Insulin receptor was identified in bands 2 and 3, with the highest scores. ‘exp score’ quantifies on a log scale (base 10) the expectation that the hit was achieved by chance, calculated using the program X!TANDEM. band exp score unique/total peptides protein 1 23 1/1 ATP-dependent RNA helicase (Rm62) 2 213 4/7 insulin-like receptor 3 213 4/7 insulin-like receptor 4 25.3 3/3 cadherin 5 22.5 2/2 transient receptor potential cation channel subfamily A (TrpA1) in poly mutant animals. In addition, the level of p-Akt at the tissue level, examined by immunofluorescence of whole mount third instar larval brains, additionally corro- borates this decrease (electronic supplementary material, figure S2). One crucial downstream effector of TOR is 4E-BP, the translation initiation factor eIF-4E-binding protein. Phos- phorylation of 4E-BP by TOR leads to its dissociation from the m7G-cap-binding protein eIF-4E, thereby allowing acti- vation of cap-dependent translation with consequent positive effects on cell growth [34]. Disruption of InR–TOR signalling causes inhibition of cap-dependent translation as a decrease in 4E-BP phosphorylation results in its binding to eIF-4E [14]. Disruption of signalling also causes increased transcription of 4E-BP owing to increased FoxO activity. 3.8. Metabolism is affected in poly mutant larvae Drosophila is frequently used to study metabolic regulation as fruitflies share the majority of metabolic functions with ver- tebrates [42]. The larval fat body is the main organ for regulation of energy homeostasis as excess energy is stored in the form of glycogen and triglycerides (TAGs; lipids). Acti- vation of signalling through the InR pathway promotes both anabolic metabolism and the storage of nutrients such as TAGs [43]. This is important for development, as the break- down of larval fat body and consequent release of energy facilitates Drosophila metamorphosis. A critical consequence of the activation of InR–TOR signal- ling is the inhibition of autophagy. Drosophila undergoes developmentally programmed autophagy at defined times to facilitate tissue remodelling during metamorphosis [35–38]. On the other hand, starvation-induced autophagy (in response to nutrient deprivation) takes place specifi- cally in the larval fat body [11]. As the larval fat body serves a similar function to the vertebrate liver by acting as a nutrient storage organ, it has been commonly used to examine autophagy during both starvation-induced and developmentally regulated autophagy [11,39,40]. While the fat body from fed wild-type larvae does not normally exhibit autophagy, autophagy is evident within a short period of amino acid starvation. In order to identify genes that were differentially expressed in poly mutant larvae, we carried out a microarray analysis. Among the list of 106 genes downregulated in poly mutants, functional enrichment group analysis identified a strong enrichment in gene ontology (GO) terms belonging to metabolic processes, suggesting that metabolism might be affected in poly mutants (table 2). We therefore investi- gated whether the decrease in InR–TOR activity was also manifest at the metabolic level in the storage of TAGs in poly mutants. TAG levels were normalized to total protein to give an accurate quantification of lipids per unit mass in wild-type and poly mutant larval extracts. Consistent with a decrease in InR–TOR signalling, we found the TAG : protein ratio to be half that detected in wild-type larvae (figure 6a). TOR directly inhibits starvation-induced autophagy, while components of InR signalling (such as InR and Akt, acting upstream of TOR) also behave as negative regulators of autophagy. We hypothesized that if poly acts in InR signal- ling, the state of autophagy as visualized with Lysotracker staining may be altered in the poly mutant fat body compared with wild-type fat body. 3.8. Metabolism is affected in poly mutant larvae As anticipated, no Lysotracker puncta were observed in fed wild-type early third instar fat body (figure 5a, fed). However, Lysotracker puncta became apparent following a 4 h amino acid starvation, owing to the activation of autophagy (figure 5a, starved). Strikingly, Lysotracker puncta were abundant even in fed poly fat bodies, demonstrating that autophagy was constitutively active in the fat body of poly mutants (figure 5b, compare fed with starved). Lipid storage droplet-2 protein (Lsd-2), a protector against lipolysis and the Drosophila perilipin homologue, is localized on the outer membrane of lipid droplets, the main storage organelle for TAGs [44,45]. We found that the level of Lsd-2 in poly larval extracts was decreased relative to wild-type fat body, consistent with the decreased TAG : protein ratio observed in poly mutants (figure 6b). Together, the reduction in TAG storage and Lsd-2 protein in poly mutant larvae suggest a decrease in anabolic metabolism, consistent with—and an expected consequence of—diminished InR–TOR signalling. rsob.royalsocietypublishing.org Open Biol 2: 110031 7 rsob.royalsocietypublishing.org Thus, several independent lines of evidence demonstrate that loss of poly leads to an activation of autophagy coupled with an increase in apoptotic cell death. Open Biol 2: 110031 Open Biol 2: 110031 3.7. Poly loss of function leads to an increase in apoptotic cell death Autophagy and apoptosis are closely related, and increased levels of autophagy can lead to apoptosis [39,41]. For example, increased autophagy resulting from the clonal over-expression of Atg1 in the wing disc resulted in elevated apoptosis, as evidenced by the appearance of cleaved caspase-3 in these clones [39]. 3.4. Poly is decreased in insulin receptor mutants Con- sistent with a decrease in InR–TOR signalling, the level of d4E-BP transcript was almost threefold greater in poly mutant larvae compared with control animals (figure 4f ). In addition, immunoblotting revealed that d4E-BP was hypo-phosphorylated in poly mutant extracts compared with wild-type extracts (figure 4g). Taken together, these results are consistent with a decrease in cap-dependent translation upon loss of Poly function. detect mitosis and apoptosis, respectively (figure 5c). Mitotic figures, though rare at the latest stages, were still normal in appearance (data not shown). As the generation of mosaic imaginal discs allows the side-by-side comparison of wild- type versus mutant cells, we generated poly loss-of-function clones in imaginal discs. Staining of mosaic discs revealed increased levels of cleaved caspase-3 in poly mutant clones, which were discernible by the lack of ß-galactosidase (figure 5d). Cleaved caspase-3 was not detectable in the adjacent wild-type cells. rsob.royalsocietypublishing.org Open Biol 2: 110031 7 7 3.9. The Poly protein relocalizes following insulin stimulation in Drosophila haemocytes and human cultured cells fed fed starved starved Lysotracker DNA Lysotracker (a) (b) poly wild-type (c) 0 1 2 3 4 5 6 7 8 6-wt 6-het 6 8 10 12 14 16 18 20 22 % cells age (days) rsob.royalsocietypublishing.org Open Biol 2: 110031 8 (b) 8 (a) Open Biol 2: 110031 caspase-3 (d) b-gal DAPI (d) Figure 5. poly mutations leads to constitutive autophagy and increased cell death. (a,b) Lysotracker (an acidic component-specific fluorescent dye) was used to monitor the state of autophagy in early third instar larval fat body. Autophagy was indicated by a punctate Lysotracker appearance. Live fat body tissues were stained with Lysotracker (red) and Hoechst 33342 to label DNA (blue). (a) In wild-type larval fat body, no autophagy is evident. As indicated by Lysotracker positive bright spots, autophagy is active upon 4 h amino acid starvation in 20 per cent sucrose solution. (b) Lysotracker puncta can be detected in fed poly fat body, as well as in starved poly fat body. (a,b) Scale bar, 50 mm. (c) Quantitation of mitosis and apoptosis in larval neuroblasts over development. While mitotic activity decreases, apoptosis increases during the extended larval stage of poly larva. Blue bars, p≏H3; red bars, TUNEL. (d) Images showing imaginal disc dissected from third instar larva of the genotype eyFLP; FRT82Bb-Gal/FRT82Bpoly05137. Immunostaining for cleaved caspase-3 (red), b-galactosidase (green) and DNA (blue) was performed. poly mutant clones are indicated by the lack of b-Gal staining. Scale bar, 30 mm. innate immune system). Prior to isolation of haemocytes, larvae were starved for 3 h in 20 per cent sucrose. Haemo- cytes were then stimulated in culture with 200 nM insulin (electronic supplementary material, figure S3). This increased staining in Poly appears to be a rapid response as it is detect- able following only 15 min of insulin stimulation, and levels of Poly remain elevated after 75 min. insulin stimulation (figure 7a). Nearly, 60 per cent of cells showed a relocalization of Poly by 60 min of insulin stimu- lation, with this level increasing to 100 per cent by 90 min. This rapid change in HsPoly behaviour was dependent on TOR signalling. Incubation of HeLa cells with rapamycin during the overnight starvation period prevented the increase in HsPoly staining following insulin stimulation (figure 7b). In rapamycin-treated cells, HsPoly remained evenly dis- tributed throughout the cell even following 90 min of insulin treatment. 3.9. The Poly protein relocalizes following insulin stimulation in Drosophila haemocytes and human cultured cells 3.9. The Poly protein relocalizes following insulin stimulation in Drosophila haemocytes and human cultured cells Given the physical interaction of Poly with the InR and func- tional links between Poly and InR–TOR signalling, we were interested to determine whether the level and/or distribution of Poly in the cell were dependent on InR activity. Because loss of poly led to constitutive activation of auto- phagy in the fat body, we investigated whether the loss of poly also resulted in increased apoptosis. Indeed, cell death increased dramatically in third instar larval neuroblasts as poly larvae aged. This was readily seen when larval neuro- blasts were stained for pS10-histone H3 and TUNEL to Strikingly, staining for Poly was noticeably increased fol- lowing insulin stimulation of larval haemocytes (cells of the fed fed starved starved Lysotracker DNA Lysotracker (a) (b) poly wild-type (c) 0 1 2 3 4 5 6 7 8 6-wt 6-het 6 8 10 12 14 16 18 20 22 % cells age (days) caspase-3 (d) b-gal DAPI ure 5. poly mutations leads to constitutive autophagy and increased cell death. (a,b) Lysotracker (an acidic component-specific fluorescent dye) was used to monitor state of autophagy in early third instar larval fat body. Autophagy was indicated by a punctate Lysotracker appearance. Live fat body tissues were stained with tracker (red) and Hoechst 33342 to label DNA (blue). (a) In wild-type larval fat body, no autophagy is evident. As indicated by Lysotracker positive bright spots, phagy is active upon 4 h amino acid starvation in 20 per cent sucrose solution. (b) Lysotracker puncta can be detected in fed poly fat body, as well as in starved poly ody. (a,b) Scale bar, 50 mm. (c) Quantitation of mitosis and apoptosis in larval neuroblasts over development. While mitotic activity decreases, apoptosis increases ng the extended larval stage of poly larva. Blue bars, p≏H3; red bars, TUNEL. (d) Images showing imaginal disc dissected from third instar larva of the genotype P; FRT82Bb-Gal/FRT82Bpoly05137. Immunostaining for cleaved caspase-3 (red), b-galactosidase (green) and DNA (blue) was performed. poly mutant clones are cated by the lack of b-Gal staining. Scale bar, 30 mm. 4. Discussion Poly is a conserved protein that plays a novel essential role in InR signalling and, crucially, promotes the effects of the TOR kinase on cell growth and metabolism. 3.9. The Poly protein relocalizes following insulin stimulation in Drosophila haemocytes and human cultured cells DAVID (the Database for Annotation, Visualization and Integrated Discovery) functional enrichment group analysis of the list of differentially expressed genes identified enrichment in gene ontology (GO) terms belonging to metabolic processes as among those genes downregulated in poly mutants. The data were analysed by Limma (see §5.10) and were for four biological replicates. Genes were selected for functional/pathway analysis if the adjusted (corrected for multiple testing) p-value was less than 0.05. Log (base 2) fold changes are given (log2FC). ‘rep’ indicates reported gene function, while ‘pred’ indicates predicted gene function according to the gene-specific FlyBase report. Table 2. Microarray analysis revealed that several metabolic functions were among the genes downregulated in poly mutants. DAVID (the Database for Annotation, Visualization and Integrated Discovery) functional enrichment group analysis of the list of differentially expressed genes identified enrichment in gene ontology (GO) terms belonging to metabolic processes as among those genes downregulated in poly mutants. The data were analysed by Limma (see §5.10) and were for four biological replicates. Genes were selected for functional/pathway analysis if the adjusted (corrected for multiple testing) p-value was less than 0.05. Log (base 2) fold changes are given (log2FC). ‘rep’ indicates reported gene function, while ‘pred’ indicates predicted gene function according to the gene-specific FlyBase report. GO TERM name of affected gene (log2FC) fold-enrichment GO:0006739≏NADP metabolic process FBgn0004057 // CG12529 (21.42) 81.9 glucose-6-phosphate dehydrogenase rep FBgn0023477 // CG2827 (21.27) transaldolase rep FBgn0004654 // CG3724 (22.15) phosphogluconate dehydrogenase rep FBgn0030239 // CG17333 (21.34) 6-phosphogluconolactonase pred FBgn0037607 // CG8036 (21.39) transketolase pred GO:0006769≏nicotinamide metabolic process same five genes 64.4 GO:0019362≏pyridine nucleotide metabolic process same five genes 60.1 GO:0006006≏glucose metabolic process same five genes, and 16.4 FBgn0002569 // CG8694 (20.89) maltase A2 rep name of affected gene (log2FC) fold-enrichment name of affected gene (log2FC) fold-enrichment GO:0006739≏NADP metabolic process GO:0006739≏NADP metabolic process GO:0006769≏nicotinamide metabolic process GO:0019362≏pyridine nucleotide metabolic process GO:0006006≏glucose metabolic process growth. Phosphorylation of Akt substrates contributes to a range of cellular processes including cell growth, proliferation and survival [46]. Dysregulation of Akt is involved in various diseases, including type-2 diabetes and cancer [47,48]. S6K kinase is one of the most downstream effectors of InR–TOR signalling, and is subject to phosphorylation and activation by TOR. Activation of S6K leads to an increase in translation through its phosphorylation and activation of ribosomal protein S6 [1]. 3.9. The Poly protein relocalizes following insulin stimulation in Drosophila haemocytes and human cultured cells A change in the appearance of Poly following insulin stimulation was conserved in human cells. Overnight serum- starved HeLa cells were stimulated with insulin for up to 90 min. As in Drosophila haemocytes, staining for HsPoly was significantly stronger following insulin stimulation, appearing maximal at 90 min. It additionally appeared that this increase in HsPoly was concentrated in or near the nucleus, with relocalization already evident by 30 min of Taken together, these observations demonstrate that the increase and nuclear relocalization of HsPoly following insu- lin treatment occurs in a TOR-dependent manner. Overall, our results identify Poly as a novel component of InR–TOR signalling that is conserved from flies to humans. (b) 51 39 poly wt Lsd-2 51 µ-tub (a) poly ** normalized triglyceride:protein 0 0.2 0.4 0.6 0.8 1.0 1.2 wt Figure 6. poly mutant larvae are characterized by reduced levels of TAGs and Lsd-2. (a) TAG levels were assessed in wild-type and poly mutant third instar larvae. The total TAG level was normalized to the total protein level. Error bars derive from the standard deviation of three independent experiments. The unpaired two- tailed t-test was used to compare the data from wild-type and poly larvae. The double asterisk represents a significant difference ( p , 0.01) in TAG : protein ratio between control and poly mutant larvae. (b) Immunoblotting was carried out on wild-type and poly mutant larval extracts with an antibody specifically recognizing Lsd2 protein. (b) 51 39 poly wt Lsd-2 51 µ-tub (a) poly ** normalized triglyceride:protein 0 0.2 0.4 0.6 0.8 1.0 1.2 wt rsob.royalsocietypublishing.org Open Biol 2: 110031 9 rsob.royalsocietypublishing.org Figure 6. poly mutant larvae are characterized by reduced levels of TAGs and Lsd-2. (a) TAG levels were assessed in wild-type and poly mutant third instar larvae. The total TAG level was normalized to the total protein level. Error bars derive from the standard deviation of three independent experiments. The unpaired two- tailed t-test was used to compare the data from wild-type and poly larvae. The double asterisk represents a significant difference ( p , 0.01) in TAG : protein ratio between control and poly mutant larvae. (b) Immunoblotting was carried out on wild-type and poly mutant larval extracts with an antibody specifically recognizing Lsd2 protein. Table 2. Microarray analysis revealed that several metabolic functions were among the genes downregulated in poly mutants. 4.1. The activity of InR signalling is decreased in poly larvae Poly acts upstream of both of these kinases in the InR signalling pathway, as levels of phosphorylated (active) dAkt and dS6K kinases were decreased in poly larvae. That Poly acts in the activation of both dAkt and dS6K is also supported by genetic data that revealed suppression of a poly-induced rough eye phenotype by mutations in dAkt and dS6K. These two kinases should only act as genetic Our data reveal an essential involvement for Poly in InR sig- nalling, an important pathway linking nutritional status to metabolism and cell growth [3]. The two kinases, Akt and S6K, are key effectors of InR–TOR signalling. The Akt kinase is located upstream of the TSC1–TSC2 complex in this pathway and has a critical role in promoting cell poly actin DNA 30 min 0 60 min 90 min + rapamycin 30 min 0 60 min 90 min (a) (b) Figure 7. Insulin stimulation of HeLa cells leads to an increase in HsPoly staining, accumulated in the nuclear area. (a) HeLa cells were serum-starved overnight and then stimulated with 100 nM insulin for 30, 60 and 90 min prior to staining for HsPoly (red), DNA (blue) and F-actin (green). Staining for HsPoly indicated peri- nuclear accumulation already by 30 min, reaching a maximum following 90 min of insulin stimulation. (b) The relocalization of HsPoly was inhibited by overnight incubation of cells with 20 nM rapamycin prior to insulin treatment. Scale bars, 50 mm. poly actin DNA 30 min 0 60 min 90 min + rapamycin 30 min 0 60 min 90 min (a) (b) ure 7. Insulin stimulation of HeLa cells leads to an increase in HsPoly staining, accumulated in the nuclear area. (a) HeLa cells were serum-starved overnight and stimulated with 100 nM insulin for 30, 60 and 90 min prior to staining for HsPoly (red), DNA (blue) and F-actin (green). Staining for HsPoly indicated peri- ear accumulation already by 30 min, reaching a maximum following 90 min of insulin stimulation. (b) The relocalization of HsPoly was inhibited by overnight bation of cells with 20 nM rapamycin prior to insulin treatment. Scale bars, 50 mm. rsob.royalsocietypublishing.org Open Biol 2: 110031 10 rsob.royalsocietypublishing.org Open Biol 2: 110031 10 10 Figure 7. Insulin stimulation of HeLa cells leads to an increase in HsPoly staining, accumulated in the nuclear area. 4.1. The activity of InR signalling is decreased in poly larvae (a) HeLa cells were serum-starved overnight and then stimulated with 100 nM insulin for 30, 60 and 90 min prior to staining for HsPoly (red), DNA (blue) and F-actin (green). Staining for HsPoly indicated peri- nuclear accumulation already by 30 min, reaching a maximum following 90 min of insulin stimulation. (b) The relocalization of HsPoly was inhibited by overnight incubation of cells with 20 nM rapamycin prior to insulin treatment. Scale bars, 50 mm. Together our data predict a decrease in InR–TOR signalling in the absence of Poly. As one read-out of TOR signalling, we examined autophagy, a multi-step, catabolic process used for nutrient recycling during development and starvation. TOR activity is responsible for inhibiting autophagy under cell growth-promoting conditions. Our finding that autophagy is constitutively active in the fat body of poly mutant larvae is in accord with the observation of reduced InR–TOR signalling, and consistent with an inhibition of cell growth and/or prolifer- ation in poly mutant animals. Furthermore, the increase in apoptotic cell death in poly mutant animals and clones generated in imaginal discs might result from increased levels of autophagy upon mutation of poly. suppressors if they are situated downstream of poly in the sig- nalling pathway, resulting in an overall decrease in InR–TOR signalling in the absence of Poly. The biochemical data presented herein revealed a phys- ical interaction of Poly with InR. Intriguingly, the level of Poly was reduced in InR mutant larval extracts indepen- dently of transcription, suggesting that a fraction of Poly may be unstable and subject to degradation in the absence of InR. Indeed, there are numerous examples of such instabil- ity if one partner in a protein complex is absent [49–51]. Whether Poly exists in a discrete complex with InR, and how such complex(es) may be regulated in response to insulin stimulation, are currently under investigation. Such an interaction may well be transient and highly dynamic, as Poly appears to accumulate in the nucleus following insulin stimulation. Considering the insulin-induced, TOR- dependent nuclear enrichment of Poly in HeLa cells, it is likely that insulin-mediated signalling via Poly is conserved from flies to humans. 5.1. Drosophila culture All fly stocks were maintained at 258C on standard medium unless otherwise stated. Fly stocks used in this study were: Canton S (wild-type), poly05137/TM6B. The following fly lines were obtained from the Bloomington Stock Center: InR05545/TM3, Akt104226/TM3, S6K07084/TM3, Cg-Gal4. All fly stocks were maintained at 258C on standard medium unless otherwise stated. Fly stocks used in this study were: Canton S (wild-type), poly05137/TM6B. The following fly lines were obtained from the Bloomington Stock Center: InR05545/TM3, Akt104226/TM3, S6K07084/TM3, Cg-Gal4. 4.4. Model for Poly action We have shown that Poly binds to InR (a transmembrane receptor) and modulates the activity of various downstream proteins. While the lack of clearly discernible functional motifs hampered a prediction of Poly’s molecular function, extensive database searches and phylogenetic analyses ident- ified Poly as a member of the Elp6 subfamily of Elongator proteins, with more distant homology to various members of the RecA/Rad51/DCM1 superfamily (such as KaiC and RadB); these observations are described in a distinct study from our laboratory. Both KaiC and Elp6 have been shown to significantly affect gene expression, pointing to a degree of functional conservation. Thus, we suggest an involvement for Poly during transcription (perhaps once relocated to the nucleus following insulin stimulation). 4.3. Comparison of poly to other mutants and Elongator Numerous aspects of the poly phenotype have been observed in other mutations, including that of InR and TOR. An extreme larval longevity, one of the most remarkable aspects of the poly mutant phenotype, has been observed in both Tor [54] and InR mutants (this study). The appearance of melano- tic masses is frequently seen in mutations with aberrant immune responses and/or haematopoietic defects [55–57]. In poly third instar larvae, melanotic masses appearing at multiple different locations along the larval body are likely to result from either an alteration in the immune response and/or a hyper-proliferation of blood cells, or haemocytes. The observation that the few InR mutant larvae that reach the third instar stage also develop melanotic masses high- lights the phenotypic similarities of poly and InR loss-of- function mutations, and further corroborates a functional relationship between poly and InR. Future research will address the detailed nature of the interaction of Poly with the InR. Does Poly interact with the InR in the absence or presence of insulin? How dynamic is this interaction? And is the level or post-translational state of Poly modified upon insulin treatment? In the light of the data presented herein, we propose that Poly is a novel mediator of InR–TOR signalling in the regulation of cell metabolism and growth in Drosophila (figure 8). We suggest that the physical interaction between Polyand the InR is followed by the translocation of Poly into the nucleus (upon insulin stimu- lation), wherein the expression of key metabolic genes is affected, thus contributing to the promotion of cell growth and metab- olism. While the detailed nature and regulation of Poly’s interaction with the InR remain to be elucidated, it is highly likely that, given the conservation of Poly, this crucial interaction and function will also hold true in human cells. Given the apparent similarity of Poly to yeast Elp6, it is therefore intriguing that the recently described mutant phe- notype for the Elongator Elp3 catalytic subunit in Drosophila resembles that of poly [28]. These observations are suggestive of a role for the Elongator holocomplex in insulin signalling— through histone and/or tubulin acetylation—or even transla- tional control through tRNA modification. In addition, as human mutations in Elp3 have been linked to familial dysautonomia [58], it is likely that the future analysis of Elongator function in model organisms will be of significant preclinical value. 4.3. Comparison of poly to other mutants and 4.3. Comparison of poly to other mutants and 4.2. Metabolism is disrupted in poly mutants A major function of the InR–TOR pathway is the modulation of metabolism. For example, 4E-BP mutant animals show increased sensitivity to starvation [16]. Interestingly, it was If Poly has a role during transcription, as the phylogenetic data suggest, how do we explain its binding to a transmem- brane receptor? One explanation is the dynamic changes in the cellular localization of Poly, occurring in a TOR-depen- dent manner. Interestingly, InR phosphorylation of IRS-1 and IRS-2 (two of the human IRS homologues) not only leads to activation of PI3K signalling, but is also associated with IRS-1 translocation to the nucleus, where it activates transcription of various genes [59–61]. Given that Poly also interacts with InR, and moves to the nucleus following insu- lin stimulation, it is possible that changes in the localization of Poly are also ultimately in response to InR signalling. Immunofluorescence on both Drosophila haemocytes and HeLa cells demonstrated that the level and/or distribution of Poly are significantly affected following insulin stimulation. Importantly, in HeLa cells, Poly relocalization to the nucleus occurred in a rapamycin-sensitive manner. Thus, we speculate that the stimulatory effects of Poly on cell growth and metab- olism may be exerted via effects on transcription. However, further analysis is required to assess whether Poly participates in an Elongator complex and, if so, in which of the myriad functions currently ascribed to Elongator. previously shown that the loss of the tumour suppressor PTEN (responsible for the dephosphorylation of PIP3) in Drosophila nurse cells results in the accumulation of activated Akt in the cytoplasm. This activated Akt drives the formation of enlarged lipid droplets along with an increase in the expression of Drosophila Lsd-2 [52]. Lsd-2 mutants are characterized by decreased TAG levels [45]. Interestingly, autophagy and lipid metabolism were shown to be two inter- linked processes, as suggested by a decrease in autophagy resulting in an increase of lipid storage in the cell [53]. Con- sistent with decreased InR–TOR signalling in the absence of poly, both Lsd-2 and the TAG : protein ratio were reduced in poly mutant animals. Therefore, we propose that Poly affects metabolism via its interaction with the InR–TOR path- way, acting as a positive regulator of anabolic metabolism. 11 rsob.royalsocietypublishing.org Open Biol 2: 110031 11 Open Biol 2: 110031 5.5. Larval manipulations Prior to dissections or protein extractions, 10–15 second instar larvae were transferred to a fresh vial of food supplemented with fresh yeast paste. Manipulations were carried out when larvae reached early third instar stage. For starvation exper- iments, early third instar larvae were starved in 20 per cent sucrose solution for 3–4 h prior to dissection. 5.2. Phylogenetic analysis Sequences homologous to Drosophila Poly were identified by multiple iterative searches using the PSI-BLAST program [62] and the HHpred interactive server [63]. Alignments between the corresponding sequences were generated using M-COFFEE [64] and manually adjusted based on predicted secondary structure according to ALI2D [65]. Identical and similar amino acids in a representative subset of aligned sequences were shaded using the BOXSHADE server (http://www.ch. embnet.org/software/BOX_form.html). Phylogenetic trees were calculated using TREE-PUZZLE [66], MRBAYES [67] and the PROML program of the PHYLIP Package [68] after clustering cytosol autophagy Akt TOR InR 4E-BP S6K Poly lsd2 transcription disrupted insulin Akt TOR autophagy InR 4E-BP S6K Poly positive regulation lsd2 transcription Poly of metabolic genes insulin (a) (b) wild-type loss of poly Figure 8. Model for the function of Poly in the InR–TOR signalling pathway. The interaction of Poly with (a) the insulin receptor allows it to fulfil a role as a positive regulator of InR–TOR signalling, which hasthe overall effect of increasing the phosphorylation and, hence, activity of numerous positive regulators of the InR–TOR pathway, such as Akt, S6K and 4E-BP, as well as inhibiting negative regulators of cell growth such as autophagy. Furthermore, Poly aids to promote anabolic metabolism resulting in an increase of TAG and Lsd-2 levels. (b) The absence of Poly leads to a decrease in the activation of positive regulators of cell growth such as Akt and S6K kinases. The 4E-BP protein becomes hypo- phosphorylated, consistent with an overall downregulation of InR–TOR signalling. Furthermore, autophagy—a negative effector of InR/TOR signalling—becomes constitutively active. Anabolic metabolism is downregulated as indicated by a decrease in TAG and Lsd-2 levels. cytosol autophagy Akt TOR InR 4E-BP S6K Poly lsd2 transcription disrupted insulin b) loss of poly rsob.royalsocietypublishing.org Open Biol 2: 110031 12 Akt TOR autophagy InR 4E-BP S6K Poly positive regulation lsd2 transcription Poly of metabolic genes insulin (a) wild-type 12 (b) (a) wild-type loss of poly Figure 8. Model for the function of Poly in the InR–TOR signalling pathway. The interaction of Poly with (a) the insulin receptor allows it to fulfil a role as a positive regulator of InR–TOR signalling, which hasthe overall effect of increasing the phosphorylation and, hence, activity of numerous positive regulators of the InR–TOR pathway, such as Akt, S6K and 4E-BP, as well as inhibiting negative regulators of cell growth such as autophagy. 5.4. Mass spectrometry The proteins in slices from sodium dodecyl sulphate poly- acrylamide gel electrophoresis (SDS–PAGE) gels were digested in-gel with trypsin by the method of Shevchenko et al. [71]. The resulting peptide mixtures were resuspended in 1 per cent formic acid and analysed by nano-electrospray liquid chromatography mass spectrometry (nano-LC MS/ 5.3. Immunoprecipitation Fifty microlitres of 0–5 h wild-type embryos (raised at 188C) were homogenized in 1 ml of cold lysis buffer (100 mM NaCl, 10 mM EDTA, 50 mM Tris–HCl pH 7.6, 0.1% Triton-X100, 10 mg ml21 each of chymostatin, leupeptin, antipain and pep- statin, and 50 mg ml21 PMSF) and briefly sonicated. Eight microlitres of rabbit pre-immune or anti-poly serum coupled to 40 ml of Dynal beads (Invitrogen) were added to pre- cleared embryo lysates overnight. Ten per cent of each sample was subjected to immunoblot analysis to verify suc- cessful immunoprecipitation of Poly, while the remaining 90 per cent of sample was resolved on precast 4–12 per cent Bis-Tris polyacrylamide gels (Novex) and stained with Colloidal Coomassie Blue (Invitrogen). Comparable molecu- lar weight regions of interest were excised from each lane (pre-immune- and immune-precipitations) and mass spec- trometry analysis performed (Dr Gerard Cagney, Dublin). 5.2. Phylogenetic analysis Furthermore, Poly aids to promote anabolic metabolism resulting in an increase of TAG and Lsd-2 levels. (b) The absence of Poly leads to a decrease in the activation of positive regulators of cell growth such as Akt and S6K kinases. The 4E-BP protein becomes hypo- phosphorylated, consistent with an overall downregulation of InR–TOR signalling. Furthermore, autophagy—a negative effector of InR/TOR signalling—becomes constitutively active. Anabolic metabolism is downregulated as indicated by a decrease in TAG and Lsd-2 levels. MS). A high-performance liquid chromatography (HPLC) instrument (Dionex, UK) was interfaced with an LTQ ion trap mass spectrometer (ThermoFinnigan, CA). Chromato- graphy buffer solutions (buffer A: 5% acetonitrile and 0.1% formic acid; buffer B: 80% acetonitrile and 0.1% formic acid) were used to deliver a 60 min gradient (35 min to 45% buffer B, 10 min to 90%, hold 10 min, 3 min to 5% and hold for 15 min). A flow rate of 2 ml min21 was used at the electro- spray source. One full scan was followed by 10 MS/MS events, and the duty cycle programmed to enforce dynamic exclusion for 2 min. In-house proteomics pipeline software (PROLINE) was used to process data. Spectra were searched using the Sequest algorithm [72] against SwissProt.2007.04.19 database restricted to Drosophila melanogaster entries. Proteins with (i) peptide prophet probability score greater than 0.99 [73] and (ii) identified by a minimum of two different peptide spectra were automatically accepted, while spectra for the minority of proteins identified by single spectra were manually checked for quality. of related sequences into smaller groups using SPLITSTREE4 [69]. Branch lengths were calculated by application of the WAG substitution matrix [70] using TREE-PUZZLE, modelling rate heterogeneity according to a gamma distribution with 16 rate categories, and bootstrap confidence intervals (provided in electronic supplementary material, figure S1) were esti- mated using the SEQBOOT program of the PHYLIP Package [68]. 5.7. Preparation of larval protein extracts for immunoblotting Open Biol 2: 110031 Fifteen to twenty wandering third instar larvae of the appro- priate genotype were placed in 1.5 ml tubes and rinsed three times with Ephrussi–Beadle Ringer’s solution (130 mM NaCl, 4.7 mM KCl, 1.9 mM CaCl2, 10 mM HEPES and pH 6.9). Next, 300ml of cold lysis buffer (Ephrussi–Beadle Ringer’s solution with 10 mM EDTA, 10 mM DTT, 1 mg ml21 of each of chymostatin, leupeptin, antipain and pepstatin [CLAP, Sigma], 1 mM phenylmethanesulphonyl fluoride [PMSF, Sigma], and 1 unit of Aprotinin [Calbiochem] was added to the tubes. The larvae were then homogenized using a motor- ized hand pestle starting at lowest speed and gently increasing the speed to the maximum for approximately 2 min. Then, 150 ml of hot (708C) 3X SDS-PAGE Sample Buffer containing 10 mM DTT was added to the homogenate and the tube placed at 1008C for 10 min. Particulate matter was pelleted at 13 000 r.p.m. for 2 min, and the supernatant transferred to a fresh tube. Samples were stored at 2208C until required. 5.10. Microarray processing and analysis Ten to fifteen second instar wild-type Canton S and poly larvae were transferred into vials supplemented with fresh yeast paste. Early third instar larvae were transferred into TRIzol 24 h later for RNAextraction. All microarray processing was by theFlychip team at the University of Cambridge (http://www.flychip.org. uk/). RNA from wild-type Canton S and poly early third instar larvae was extracted (medium scale) using TRIzol (http:// www.flychip.org.uk/protocols/gene_expression/standard_ extraction.php). RNAwas reverse transcribed and labelled using Klenow labelling: 5 mg of total RNA were reverse transcribed to cDNA (anchored oligo (dT)23 (Sigma), Superscript III (Invitro- gen)) and second strand synthesis was then performed (Second strand buffer (Invitrogen), DNA Polymerase I (Invitrogen), RNaseH (New England Biolabs), Escherichia coli DNA ligase (GE Healthcare)) to obtain double-stranded DNA. Random primers are annealed to 500 ng of this denatured DNA template and extended by Klenow fragment using the Bioprime DNA Labeling System (Invitrogen), while fluorescent dyes Cy3-dCTP or Cy5-dCTP (GE Healthcare) are incorpora- ted (http://www.flychip.org.uk/protocols/gene_expression/ klenow_v2.php).HybridizationtoFL002microarrays:hybridiz- ation to amino-modified long oligonucleotide microarrays using a Genomic Solutions hybridization station with the Bioso- lutions hybridization buffer (http://www.flychip.org.uk/ protocols/gene_expression/hyb_oligoMWG.php). Scanning was with the Genepix 4000B dual laser scanner at 5 mM pixel resolution (http://www.flychip.org.uk/protocols/gene_ expression/scanning2.php). Spot finding and quantitation were via the DAPPLE package. Raw data were normalized by the vsn method in BIOCONDUCTOR (http://www.bioconductor. org/packages/2.8/bioc/html/vsn.html) to generate log (base 2) fold changes and average signals. Differential expres- sion was tested using Limma, also in BIOCONDUCTOR (http:// www.bioconductor.org/packages/2.8/bioc/html/limma.html). GO and KEGG biological pathway enrichment in the differen- tially expressed gene set were assessed using the DAVID functional annotation bioinformatics microarray analysis tool (http://david.abcc.ncifcrf.gov/). Microarray data were deposited in the gene expression omnibus (GEO) under the accession number GSE32637 (http://www.ncbi.nlm.nih.gov/ geo/query/acc.cgi?acc=GSE32637). 5.11. Triglyceride assay on larvae TAG assay on larvae was carried out as described by Gronke et al. [75]. Briefly, six whole larvae corresponding to each gen- otype were collected in 500 ml of 0.05 per cent Tween 20 and homogenized using a Polytron apparatus, followed by treat- ment at 708C for 5 min. Samples were centrifuged for 1 min at 3500 r.p.m. and 350 ml of the supernatant were transferred to a new vial and centrifuged for 3 min at 2500 r.p.m. Six hun- dred microlitres of Thermo Infinity Trig solution (Thermo Electron, 981786) were added to 75 ml of isolated supernatant 5.8. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting Protein samples were resolved at 170 V by SDS-PAGE on precast 4–12% Bis-Tris polyacrylamide gels (Novex) and transferred onto nitrocellulose membranes in a Trans-Blot apparatus (Biorad). Membranes were blocked in TBSTw (Tris- buffered saline (150 mM NaCl, 20 mM Tris pH 7.5) þ 0.05% Tween 20) and 5% (w/v) semi-skimmed milk for 1 h at room temperature and then incubated for 1 h with primary antibody in TBSTw. After washing three times for 5 min with TBSTw, the membranes were incubated in an appropriate horseradish per- oxidase-linked secondary antibody (1 : 10 000) for 1 h in TBSTw at room temperature. Finally, the membranes were washed as above in Tris-buffered saline plus 0.2% Triton X and immune- complexes detected by enhanced chemiluminescence (ECL; Amersham Biosciences). Primary antibodies and dilutions used in immunoblotting experiments were as follows: DmPoly 504 (1 : 1000), anti-Isd2 (1 : 2000), p-Akt antibody (1 : 1000), p-S6K antibody (1 : 1000) and p-4E-BP antibody (1 : 200). The anti-Isd2 antibody was a generous gift from Ronald Ku¨hnlein (MPI, Go¨ttingen), while the last three were purchased from Cell Signaling Technology. 5.6. RNA extraction and quantitative reverse transcriptase–polymerase chain reaction Larvae were transferred to RNase-free Eppendorf tubes (Ambion). RNA was extracted with the Qiagen RNeasy (Molecular Probes), with 1 mM Hoechst 33342 (Molecular Probes) in PBS. Fat bodies were mounted with phosphate- buffered saline (PBS) on a glass slide and visualized using an Olympus AX-70 Provis epifluorescence microscope and Hamamatsu Orca II charge coupled device (CCD) camera. Images were captured using SMARTCAPTURE 3 and processed using Adobe PHOTOSHOP. Plus Kit (Qiagen Hilden, Germany) according to manufac- turer’s instructions. cDNA synthesis was carried out by using the SuperScript III (Invitrogen) following the manufac- turer’s instructions. Real-time quantitative RT-PCR analysis was performed on the LightCycler system (Roche) using the universal probe library. The reactions were set up following manufacturer’s recommendation with the LightCycler master mix kit. The relative cDNA ratio was calculated with Lightcycler software 480. Actin5C was used as control to normalize equal loading of template cDNA. rsob.royalsocietypublishing.org Open Biol 2: 110031 13 13 6. Acknowledgements The authors would like to express their gratitude to Dr Gerard Cagney (Conway Institute, University College Dublin) for invaluable assistance with the mass spectrometry experiments. We also thank Kasper Pedersen and the Conway Institute Mass Spectrometry Resource for exper- imental support, and Andreas de Stefani for bioinformatics support. Emanuela Giacometti, Josefin Fernius, Shubha Gururaja Rao, Swati Naidu and Mei Xuan Lye made valuable contributions at various stages of this project. We thank William Earnshaw and Paul Hartley for critical feedback on the manuscript. E.B., S.V., N.C. and M.M.S.H. were sup- ported by The Wellcome Trust. B.N. was supported by the Natural Sciences and Engineering Research Council of Canada, while V.S. was an MSc student at the University of Edinburgh. D.R.D. was supported by a British Heart Foun- dation Centre of Research Excellence Award. Primary rabbit anti-DmPoly serum was used in immuno- fluorescence experiments at a 1 : 1000 dilution. Alexa Fluor-488 anti-rabbit (1 : 500) and Alexa Fluor-594 conjugated phalloidin (1 : 500) were purchased from Molecular Probes. 5.14. Immunofluorescence on HeLa cells 5.14. Immunofluorescence on HeLa cells and the absorbance at 540 nm was measured following incu- bation for 30 min. Similarly, protein content was determined by adding 10 ml of isolated supernatant to Bradford reagent (Sigma, B6916) and reading the absorbance at 595 nm. TAG levels were normalized to corresponding protein levels. HeLa cells were cultured on coverslips in six-well plates over- night. Cells were rinsed in PBS for 3 min followed by a 3 min fixation in PBS containing 4 per cent paraformaldehyde (diluted from 16% ampoules). The fixative was removed by a 2 min wash in PBS. Cells were permeabilized by a 5 min incubation in PBS þ 0.5% Triton X-100 followed by a 1 h block in PBS containing 3 per cent BSA. Subsequently, cells were washed in PBS þ 0.1% Triton X-100 for 5 min. Primary antibody was diluted in PBS þ 0.3% BSA þ 0.1% Triton X-100 and incubated on the cells for 1 h at room temperature. Cells were washed twice for 5 min followed by a 1 h second- ary antibody incubation diluted in PBS þ 0.3% BSA þ 0.1% Triton X-100. Subsequently, 45 min washes were performed in PBS þ 0.1% Triton X-100. DAPI was included in the penultimate wash at 0.1 mg ml21 concentration. 5.12. Antibody staining of haemocytes Third instar larvae were bled on multispot microscope slides (Hendley-Essex) using a pair of forceps and a 25-gauge needle in 20 ml of PBS. Cells were left to settle on the slide for 1 h at room temperature in a humidified chamber to allow adherence to the slide. Cells were fixed with 20 ml of 3.7 per cent paraformaldehyde in PBS for 5 min. Cells were washed with PBS for 5 min, followed by a 5 min permeabilization in PBS þ 0.1% Triton X-100. Following an additional 5 min wash in PBS, blocking was performed by incubating cells in PBS þ 3% BSA for 1 h. Cells were then incubated with primary anti- body diluted in PBS þ 3% BSA overnight at 48C. The following day cells were washed in PBS three times and incu- bated at room temperature with secondary antibody diluted in PBS þ 3% BSA. Following the secondary antibody, incu- bation cells were washed twice in PBS for 5 min. Cells were incubated with DAPI diluted in PBS (1 : 5000 dilution) for 5 min followed by a final 5 min wash in PBS. Coverslips were mounted with mowiol on top of the slide. Open Biol 2: 110031 Primary rabbit anti-HsPoly serum was used in immuno- fluorescence experiments at a 1 : 1000 dilution. Texas-Red conjugated goat anti-rabbit (1 : 500) secondary antibody and Alexa Fluor-488-conjugated phalloidin (1 : 500) were purchased from Molecular Probes. 5.13. HeLa cell culture HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Sigma) supplemented with 10 per cent foetal bovine serum (FBS; Gibco). Cells were grown to 80–90% conflu- ence before overnight serum withdrawal. For insulin stimulation, serum-starved cells were treated with 100 nM insulin (Sigma). Cells were treated with 20 nM rapamycin (Cell Signaling Technology) during an overnight serum starvation. rsob.royalsocietypublishing.org Open Biol 2: 110031 14 14 5.9. Lysotracker staining Lysotracker staining was performed as described [74]. 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Comparison of mitral annulus geometry between patients with ischemic and non-ischemic functional mitral regurgitation: implications for transcatheter mitral valve implantation
Cardiovascular ultrasound
2,018
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RESEARCH Open Access Comparison of mitral annulus geometry between patients with ischemic and non- ischemic functional mitral regurgitation: implications for transcatheter mitral valve implantation Patrizia Aruta1, Denisa Muraru1, Andrada Camelia Guta1,2, Sorina Mihaila2, Niccolò Ruozi1, Chiara Palermo1, Basma Elnagar3, Sabino Iliceto1 and Luigi P. Badano1* Abstract Background: Transcatheter mitral valve replacement (TMVR) is a new therapeutic option for high surgical risk patients with mitral regurgitation (MR). Mitral valve (MV) geometry quantification is of paramount importance for success of the procedure and transthoracic 3D echocardiography represents a useful screening tool. Accordingly, we sought to asses MV geometry in patients with functional MR (FMR) that would potentially benefit of TMVR, focusing on the comparison of mitral annulus (MA) geometry between patients with ischemic (IMR) and non ischemic mitral regurgitation (nIMR). Methods: We retrospectively selected 94 patients with severe FMR: 41 (43,6%) with IMR and 53 (56,4%) with nIMR. 3D MA analysis was performed on dedicated transthoracic 3D data sets using a new, commercially-available software package in two moments of the cardiac cycle (early-diastole and mid-systole). We measured MA dimension and geometry parameters, left atrial and left ventricular volumes. Results: Maximum (MA area 10.7 ± 2.5 cm2 vs 11.6 ± 2.7 cm2, p > 0.05) and the best fit plane MA area (9.9 ± 2.3 cm2 vs 10.7 ± 2.5 cm2, p > 0.05, respectively) were similar between IMR and nIMR. nIMR patients showed larger mid-systolic 3D area (9.8 ± 2.3 cm2 vs 10.8 ± 2.7 cm2, p < 0.05) and perimeter (11.2 ± 1.3 cm vs 11.8 ± 1.5 cm, p < 0.05) with longer and larger leaflets, and wider aorto-mitral angle (135 ± 10° vs 141 ± 11°, p < 0.05). Conversely, the area of MA at the best fit plane did not differ between IMR and nIMR patients (9 ± 1.1 cm2 vs 9.9 ± 1.5 cm2, p > 0.05). Conclusions: Patients with ischemic and non-ischemic etiology of FMR have similar maximum dimension, yet systolic differences between the two groups should be taken into account to tailor prosthesis’s selection. Aruta et al. Cardiovascular Ultrasound (2018) 16:27 https://doi.org/10.1186/s12947-018-0145-8 Aruta et al. Cardiovascular Ultrasound (2018) 16:27 https://doi.org/10.1186/s12947-018-0145-8 Trial registration: N.A. Keywords: Mitral valve, Transcatheter procedure planning, Functional mitral regurgitation, Transcatheter mitral valve replacement, 3D echocardiography, Mitral valve prosthesis * Correspondence: lpbadano@gmail.com 1Department of Cardiac, Thoracic and Vascular Science, University of Padua, Via Giustiniani 2, 35128 Padua, Italy Full list of author information is available at the end of the article * Correspondence: lpbadano@gmail.com 1Department of Cardiac, Thoracic and Vascular Science, University of Padua, Via Giustiniani 2, 35128 Padua, Italy Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background of significant coronary artery diseases. The study was ap- proved by the University of Padua Ethics Committee (protocol no. 70299). In Europe, mitral regurgitation (MR) represents the sec- ond most frequent heart valve disease after aortic valve stenosis [1]. Among patients with moderate and severe MR, 30% are affected by functional MR (FMR) with high prevalence of the ischemic etiology [2]. Despite clinical indication, 49% of patients with MR are denied for sur- gery due to advanced age, reduced ejection fraction or multiple comorbidities [3] and, among them, the vast majority is represented by patients with FMR [4]. In the last decade, percutaneous transcatheter procedures, simu- lating surgical techniques, have been developed to extend the therapeutic options for high surgical risk patients with MR. Among them, transcatheter mitral valve replacement (TMVR) represents the newest option [5–7]. Mitral valve analysis software package validation Aruta et al. Cardiovascular Ultrasound (2018) 16:27 Page 2 of 10 Echocardiography and quantitative image analysis g p y q g y All transthoracic examinations were performed using a commercially available Vivid E9 system (GE Vingmed Ultrasound AS, Horten, Norway) equipped with a 4 V probe for 3DE acquisitions according to a standardized protocol. Image analysis was performed on a dedicated workstation equipped with a commercially available software package for offline analysis of 3D datasets (EchoPac 2.02). Quantitation of LV volumes and ejec- tion fraction (LVEF) was performed using 4D Auto-LVQ software [17] (GE Vingmed Ultrasound AS, Horten, Norway). Left atrium (LA) maximum volume was mea- sured using the biplane disk summation method, at LV end-systole [18]. MR severity and conventional MV geom- etry parameters —antero-posterior (AP) and commissural (CC) diameters, tenting height and tenting area— were assessed according to current recommendations [14]. 3D MA analysis was performed on dedicated datasets by a single experienced observer (P.A.), using a new, commer- cially available, software package (4D Auto MVQ, GE Vingmed Ultrasound AS, Horten, Norway), in two mo- ments of the cardiac cycle: early-diastole and mid-systole. Firstly, two time points were identified in the way that the selected frame of the analysis was midway among them. For mid-systolic analysis, the two time-points were early-systole (the frame after MV closure) and end-systole (the frame before MV begins to open). For early diastolic analysis, after identification of early-diastolic frame (first frame when MV start to open), the two time-points were placed 8 frames before and after the selected early-dia- stolic frame. The two orthogonal planes were adjusted to visualize the commissural and longitudinal view of MV (the longitudinal plane intersected the MV at the level of The aim of this study was to asses MV geometry in pa- tients with FMR that would potentially benefit of TMVR, focusing on the comparison of MA geometry between IMR and nIMR patients in two key moments of the car- diac cycle —mid-systole and early-diastole. Mitral valve analysis software package validation y Two sub-studies were carried on to validate the software package used to quantitate MV geometry (4D Auto MVQ, GE Vingmed Ultrasound AS, Horten, Norway). First, the same operator (P.A.) performed the quantita- tive analysis of the MV in a blinded fashion, and after a time interval of one month form each other, using the same TTE data sets and both the new and a previously validated [15, 16] (4D MV Analysis; Tomtec Imaging Systems, Unterschleissheim, Germany) software packages. Second, 3D TTE and transesophageal (TEE) echocardio- graphic MV data sets were analyzed using the same soft- ware package for MV quantitative analysis (4D Auto MVQ, GE Vingmed Ultrasound AS, Horten, Norway) by the same operator (P.A.) in a blinded fashion, after a time interval of one week. Mitral valve (MV) geometry quantification is of para- mount importance for the success of TMVR, and trans- thoracic (TTE) three-dimensional echocardiography (3DE) represents a useful tool to select the patients with the high- est likelihood of uncomplicated implant [8]. It has been previously reported that MV geometry may differ in ische- mic and non-ischemic FMR. In patients with ischemic MR (IMR), regional wall motion abnormalities and left ven- tricular [9] remodeling are more often associated with mi- tral annulus (MA) asymmetric dilatation [10]. Conversely, in non-ischemic MR (nIMR) global LV remodeling leads to symmetric MA dilatation [11]. Yet, MV geometry in FMR has been mainly compared to organic MR, and only few small echocardiographic studies analyzed MV geometry differentiating between IMR and nIMR [10–13]. However, none of them provided MA geometry characterization framed to pre-procedural screening for TMVR [8]. Study population Using the electronical database of the echocardiography laboratory of the department of cardiac, thoracic and vascular sciences of the University of Padua, 94 patients with severe FMR and complete transthoracic echocardi- ography performed between November 2010 and March 2018, have been retrospectively selected. Inclusion cri- teria were: age > 18 years; severe FMR according to current guidelines [14]; availability of good quality 3D data sets of both the left ventricle (LV) and the MV. We excluded patients with organic MR, mitral stenosis, aor- tic stenosis, more than moderate aortic regurgitation, or those with valve prostheses. Each patient was assigned to the IMR or nIMR subgroup according to his/her clin- ical history and the documentation of presence/absence Aruta et al. Cardiovascular Ultrasound (2018) 16:27 Page 3 of 10 Page 3 of 10 A2 and P2 scallops). For initialization, anatomic land- marks have to be added at the level of MA in the longitu- dinal view (posterior, P; anterior, A; leaflets coaptation point, Coap; and aortic valve, Ao) and commissural view (MA1 and MA2). The software package automatically cre- ated a 3D model of the MV in the selected frame which could eventually be edited manually, if needed (Fig. 1). Automatic quantitative parameters of the MV geometry were: MA 3D area; MA 2D area (projected 2D area at the level of the best fit plane); MA perimeter; MA AP diam- eter, as the distance between the two landmarks A and P; MA anterolateral-posteromedial diameter (ALPM), as the longest diameter of MA perpendicular to AP diameter; sphericity index (as the ratio between AP and ALPM diam- eters); MA CC diameter, as the distance between the two commissure; MA inter-trigonal distance, measured between the two automatically identified trigons; MA height, as the distance between the lowest and the highest points of MA; the non-planimetry angle, that assesses the saddle shape of MA; mitral-aortic angle, as the angle between the aortic valve and the MA (along the AP direction) planes; anterior and posterior leaflets area and length, MV tenting height, tenting area and tenting volume. and mid-systolic and early-diastolic parameters obtained from TTE and TEE data sets in the same patient. In addition, Bland–Altman plots were used to assess the mean difference and the limits of agreement between them. Validation study The TTE validation cohort included 30 patients (15 with IMR; 22 men; mean age 64 ± 2 year) with good image quality. The temporal resolution of the 3D dataset for MV quantification was 35 ± 3 volumes per second (vps). Close correlations and good agreements were found be- tween the measurements obtained with the two software packages (Figs. 2 and 3). Study population Paired t test or Wilcoxon rank test were used, as appropriate, for comparing the MV dimension obtained by TTE and TEE data set in the same patient. Variables were compared between IMR and nIMR pa- tients using the unpaired t or the Mann-Whitney tests, as appropriate. Chi-square was used to compare the cat- egorical variables. A paired t test or Wilcoxon signed rank test was used to compare systolic and diastolic di- mensions within the same subgroups, as appropriate. Percentage change of the systo-diastolic measurements was also calculated. Data analyses was performed using SPSS version 20.0 (SPSS, Inc., Chicago, IL) and GraphPad Prism V 7 (GraphPad Software, La Jolla, NY). Differences among variables were considered significant at p value < 0.05. Statistical analysis The normal distribution of the variables was checked using the Kolmogorov-Smirnov test. Continuous data were presented as mean ± standard deviation (SD) or Median (25°-75°) and categorical variables as absolute numbers and percentages, as appropriate. In the valid- ation study, we used Pearson or Spearman correlation to test the relationships between TTE mid-systolic MA pa- rameters, measured using the two software packages, The TEE validation cohort included 15 patients (8 with IMR; 14 men; mean age 63 ± 15 year). As ex- pected, both image quality (excellent quality in 75% Fig. 1 Mitral annulus parameters automatically analyzed at mid-systolic frame. Legend: Panel a 3D mitral annulus area, b mitral annulus area at the best fit plane, c Inter-trigonal distance, d Aorto-Mitral angle Fig. 1 Mitral annulus parameters automatically analyzed at mid-systolic frame. Legend: Panel a 3D mitral annulus area, b mitral annulus area at the best fit plane, c Inter-trigonal distance, d Aorto-Mitral angle Aruta et al. Cardiovascular Ultrasound (2018) 16:27 Page 4 of 10 Fig. 2 Comparisons of mitral annulus diameter measured by GE and TomTec software using Pearson correlation (top) and Bland–Altman (bottom) analyses (Table 1). However, there was a close correlation be- tween the two techniques and the differences were not clinically relevant. Among linear dimension, ALPM, commissural diameter and diastolic inter-trigonal dis- tance are the most similar in TEE and TTE data sets, while tenting area, tenting volume and non-planar showed the largest differences (Table 1). versus 25%, respectively, p = 0.009) and temporal resolution (34 ± 15 vps versus 29 ± 10 vps, respectively, p < 0.05) were higher for TEE than TTE data sets. The mean time lapse between TTE and TEE data set acquisitions was 1(0–6) day. Measurements obtained from TEE data sets resulted in slightly larger area, perimeter and AP diameter Fig. 3 Comparisons of mitral annulus area and perimeter measured by GE and TomTec software using Pearson correlation (top) and Bland–Altman (bottom) analyses Fig 3 Comparisons of mitral annulus area and pe Fig. 3 Comparisons of mitral annulus area and perimeter measured by GE and TomTec software using Pearson correlation (top) and Bland–Altman (bottom) analyses Aruta et al. Data are expressed as Mean ± Standard Deviation Statistical analysis Cardiovascular Ultrasound (2018) 16:27 Page 5 of 10 Table 1 Comparison of mitral annulus parameter among transthoracic and transoesophageal data sets Transthoracic N = 15 Transoesophageal N = 15 p r Diastolic dimension Annulus area (3D) (cm2) 11.5 ± 3.1 12.4 ± 3.2 0.031 0.879** Annulus best fit plane (cm2) 10.6 ± 3.0 11.7 ± 3.1 0.016 0.869** Annulus perimeter (cm) 12.1 ± 1.7 12.6 ± 1.6 0.036 0.883** AP diameter (cm) 3.5 ± 0.5 3.8 ± 0.5 0.012 0.799** ALPM diameter (cm) 3.7 ± 0.5 3.8 ± 0.5 0.342 0.840** Commissural diameter (cm) 3.7 ± 0.4 3.8 ± 0.5 0.231 0.777** Itertrigonal distance (cm) 2.8 ± 0.3 3.0 ± 0.4 0.094 0.715** Sphericity index 0.9 ± 0.1 1.0 ± 0.1 0.150 0.157 Annulus height (mm) 7.3 ± 1.7 6.8 ± 1.6 0.343 0.559* Non planar angle 156 ± 13 152 ± 11 0.314 0.474 Mitro-aortic angle 131 ± 8 125 ± 10 0.039 0.418 Systolic dimension Annulus area (3D) (cm2) 10.2 ± 2.6 11.0 ± 2.6 0.006 0.936** Annulus best fit plane (cm2) 9.3 ± 2.5 10.2 ± 2.5 0.001 0.959** Annulus perimeter (cm) 11.4 ± 1.5 11.8 ± 1.4 0.010 0.943** AP diameter (cm) 3.2 ± 0.4 3.4 ± 0.6 0.002 0.624* ρ ALPM diameter (cm) 3.6 ± 0.5 3.6 ± 0.4 0.329 0.857** Commissural diameter (cm) 3.5 ± 0.4 3.6 ± 0.4 0.151 0.889** Itertrigonal distance (cm) 2.6 ± 0.4 3.0 ± 0.4 0.000 0.830** Sphericity index 0.9 ± 0.1 0.9 ± 0.1 0.062 0.321ρ Annulus height (mm) 7.0 ± 1.3 7.0 ± 1.4 0.910 0.300 Non planar angle (°) 153 ± 10 153 ± 9 0.893 0.240 Aorto-mitral angle (°) 139 ± 10 129 ± 8 0.005 0.367 Tenting height (mm) 10.3 ± 2.0 7.2 ± 3.6 0.02 - 0.108 Tenting area (cm2) 2.3 ± 0.6 2.3 ± 0.7 0.934 0.542* Tenting volume (mL) 4.2 ± 1.3 4.1 ± 1.5 0.742 0.747** Data are expressed as Mean ± Standard Deviation Abbreviations: ALPM anterolateral-posteromedial, AP antero-posterior diameter * f l ti ith 0 05 ** f l ti ith 0 001 ρ l t d ith S ’ l ti Comparison of mitral annulus geometry between ischemic and non-ischemic mitral regurgitation p Abbreviations: ALPM anterolateral-posteromedial, AP antero-posterior diameter * for correlation with p < 0.05; ** for correlation with p < 0.001; ρ evaluated with Sperman’s correlation Comparison of mitral annulus geometry between ischemic and non-ischemic mitral regurgitation patients (33 ± 14 vps versus 40 ± 16 vps, p = 0.023). All data sets had enough good quality for the quantitative analysis. The image quality was graded excellent in 47 patients (50%), good in 32 (34%), and fair in 15 (16%) and it was comparable between IMR and nIMR pa- tients (p = 0.634). g g We enrolled 94 patients, 41 (43,6%) with IMR and 53 (56,4%) with nIMR. Patients with IMR were more fre- quently male and had a higher incidence of hyperten- sion, diabetes and dyslipidemia (Table 2). The severity of MR was comparable between the two groups (Table 3). Although patients in both groups showed severe LV dila- tation and dysfunction, patients with IMR had a higher LVEF (31 (26–38)% versus 28 (22–32)%, p = 0.030) and LV wall motion score index (2.1 ± 0.3 versus 1.9 ± 0.6, p = 0.021) (Table 3). Using conventional two-dimensional echocardiog- raphy MV geometry parameters, patients with nIMR showed larger AP diameter both in diastole (41 ± 7 mm in nIMR versus 38 ± 6 mm in IMR, p = 0.029) and in systole (37 ± 6 mm in nIMR versus 34 ± 4 mm in IMR, p = 0.024). Conversely, CC diameter (43 ± 8 mm in nIMR versus 39 ± 9 mm in IMR, p = 0.088), tenting height (9 ± 3 mm in nIMR versus 8.5 ± 3 mm in IMR, p = 0.180) Temporal resolution of the 3D dataset dedicated for MV quantification was higher in IMR than in nIMR Aruta et al. Cardiovascular Ultrasound (2018) 16:27 Page 6 of 10 Table 2 Demographics and clinical characteristics Table 2 Demographics and clinical characteristics Ischemic mitral regurgitation N = 41 Non ischemic mitral regurgitation N = 53 p Age (years) 69 (63–75) 64 (55–72) 0.081 Men (%) 35 (85) 35 (66) 0.033 Body surface area (m2) 1.8 (1.7–1.9) 1.9 (1.7–2.0) 0.374 Heart rate (bpm) 71 (59–85) 75 (65–86) 0.237 Systolic blood pressure (mmHg) 110 (100–120) 100 (95–115) 0.340 Diastolic blood pressure(mmHg) 65 (60–71) 65 (60–70) 0.233 Hypertension 32 (80%) 25 (48.1%) 0.002 Diabetes 15 (37.5%) 8 (15.4%) 0.015 Dyslipidemia 30 (75%) 22 (42.3%) 0.002 Smokers 23 (57.5%) 24 (46.2%) 0.280 Resynchronization therapy 8 (20%) 17 (32.7%) 0.175 Data are expressed as Median (25°-75°) or Number (%). Data are expressed as Median (25 -75 ) or Number (%) Abbreviations: AR aortic regurgitation, EROA effective regurgitant orifice area, LA left atrial, LV EDV left ventricular end-diastolic volume, LV ESV left ventricular end-systolic volume, MR mitral regurgitation, PISA proximal isovelocity surface area, R Vol regurgitant volume, sPAP systolic pulmonary artery pressure, TR tricuspid regurgitation. Italicized values highlight statistically significant differences Data are expressed as Median (25°-75°) or Number (%) Comparison of mitral annulus geometry between ischemic and non-ischemic mitral regurgitation Italicized values highlight statistically significant differences and tenting area (1.9 ± 0.7 cm2 in nIMR versus 1.7 ± 0.6 cm2 in IMR, p = 0.189) were similar between the two groups. longer leaflets. However, the area of the annulus at the best fit plane, and all diameters (AP, CC, ALPM diam- eter and trans-trigonal distance) did not differ between IMR and nIMR patients. Tenting height and area did not differ between IMR and nIMR patients, whereas tenting volume, annulus height and aorto-mitral angle were larger in nIMR patients (Table 4). g p At 3DE analysis, both subgroups had similar diastolic geometry of MA, even though all MA dimensions were slightly larger in nIMR. nIMR patients showed larger mid-systolic 3D area and perimeter of the MA with Table 3 Echocardiography characteristics Ischemic mitral regurgitation N = 41 Non ischemic mitral regurgitation N = 53 p MR Vena contracta (mm) 7 (6–8) 7 (6–8) 0.658 MR PISA radius (mm) 7 (6–8) 8 (7–9) 0.138 MR EROA (mm2) 2 (2–3) 2.1(2–3) 0.421 MR R Vol (mL) 38 (28–58) 38.5 (29–47.7) 0.803 sPAP 47 (35–56) 44 (35–49) 0.211 TR severity Trivial 6 (11.3%) Trivial 6 (15.4%) 0.753 Mild 29 (54.7%) Mild 20 (51.3%) Moderate11 (20.8%) Moderate10 (25.6%) Severe 7 (13.2%) Severe3 (7.7%) AR severity None 25 (49%) None 25 (65%) 0.437 Trivial 13 (25%) Trivial 6 (15%) Mild 12 (23.5%) Mild 9 (22.5%) Moderate 1 (2%) Moderate 0 (0%) LV EDV (ml/m2) 134 (114–153) 143 (116–178) 0.078 LV ESV (mL/m2) 96 (68–109) 105 (78–135) 0.075 Ejection Fraction (%) 31 (26–38) 28 (22–32) 0.030 Indexed LA volume (mL/m2) 60 (51–68) 70 (53–91) 0.031 Data are expressed as Median (25°-75°) or Number (%) Table 3 Echocardiography characteristics Data are expressed as Median (25°-75°) or Number (%) Data are expressed as Median (25°-75°) or Number (%) Abbreviations: AR aortic regurgitation, EROA effective regurgitant orifice area, LA left atrial, LV EDV left ventricular end-diastolic volume, LV ESV left ventricular end-systolic volume, MR mitral regurgitation, PISA proximal isovelocity surface area, R Vol regurgitant volume, sPAP systolic pulmonary artery pressure, TR tricuspid regurgitation. Italicized values highlight statistically significant differences Aruta et al. Comparison of mitral annulus geometry between ischemic and non-ischemic mitral regurgitation Cardiovascular Ultrasound (2018) 16:27 Page 7 of 10 Table 4 Three-dimensional mitral valve dimension Ischemic mitral regurgitation N = 41 Non ischemic mitral regurgitation N = 53 p Diastolic dimension Annulus area (3D) (cm2) 10.7 ± 2.5a 11.6 ± 2.7a 0.124 Annulus best fit plane (cm2) 9.9 ± 2.3a 10.7 ± 2.5a 0.135 Annulus perimeter (cm) 11.7 ± 1.4a 12.2 ± 1.4a 0.111 AP diameter (cm) 3.3 ± 0.4a 3.5 ± 0.5a 0.072 ALPM diameter (cm) 3.6 ± 0.4a 3.8 ± 0.5a 0.129 Commissural diameter(cm) 3.6 ± 0.4 3.7 ± 0.4 0.300 Itertrigonal distance (cm) 2.7 ± 0.4a 2.8 ± 0.3a 0.374 Annulus height (mm) 6.3 ± 1.7a 6.8 ± 1.7a 0.144 Sphericity index 0.9 ± 0.1a 0.9 ± 0.1a 0.963 Non planar angle (°) 156 ± 11a 153 ± 10a 0.232 Anterior leaflet area (cm2) 7.5 ± 1.6a 8.0 ± 1.6a 0.142 Posterior leaflet area (cm2) 7.2 ± 2.3a 7.5 ± 2.1a 0.413 Anterior leaflet length (cm) 2.9 ± 0.4 3.3 ± 0.9a 0.102 Posterior leaflet length (cm) 1.6 ± 0.4a 1.7 ± 0.6a 0.319 Aorto-mitral angle (°) 131 ± 9a 135 ± 11a 0.115 Systolic dimension Annulus area (3D) (cm2) 9.8 ± 2.3 10.8 ± 2.7 0.046 Annulus best fit plane (cm2) 9 ± 2.1 9.9 ± 2.5 0.063 Annulus perimeter (cm) 11.2 ± 1.3 11.8 ± 1.5 0.048 AP diameter (cm) 3.1 ± 0.4 3.2 ± 0.5 0.063 ALPM diameter (cm) 3.5 ± 0.4 3.7 ± 0.5 0.065 Commissural diameter(cm) 3.5 ± 0.4 3.7 ± 0.4 0.130 Itertrigonal distance (cm) 2.5 ± 0.3 2.7 ± 0.3 0.051 Annulus height (mm) 6.7 ± 1.6 7.5 ± 1.9 0.047 Sphericity index 0.9 ± 0.08 0.9 ± 0.1 0.598 Non planar angle (°) 153 ± 11 150 ± 10 0.268 Anterior leaflet area (cm2) 6.5 ± 1.6 7.4 ± 1.7 0.006 Posterior leaflet area (cm2) 5.7 ± 1.7 6.5 ± 1.9 0.049 Anterior leaflet length (cm) 2.8 ± 0.6 3 ± 0.4 0.022 Posterior leaflet length (cm) 1.3 ± 0.4 1.5 ± 0.8 0.022 Aorto-mitral angle (°) 135 ± 10 141 ± 11 0.011 Tenting height (mm) 9.3 ± 2.6 10.3 ± 2.8 0.082 Tenting area (cm2) 2.2 ± 0.7 2.4 ± 0.8 0.141 Tenting volume (mL) 4 ± 1.7 4.7 ± 1.7 0.047 Data are expressed as Mean ± Standard Deviation Abbreviations: ALPM anterolateral-posteromedial, AP antero.posterior diameter Italicized values highlight statistically significant differences aStatistical difference vs systolic dimension Table 4 Three-dimensional mitral valve dimension Data are expressed as Mean ± Standard Deviation Abbreviations: ALPM anterolateral-posteromedial, AP antero.posterior diameter Italicized values highlight statistically significant differences aStatistical difference vs systolic dimension Data are expressed as Mean ± Standard Deviation Abbreviations: ALPM anterolateral-posteromedial, AP antero.posterior diameter Italicized values highlight statistically significant differences aStatistical difference vs systolic dimension Mitral annulus dynamics In the present study, we used 3D TTE to compare MA geometry in patients with severe ischemic and non-is- chemic FMR, who are potential candidates for TMVR. In both groups, MA significantly reduced its dimensions in systole (except for the CC diameter) with similar percent- age change of the measurement in both groups (p > 0.05) (Tables 4 and 5). During systole, the MA mitral-aortic angle flattens, while the non-planarity angle becomes more acute. The main findings of our study were in patients with FMR: i, diastolic MA geometry is similar in both nIMR Aruta et al. Cardiovascular Ultrasound (2018) 16:27 Page 8 of 10 Table 5 Fractional changes of the mitral annulus parameters between diastole and systole Ischemic mitral regurgitation N = 41 Non ischemic mitral regurgitation N = 53 p MA area (3D) fraction (%) -6 (-11.7 — -1.8) -4.3 ( -9.8 — -1.3) > 0.05 MA best fit plane fraction (%) -6.3 (-13 — -4) -6.7 (-11.4 — -1.8) > 0.05 MA perimeter fraction (%) -3.2 (-5.8 — -0.4) -2.1 (-4.6 — -0.8) > 0.05 AP diameter fraction (%) -7.4 (-11.4 — -2.4) -5.9 (-11.2 — -1.3) > 0.05 ALPM diameter fraction (%) -2.9 (-6.9 — 0.0) 0.0 (-7.3 — 0.0) > 0.05 CC diameter fraction (%) -2.6 (-5.5 — 2.9) 0.0 (-4.7 — 2.7) > 0.05 TT distance fraction (%) -4.8 (-12.4 — 0.0) -3.1 (-10 — 3.8) > 0.05 Non planar angle fraction (%) -2 (-6.3 — 2.5) -2.4 (-5.6 — 2) > 0.05 Aorto-mitral angle fraction (%) 3.7 (-2.5 — 8.4) 3.4 (0–8.4) > 0.05 Data are expressed as Median (25°-75°) Abbreviations: ALPM anterolateral-posteromedial, AP antero-posterior diameter, CC commissural, MA mitral annulus, TT, trans-trigonal Table 5 Fractional changes of the mitral annulus parameters between diastole and systole and IMR patients; ii, systolic MV geometry significantly differs between the groups. and IMR patients; ii, systolic MV geometry significantly differs between the groups. In this study, we reported all MV anatomical and geomet- rical features that should be assessed before TMVR [8, 22] and demonstrated that patients with severe IMR and nIMR have similar, symmetrical, diastolic (maximal) MA dimen- sion. The 3D MA area obtained from our patients were comparable with the maximum MA surface area reported by Veronesi et al. [12] in a smaller group of patients using TTE 3DE datasets. Our results are also in agreement with those reported by Daimon et al. Validation study y Multimodality imaging represents the gold standard for planning transcatheter mitral valve procedures, TEE and multi-slice computed tomography (MSCT) playing the major role [19]. Due to longer survival of patients with chronic heart diseases and progressive aging of the general population, the number of patients who could benefit of TMVR is likely to increase, and 3DTTE will be of para- mount importance as a screening tool for the analysis of MV geometry. Previous clinical studies assessing MA geometry used 3DTEE data sets [20, 21] to obtain adequate spatial and temporal resolution for quantitative analysis of the MV. However, the progressive improvement of 3DE technology allows to obtain better and better quality 3DE data sets with TTE, too. Moreover, feasibility and cost/ef- fectiveness considerations suggest that TTE approach would be better suited to screen potential candidates to TMVR. Accordingly, we decided to explore the use of 3DTTE data sets perform quantitative analysis of the MV in patients with FMR. In our patients, MA dimensions ob- tained from TTE datasets were similar to those obtained with the 3D TEE approach in the validation study. While in our study mid-systolic 3D annulus area and perimeter are significantly larger in patients with nIMR, MA area at the best fit plane and MA diameter were similar. It has been suggested that the projected 3D MA area at the level of the best fit plane is the most reliable parameter of MA geometry to be used for planning TMVR compared to the saddle-shaped 3D area [22]. Though, our MA area at the best fit plane resulted smaller than the mean projected MA area measured in a recent MSCT study [23] on 32 patients with FMR of dif- ferent etiologies and severity, it is already known that 3DE can underestimate measurement compared with MSCT due to its suboptimal lateral resolution in the coronal plane [24]. Mitral annulus dynamics [10] who showed that dia- stolic MA diameters did not differ among IMR and nIMR. However, the actual MA sizes in our patients were slightly larger than in their cohorts. This finding could be partially explained by the different time point se- lected for the analysis (mid-diastolic phase, compared to early-diastole in our study). Comparison of mitral annulus dimension between ischemic and non-ischemic mitral regurgitation A new D-shaped MA segmentation developed by Blanke et al. [25], with the truncation of anterior saddle horn at the level of inter-trigonal line, has been used to select candidates to Tiara [5], Tendyne [7] and Intrepid [6] valve implants. This method was also recently ap- plied by Mak et al. [26] using 3D TEE with comparable results, but it is unclear at this early stage of TMVR ex- perience whether this is the best parameter to size the prosthesis for TMVR interventions [27]. We focused our study on patients with FMR because they represent the main potential target of new TMVR. The few previous studies that analyzed the possible dif- ferences between IMR and nIMR [10–13] included a limited number of patients and were focused only on MA size (annulus area and diameters), without any in- formation about the MV geometry (MA area at the best fit plane, mitro-aortic angle, length of the anterior leaf- let) which are crucial to select patients for TMVR [19]. Page 9 of 10 Page 9 of 10 Aruta et al. Cardiovascular Ultrasound (2018) 16:27 Left ventricular out flow tract (LVOT) obstruction is a possible complication related to TMVR that can be pre- dicted during procedure planning because it is related to the design of the prosthesis and patient anatomy (inter- ventricular septal dimension, LV size, aorto-mitral angle, anterior leaflet length). 3DE allows the measurement of both the aorto-mitral angle (the angle between the aortic valve and the MA along the AP direction) and anterior leaflet length. None of the previous MSCT nor the 3DE studies reported these parameters in patients considered for TMVR. In our study we found that nIMR group pre- sented significantly wider aorto-mitral angle that balance the potential higher risk of LVOT obstruction due to longer and larger anterior leaflets in these patients. Study limitations We acknowledge several limitations of our study. First, to obtain all the measurements needed to plan TMVR from 3DE data sets, we used a new MV software package that was not previously validated. To overcome this limi- tation, we compared the measurements obtained with the new software package with those obtained from the same data sets using a validated software [16] with a close corre- lations and good agreement. However, we did not com- pare our measurement with MSCT, which represents the current gold standard to select patients for TMVR. Secondly, currently available 3DE software packages allow MV dynamic analysis only during the systolic phase of the cardiac cycle. While mid-systole could be defined by the operator according to MV opening and closure or automatically by the software (as mid-way be- tween R and T waves on the ECG tracing), early-diastole has to be manually identified by the operator with an in- creased possibility of errors. Current literature reports contradicting data about the moment when MA reaches its maximum and minimum sizes, however the importance of definition of maximum MA dimension is of paramount importance for accurate device’s sizing and emphasizes the need of multiphasic annular measurement. Availability of data and materials The datasets used and analyzed during the current study are available from the first author on reasonable request. All data generated or analyzed during this study are included in this published article. Conclusion The reported MA geometry in a relatively large group of patients with severe FMR, potentially candidates for TMVR, represents useful information for transcatheter MV prosthesis design and patient selection. Patients with ischemic and non-ischemic aetiologies of FMR have similar maximum dimensions, yet systolic differences between the two groups should be taken into account to tailor prosthesis’s selection. Implications for trans-catheter mitral valve selection TMVR represents a promising option for patients with severe FMR, and assessment of MA dimension and geometry is of paramount importance to size the device and also to plan future development of new prostheses. We found that, patients with IMR and nIMR have simi- lar MA geometry, supporting the concept that there is no need of different prosthesis sizing according to eti- ology of the FMR. However, we found that nIMR pa- tients had significantly larger and longer anterior mitral leaflet, that could increase the risk of LVOT obstruction. Therefore, for nIMR patients it could be more appropri- ate to select a device that has an anterior hook to fix the anterior leaflet of the native MV. On the other hand, nIMR patients showed a wider aorto-mitral angle that could counterbalance the higher risk of LVOT obstruc- tion carried by longer anterior leaflet. Probably, this sub-group of patients would be eligible also for devices that have larger protrusion or flaring into LV. Mitral annulus dynamics MA is dynamic structure characterized by contraction and expansion phase during cardiac cycle [12, 28]. These changes, although less pronounced than in normal sub- jects, have been reported also in patients with IMR [20, 29] and nIMR [12]. We found that in patients with severe FMR, MA is significantly smaller in mid-systole compared to early diastolic phase. 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Quantitative analysis of mitral annular geometry and function in healthy volunteers using transthoracic three-dimensional echocardiography. J Am Soc Echocardiogr. 2014;27:846–57. Publisher’s Note 17. Muraru D, Cecchetto A, Cucchini U, Zhou X, Lang RM, Romeo G, et al. Intervendor consistency and accuracy of left ventricular volume measurements using three-dimensional echocardiography. J Am Soc Echocardiogr. 2018;31:158–68 e1. 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. 18. Lang RM, Badano LP, Mor-Avi V, Afilalo J, Armstrong A, Ernande L, et al. Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging. Eur Heart J Cardiovasc Imaging. 2015;16:233–70. 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Establishing Consensus Among Modalities and Disciplines JACC Cardiovasc Imaging. 2015;8:1191–208. 26. Mak GJ, Blanke P, Ong K, Naoum C, Thompson CR, Webb JG, et al. Three- dimensional echocardiography compared with computed tomography to determine mitral annulus size before Transcatheter mitral valve implantation. Circ Cardiovasc Imaging. 2016;9:1–9. 9. Boekstegers P, Hausleiter J, Baldus S, von Bardeleben RS, Beucher H, Butter C, et al. Percutaneous interventional mitral regurgitation treatment using the Mitra-clip system. Clin Res Cardiol. 2014;103:85–96. 27. Khalique OK, Hahn RT. Multimodality imaging in Transcatheter mitral interventions: buzzword or modern age toolbox? Circ Cardiovasc Imaging. 2016;9:1–3. 10. Daimon M, Saracino G, Gillinov AM, Koyama Y, Fukuda S, Kwan J, et al. Local dysfunction and asymmetrical deformation of mitral annular geometry in ischemic mitral regurgitation: a novel computerized 3D echocardiographic analysis. Echocardiography. 2008;25:414–23. 28. Jiang L, Owais K, Matyal R, Khabbaz KR, Liu DC, Montealegre-Gallegos M, et al. Dynamism of the mitral annulus: a spatial and temporal analysis. J Cardiothorac Vasc Anesth. 2014;28:1191–7. 29. Owais K, Montealegre-Gallegos M, Jeganathan J, Matyal R, Khabbaz KR, Mahmood F. Dynamic changes in the ischemic mitral annulus: implications for ring sizing. Ann Card Anaesth. 2016;19:15–9. 11. Nagasaki M, Nishimura S, Ohtaki E, Kasegawa H, Matsumura T, Nagayama M, et al. The echocardiographic determinants of functional mitral regurgitation differ in ischemic and non-ischemic cardiomyopathy. Int J Cardiol. 2006;108: 171–6. 12. Veronesi F, Corsi C, Sugeng L, Caiani EG, Weinert L, Mor-Avi V, et al. Quantification of mitral apparatus dynamics in functional and ischemic mitral regurgitation using real-time 3-dimensional echocardiography. J Am Soc Echocardiogr. 2008;21:347–54. 13. Authors’ contributions ll h h b All authors have contributed significantly to the submitted work: LPB: study design, critical revision of the first draft and final approval of the manuscript; PA: study design, image acquisition, data analysis, first draft and final approval of the manuscript; BE and NR: data collection, image analysis and final approval of the manuscript; SM and CP: image acquisition and final approval of the manuscript; ACG, DM and SI: critical revision of the first draft and final approval of the manuscript. The significant change of MA during the cardiac cycle, also preserved in patients with severe FMR, stresses the need to evaluate accurately the smallest MA dimension in order to reduce the risk of excessive stress of the prosthesis frame by MA. Page 10 of 10 Page 10 of 10 Page 10 of 10 Aruta et al. Cardiovascular Ultrasound (2018) 16:27 Author details 1 1Department of Cardiac, Thoracic and Vascular Science, University of Padua, Via Giustiniani 2, 35128 Padua, Italy. 2University of Medicine and Pharmacy “Carol Davila”, Bucharest, Romania. 3Tanta University Hospital, Tanta, Egypt. 19. Mackensen GB, Lee JC, Wang DD, Pearson PJ, Blanke P, Dvir D, et al. Role of echocardiography in Transcatheter mitral valve replacement in native mitral valves and mitral rings. J Am Soc Echocardiogr. 2018;31:475–90. Received: 13 September 2018 Accepted: 24 September 2018 Ethics approval and consent to participate cardiomyopathy with significant mitral regurgitation: real-time three- dimensional echocardiography study. Circulation. 2003;107:1135–40. cardiomyopathy with significant mitral regurgitation: real-time three- dimensional echocardiography study. Circulation. 2003;107:1135–40. Ethics approval and consent to participate The study was approved by the University of Padua Ethics Committee (protocol no. 70299). 14. Lancellotti P, Moura L, Pierard LA, Agricola E, Popescu BA, Tribouilloy C, et al. European Association of Echocardiography recommendations for the assessment of valvular regurgitation. Part 2: mitral and tricuspid regurgitation (native valve disease). Eur J Echocardiogr. 2010;11:307–32. Consent for publication “Not applicable” in this section. Consent for publication “Not applicable” in this section. References Kwan J, Shiota T, Agler DA, Popovic ZB, Qin JX, Gillinov MA, et al. Geometric differences of the mitral apparatus between ischemic and dilated
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Multidimensional analysis of Gammaherpesvirus RNA expression reveals unexpected heterogeneity of gene expression
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. CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint Multidimensional analysis of Gammaherpesvirus RNA expression reveals 1 unexpected heterogeneity of gene expression 2 3 Lauren M. Oko1, Abigail K. Kimball2, Rachael E. Kaspar2, Ashley N. Knox1, Carrie B. 4 Coleman1, Rosemary Rochford1, Tim Chang3, Benjamin Alderete4, Linda F. van Dyk1*, 5 Eric T. . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint Clambey2*# 6 7 8 Affiliations: 9 1 Department of Immunology and Microbiology, 2 Department of Anesthesiology, 10 University of Colorado Denver | Anschutz Medical Campus, Aurora, CO, 80045, USA 11 3 MilliporeSigma, a business of Merck KGaA, Darmstadt, Germany (Seattle, WA) 12 4 Luminex Corporation, Austin, TX, USA 13 14 15 * Co-Corresponding authors: 16 Eric T. Clambey, eric.clambey@ucdenver.edu, 303-724-7783 (phone) 17 Linda F. van Dyk, linda.vandyk@ucdenver.edu, 303-724-4207 (phone) 18 19 # Lead author for MS correspondence 20 21 22 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. Multidimensional analysis of Gammaherpesvirus RNA expression reveals 1 unexpected heterogeneity of gene expression 2 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint ABSTRACT 24 ABSTRACT 24 Virus-host interactions are frequently studied in bulk cell populations, obscuring 25 cell-to-cell variation. Here we investigate endogenous herpesvirus gene expression at 26 the single-cell level, combining a sensitive and robust fluorescent in situ hybridization 27 platform with multiparameter flow cytometry, to study the expression of 28 gammaherpesvirus non-coding RNAs (ncRNAs) during lytic replication, latent infection 29 and reactivation in vitro. This method allowed robust detection of viral ncRNAs of 30 murine gammaherpesvirus 68 (γHV68), Kaposi’s sarcoma associated herpesvirus and 31 Epstein-Barr virus, revealing variable expression at the single-cell level. By quantifying 32 the inter-relationship of viral ncRNA, viral mRNA, viral protein and host mRNA 33 regulation during γHV68 infection, we find heterogeneous and asynchronous gene 34 expression during latency and reactivation, with reactivation from latency identified by a 35 distinct gene expression profile within rare cells. Further, during lytic replication with 36 γHV68, we find many cells have limited viral gene expression, with only a fraction of 37 cells showing robust gene expression, dynamic RNA localization, and progressive 38 infection. Lytic viral gene expression was enhanced in primary fibroblasts and by 39 conditions associated with enhanced viral replication, with multiple subpopulations of 40 cells present in even highly permissive infection conditions. These findings, powered by 41 single-cell analysis integrated with automated clustering algorithms, suggest inefficient 42 or abortive γHV infection in many cells, and identify substantial heterogeneity in viral 43 gene expression at the single-cell level. 44 45 2 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint AUTHOR SUMMARY 46 The gammaherpesviruses are a group of DNA tumor viruses that establish 47 lifelong infection. How these viruses infect and manipulate cells has frequently been 48 studied in bulk populations of cells. While these studies have been incredibly insightful, 49 there is limited understanding of how virus infection proceeds within a single cell. Here 50 we present a new approach to quantify gammaherpesvirus gene expression at the 51 single-cell level. This method allows us to detect cell-to-cell variation in the expression 52 of virus non-coding RNAs, an important and understudied class of RNAs which do not 53 encode for proteins. By examining multiple features of virus gene expression, this 54 method further reveals significant variation in infection between cells across multiple 55 stages of infection, even in conditions generally thought to be highly uniform. These 56 studies emphasize that gammaherpesvirus infection can be surprisingly heterogeneous 57 when viewed at the level of the individual cell. Because this approach can be broadly 58 applied across diverse viruses, this study affords new opportunities to understand the 59 complexity of virus infection within single cells. 60 INTRODUCTION 62 The Herpesviridae are a family of large dsDNA viruses that include multiple 63 prominent human and animal pathogens [1]. Although these viruses infect different cell 64 types, and are associated with diverse pathologies, they share conserved genes and 65 two fundamental phases of infection: lytic replication and latent infection [1]. Lytic 66 replication is characterized by a cascade of viral gene expression, active viral DNA 67 replication and the production of infectious virions. Conversely, latency is characterized 68 3 3 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint by limited viral gene expression and the absence of de novo viral replication. While 69 latent infection is a relatively quiescent form of infection, the herpesviruses can 70 reactivate from latency, to reinitiate lytic replication. 71 by limited viral gene expression and the absence of de novo viral replication. While 69 latent infection is a relatively quiescent form of infection, the herpesviruses can 70 reactivate from latency, to reinitiate lytic replication. 71 by limited viral gene expression and the absence of de novo viral replication. While 69 latent infection is a relatively quiescent form of infection, the herpesviruses can 70 reactivate from latency, to reinitiate lytic replication. 71 Among the herpesviruses, the gammaherpesviruses (γHV) are lymphotropic 72 viruses that include the human pathogens Epstein-Barr virus (EBV) [2] and Kaposi’s 73 sarcoma associated herpesvirus (KSHV) [3]. Murine gammaherpesvirus 68 (γHV68, or 74 MHV-68; ICTV nomenclature Murid herpesvirus 4, MuHV-4), is a well-described small 75 animal model for the γHVs [4]. While these viruses establish a lifelong infection that is 76 often clinically inapparent, immune-suppressed individuals are particularly at risk for 77 γHV-associated malignancies [5]. 78 Herpesvirus gene expression is extremely well-characterized in bulk population 79 Despite increasing evidence for single-cell heterogeneity in gene expression [6-8], ther 80 remains limited understanding of herpesvirus infection at the single-cell level [9-12]. 81 Here, we tracked endogenous viral and host RNAs using a sensitive, robust fluorescen 82 in situ hybridization assay combined with multiparameter flow cytometry (PrimeFlowTM 83 [13] to analyze the expression and inter-relationships of viral ncRNA, viral mRNA and 84 cellular mRNA at the single-cell level during γHV latency, reactivation and lytic 85 replication. These studies revealed unanticipated heterogeneity of infection, 86 emphasizing how single-cell analysis of virus infection can afford significant new 87 insights into the complexity of γHV infection. 88 89 Among the herpesviruses, the gammaherpesviruses (γHV) are lymphotropic 72 viruses that include the human pathogens Epstein-Barr virus (EBV) [2] and Kaposi’s 73 sarcoma associated herpesvirus (KSHV) [3]. Murine gammaherpesvirus 68 (γHV68, or 74 MHV-68; ICTV nomenclature Murid herpesvirus 4, MuHV-4), is a well-described small 75 animal model for the γHVs [4]. While these viruses establish a lifelong infection that is 76 often clinically inapparent, immune-suppressed individuals are particularly at risk for 77 γHV-associated malignancies [5]. 78 Herpesvirus gene expression is extremely well-characterized in bulk populations. 79 Despite increasing evidence for single-cell heterogeneity in gene expression [6-8], there 80 remains limited understanding of herpesvirus infection at the single-cell level [9-12]. 81 remains limited understanding of herpesvirus infection at the single-cell level [9-12]. RESULTS 90 RESULTS 90 Single-cell analysis of viral RNAs during lytic infection. 91 Traditional measurements of gene expression frequently rely on pooled cellular 92 material, obscuring intercellular variation in gene expression. To better define 93 expression of γHV RNAs at the single cell level, we employed the PrimeFlowTM RNA 94 assay [13] to study viral gene expression during murine gammaherpesvirus 68 (γHV68) 95 infection, a small animal γHV [4, 13]. This method is a highly sensitive, extremely 96 specific in situ hybridization assay, integrating Affymetrix-designed branched DNA 97 technology with single-cell analysis powered by multiparameter flow cytometry. This 98 method has been successfully used to detect both virus and host RNAs (e.g. in the 99 context of HIV infected individuals [13, 14]). 100 We first tested the ability of PrimeFlowTM to measure multiple viral RNAs during 101 lytic infection with γHV68, including small non-coding RNAs (tRNA-miRNA encoding 102 RNAs or TMERs [15]) and mRNAs. Mouse 3T12 fibroblasts were infected with a 103 multiplicity of infection (MOI=5 plaque forming units (PFU) of virus/cell). Under these 104 conditions, TMER-5, one of the eight γHV68 TMERs, and the γHV68 ORF73, were 105 readily detectable by conventional real-time PCR in γHV68-infected, but not mock- 106 infected, cultures (Fig. 1A, C). Parallel cultures were analyzed for RNA expression by 107 PrimeFlowTM. Whereas mock-infected cells had no detectable expression of either the 108 γHV68 TMERs or ORF73, WT γHV68-infected fibroblasts had a prominent population of 109 TMER+ and ORF73+ cells, respectively (Fig. 1B and 1D). Infection of cells with a 110 TMER-deficient γHV68 (TMER-TKO [16]), in which TMER expression is ablated through 111 promoter disruption, revealed no detectable TMER expression (Fig. 1B), yet robust 112 Single-cell analysis of viral RNAs during lytic infection. 91 Traditional measurements of gene expression frequently rely on pooled cellular 92 material, obscuring intercellular variation in gene expression. To better define 93 expression of γHV RNAs at the single cell level, we employed the PrimeFlowTM RNA 94 assay [13] to study viral gene expression during murine gammaherpesvirus 68 (γHV68) 95 infection, a small animal γHV [4, 13]. This method is a highly sensitive, extremely 96 specific in situ hybridization assay, integrating Affymetrix-designed branched DNA 97 technology with single-cell analysis powered by multiparameter flow cytometry. This 98 method has been successfully used to detect both virus and host RNAs (e.g. in the 99 context of HIV infected individuals [13, 14]). by limited viral gene expression and the absence of de novo viral replication. While 69 latent infection is a relatively quiescent form of infection, the herpesviruses can 70 reactivate from latency, to reinitiate lytic replication. 71 81 Here, we tracked endogenous viral and host RNAs using a sensitive, robust fluorescent 82 in situ hybridization assay combined with multiparameter flow cytometry (PrimeFlowTM) 83 [13] to analyze the expression and inter-relationships of viral ncRNA, viral mRNA and 84 cellular mRNA at the single-cell level during γHV latency, reactivation and lytic 85 replication. These studies revealed unanticipated heterogeneity of infection, 86 emphasizing how single-cell analysis of virus infection can afford significant new 87 insights into the complexity of γHV infection. 88 89 4 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint RESULTS 90 100 Single-cell analysis of viral RNAs during lytic infection. 91 Single-cell analysis of viral RNAs during lytic infection. 91 Traditional measurements of gene expression frequently rely on pooled cellular 92 material, obscuring intercellular variation in gene expression. To better define 93 expression of γHV RNAs at the single cell level, we employed the PrimeFlowTM RNA 94 assay [13] to study viral gene expression during murine gammaherpesvirus 68 (γHV68) 95 infection, a small animal γHV [4, 13]. This method is a highly sensitive, extremely 96 specific in situ hybridization assay, integrating Affymetrix-designed branched DNA 97 technology with single-cell analysis powered by multiparameter flow cytometry. This 98 method has been successfully used to detect both virus and host RNAs (e.g. in the 99 context of HIV infected individuals [13, 14]). 100 We first tested the ability of PrimeFlowTM to measure multiple viral RNAs during 101 lytic infection with γHV68, including small non-coding RNAs (tRNA-miRNA encoding 102 RNAs or TMERs [15]) and mRNAs. Mouse 3T12 fibroblasts were infected with a 103 multiplicity of infection (MOI=5 plaque forming units (PFU) of virus/cell). Under these 104 conditions, TMER-5, one of the eight γHV68 TMERs, and the γHV68 ORF73, were 105 readily detectable by conventional real-time PCR in γHV68-infected, but not mock- 106 infected, cultures (Fig. 1A, C). Parallel cultures were analyzed for RNA expression by 107 PrimeFlowTM. Whereas mock-infected cells had no detectable expression of either the 108 γHV68 TMERs or ORF73, WT γHV68-infected fibroblasts had a prominent population of 109 TMER+ and ORF73+ cells, respectively (Fig. 1B and 1D). Infection of cells with a 110 TMER-deficient γHV68 (TMER-TKO [16]), in which TMER expression is ablated through 111 promoter disruption, revealed no detectable TMER expression (Fig. 1B), yet robust 112 5 5 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint ORF73 expression (Fig. 1D). RESULTS 90 Parallel studies revealed ready detection of ORF18, 113 another γHV68 gene product (Fig. 1E). These studies show that PrimeFlowTM is a 114 sensitive, robust and specific method to detect both viral non-coding and messenger 115 RNAs during lytic infection, quantifying both the frequency of gene expression and 116 expression on a per cell basis. 117 118 Heterogeneous gene expression during γHV latency and reactivation from latency. 119 γHV latency is characterized by limited gene expression. We next 120 measured viral RNAs during latency and reactivation using the γHV68-infected 121 A20 HE2.1 cell line (A20.γHV68), a drug-selected latency model with restricted viral 122 gene expression that can reactivate following stimulation [17]. A20.γHV68 cells are 123 characterized by restricted viral gene expression, yet remain competent for reactivatio 124 from latency and the production of infectious virions following chemical stimulation wit 125 the phorbol ester, TPA [17, 18]. 126 ORF73 expression (Fig. 1D). Parallel studies revealed ready detection of ORF18, 113 another γHV68 gene product (Fig. 1E). These studies show that PrimeFlowTM is a 114 sensitive, robust and specific method to detect both viral non-coding and messenger 115 RNAs during lytic infection, quantifying both the frequency of gene expression and 116 expression on a per cell basis. 117 Heterogeneous gene expression during γHV latency and reactivation from latency. 119 γHV latency is characterized by limited gene expression. We next 120 measured viral RNAs during latency and reactivation using the γHV68-infected 121 A20 HE2.1 cell line (A20.γHV68), a drug-selected latency model with restricted viral 122 gene expression that can reactivate following stimulation [17]. A20.γHV68 cells are 123 characterized by restricted viral gene expression, yet remain competent for reactivation 124 from latency and the production of infectious virions following chemical stimulation with 125 the phorbol ester, TPA [17, 18]. 126 When we compared TMER expression between uninfected (parental, virus- 127 negative A20) and infected (A20.γHV68) cells by qRT-PCR, the viral ncRNA TMER-5 128 was readily detectable in A20.γHV68 cells above background signals in parental A20 129 cells, with minimal changes between untreated and chemically-stimulated conditions 130 (Fig. 2A). PrimeFlowTM analysis of TMER expression in untreated A20.γHV68 cells 131 revealed that a majority of these cells expressed the TMERs, as defined by a positive 132 signal in samples subjected to the TMER probe relative to unstained cells (Fig. 2B). 133 Untreated A20.γHV68 cells contained a high frequency of cells expressing intermediate 134 levels of TMERs (i.e. RESULTS 90 TMERmid cells), with a significant signal enrichment above 135 Untreated A20.γHV68 cells contained a high frequency of cells expressing intermediate 134 levels of TMERs (i.e. TMERmid cells), with a significant signal enrichment above 135 6 6 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint parental, virus-negative A20 cells (Fig. 2C). While the frequency of TMERmid cells 136 remained relatively constant following treatment with TPA (compare “Untreated” versus 137 “TPA stimulated”, Fig. 2C), TPA stimulated A20.γHV68 cultures also contained a small 138 fraction of cells with high levels of TMERs (i.e. TMERhigh cells), not present in untreated 139 cultures (Fig. 2C-D). Chemical stimulation is known to result in variable penetrance of 140 reactivation in latently infected cell lines [17]. Based on this, we hypothesized that these 141 rare, TMERhigh cells may represent a subset of cells that are undergoing reactivation 142 from latency. 143 To test this, we analyzed the properties of TMERmid and TMERhigh cells, 144 comparing viral protein expression in untreated and stimulated A20.γHV68 cells. We 145 analyzed: 1) a γHV68 expressed GFP-hygromycin resistance fusion protein 146 (HygroGFP), under the control of a heterologous viral promoter (the CMV immediate 147 early promoter) [17], and 2) the γHV68 regulator of complement activation (RCA), a viral 148 protein encoded by the γHV68 ORF4, an early-late transcript [19]. The vast majority of 149 TMERmid cells were negative for HygroGFP and RCA (i.e. HygroGFP- RCA-), 150 7 7 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . RESULTS 90 CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint fluorescence (Fig. 2G). These data demonstrate that the TMERs are expressed during 159 latency, and that following reactivation-inducing stimulation, TMERs are further induced 160 in a rare subset of cells which are characterized by increased viral transcription and 161 translation. 162 163 Detection of endogenous viral gene expression during KSHV latency and reactivation. 164 To extend these findings, we analyzed viral gene expression in the KSHV 165 infected B cell tumor line, BCBL-1, focused on detection of an abundant viral ncRNA, 166 the KSHV polyadenylated nuclear RNA (PAN, nut1, or T1.1) [20]. PAN RNA is known to 167 be highly inducible upon induction of reactivation in KSHV latently infected B cell 168 lymphoma cell lines [10, 20]. The frequency of PAN RNA+ cells was low in untreated 169 BCBL-1 cells, with ~1% of cells spontaneously expressing this ncNRA (Fig. 3A-B). 170 Despite the low frequency, this hybridization was clearly above background, as ncRNA 171 defined on the KSHV- and EBV-negative B cell lymphoma cell line BL41 [21, 22] (Fig. 172 3A-B). Upon stimulation of BCBL-1 cells with the reactivation-inducing stimuli TPA and 173 sodium butyrate, the frequency of PAN RNA+ cells significantly increased with 174 expression in ~25% of cells (Fig. 3A-B). Although stimulation of BCBL-1 cells 175 significantly increased the frequency of PAN RNA+ events compared to untreated 176 cultures, PAN RNA expression on an individual cell basis was comparable between 177 cells from untreated or stimulated cultures (Fig. 3C). As anticipated, stimulation of 178 BCBL-1 cells was associated with increased viral DNA, consistent with stimulated 179 cultures undergoing reactivation from latency (Supplemental Fig. 1). 180 latency, and that following reactivation-inducing stimulation, TMERs are further induced 160 in a rare subset of cells which are characterized by increased viral transcription and 161 translation. 162 163 Detection of endogenous viral gene expression during KSHV latency and reactivation. 164 To extend these findings, we analyzed viral gene expression in the KSHV 165 infected B cell tumor line, BCBL-1, focused on detection of an abundant viral ncRNA, 166 the KSHV polyadenylated nuclear RNA (PAN, nut1, or T1.1) [20]. RESULTS 90 CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint We next analyzed the properties of BCBL-1 cells as a function of PAN RNA 181 expression. In untreated cells, PAN RNA+ or RNA- cells had comparable cell size 182 (define by forward scatter, FSC) and granularity (defined by side scatter, SSC). ORF73 183 RNA expression was low in untreated BCBL-1 samples, with signal intensity in PAN 184 RNA- cells close to the background fluorescence observed in unstained samples. PAN 185 RNA+ cells in untreated cultures had a modest increase in ORF73 RNA expression 186 relative to PAN RNA- cells (Fig. 3D-E). In stimulated BCBL-1 cultures, PAN RNA+ cells 187 had a modest decrease in cell size (defined by forward scatter) and a trend towards 188 reduced granularity (defined by side scatter) compared to PAN RNA- cells (Fig. 3F-G). 189 Stimulated BCBL-1 cultures also had an increased ORF73 signal when compared to 190 unstained samples (Fig. 3F), with PAN RNA+ cells again showing ~2-fold increase 191 compared to PAN RNA- cells (Fig. 3F-G). These data demonstrate robust detection of 192 PAN RNA by PrimeFlowTM, and further identify PAN RNA expression in a subset of both 193 untreated and reactivation-induced BCBL-1 cells. 194 195 Detection of endogenous viral gene expression during EBV latency and reactivation. 196 EBV encodes two abundant non-coding RNAs, the EBV-encoded RNAs (EBERs) 197 EBER1 and EBER2. We tested the ability of the PrimeFlowTM method to detect EBER in 198 an EBV positive, Burkitt lymphoma type I latency cell line, Mutu I [23]. EBER expression 199 was detected in ~45% of Mutu I cells in either untreated or TPA stimulated conditions, 200 with EBER+ cells defined relative to background probe hybridization in the KSHV- and 201 EBV-negative BL41 cell line, a conservative measurement (Fig. 4A). TPA stimulated 202 Mutu I cells showed a modest, 2-fold increase in EBER expression on an individual cell 203 We next analyzed the properties of BCBL-1 cells as a function of PAN RNA 181 expression. In untreated cells, PAN RNA+ or RNA- cells had comparable cell size 182 (define by forward scatter, FSC) and granularity (defined by side scatter, SSC). RESULTS 90 PAN RNA is known to 167 be highly inducible upon induction of reactivation in KSHV latently infected B cell 168 lymphoma cell lines [10, 20]. The frequency of PAN RNA+ cells was low in untreated 169 BCBL-1 cells, with ~1% of cells spontaneously expressing this ncNRA (Fig. 3A-B). 170 Despite the low frequency, this hybridization was clearly above background, as ncRNA 171 defined on the KSHV- and EBV-negative B cell lymphoma cell line BL41 [21, 22] (Fig. 172 3A-B). Upon stimulation of BCBL-1 cells with the reactivation-inducing stimuli TPA and 173 sodium butyrate, the frequency of PAN RNA+ cells significantly increased with 174 expression in ~25% of cells (Fig. 3A-B). Although stimulation of BCBL-1 cells 175 Detection of endogenous viral gene expression during KSHV latency and reactivation. 164 To extend these findings, we analyzed viral gene expression in the KSHV 165 infected B cell tumor line, BCBL-1, focused on detection of an abundant viral ncRNA, 166 the KSHV polyadenylated nuclear RNA (PAN, nut1, or T1.1) [20]. PAN RNA is known to 167 be highly inducible upon induction of reactivation in KSHV latently infected B cell 168 lymphoma cell lines [10, 20]. The frequency of PAN RNA+ cells was low in untreated 169 BCBL-1 cells, with ~1% of cells spontaneously expressing this ncNRA (Fig. 3A-B). 170 Despite the low frequency, this hybridization was clearly above background, as ncRNA 171 defined on the KSHV- and EBV-negative B cell lymphoma cell line BL41 [21, 22] (Fig. 172 3A-B). Upon stimulation of BCBL-1 cells with the reactivation-inducing stimuli TPA and 173 sodium butyrate, the frequency of PAN RNA+ cells significantly increased with 174 expression in ~25% of cells (Fig. 3A-B). Although stimulation of BCBL-1 cells 175 significantly increased the frequency of PAN RNA+ events compared to untreated 176 cultures, PAN RNA expression on an individual cell basis was comparable between 177 cells from untreated or stimulated cultures (Fig. 3C). As anticipated, stimulation of 178 BCBL-1 cells was associated with increased viral DNA, consistent with stimulated 179 cultures undergoing reactivation from latency (Supplemental Fig. 1). 180 8 8 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . RESULTS 90 Based on these data, EBER 204 expression in Mutu I cells appears to be constitutive, with stimulation under these 205 conditions resulting in minimal consequences on either the frequency or per-cell 206 expression of the EBERs. 207 To extend these findings, we further analyzed EBER expression in a panel of 208 LCLs and during in vitro infection of primary human B cells. EBER expression was 209 readily detected in LCL cultures, with EBER+ cells also found in a subset of human 210 primary B cells following in vitro EBV infection (Supplemental Fig. 2, 3). During EBV 211 infection of human primary B cells, EBER+ B cells had an increased cell size and 212 granularity relative to EBER- B cells, with increased expression of both CD69 and acti 213 mRNA, an activated cell phenotype (Supplemental Fig. 3D). These data demonstrate 214 cell to cell variation in EBER expression and suggest EBER expression as a potential 215 discriminator to investigate variability during EBV infection. 216 basis, relative to untreated EBER+ cells (Fig. 4B). Based on these data, EBER 204 expression in Mutu I cells appears to be constitutive, with stimulation under these 205 conditions resulting in minimal consequences on either the frequency or per-cell 206 expression of the EBERs. 207 To extend these findings, we further analyzed EBER expression in a panel of 208 LCLs and during in vitro infection of primary human B cells. EBER expression was 209 readily detected in LCL cultures, with EBER+ cells also found in a subset of human 210 primary B cells following in vitro EBV infection (Supplemental Fig. 2, 3). During EBV 211 infection of human primary B cells, EBER+ B cells had an increased cell size and 212 granularity relative to EBER- B cells, with increased expression of both CD69 and actin 213 mRNA, an activated cell phenotype (Supplemental Fig. 3D). These data demonstrate 214 cell to cell variation in EBER expression and suggest EBER expression as a potential 215 discriminator to investigate variability during EBV infection. 216 basis, relative to untreated EBER+ cells (Fig. 4B). Based on these data, EBER 204 expression in Mutu I cells appears to be constitutive, with stimulation under these 205 conditions resulting in minimal consequences on either the frequency or per-cell 206 expression of the EBERs. RESULTS 90 ORF73 183 RNA expression was low in untreated BCBL-1 samples, with signal intensity in PAN 184 RNA- cells close to the background fluorescence observed in unstained samples. PAN 185 RNA+ cells in untreated cultures had a modest increase in ORF73 RNA expression 186 relative to PAN RNA- cells (Fig. 3D-E). In stimulated BCBL-1 cultures, PAN RNA+ cells 187 had a modest decrease in cell size (defined by forward scatter) and a trend towards 188 reduced granularity (defined by side scatter) compared to PAN RNA- cells (Fig. 3F-G). 189 Stimulated BCBL-1 cultures also had an increased ORF73 signal when compared to 190 unstained samples (Fig. 3F), with PAN RNA+ cells again showing ~2-fold increase 191 compared to PAN RNA- cells (Fig. 3F-G). These data demonstrate robust detection of 192 PAN RNA by PrimeFlowTM, and further identify PAN RNA expression in a subset of both 193 untreated and reactivation-induced BCBL-1 cells. 194 Detection of endogenous viral gene expression during EBV latency and reactivation. 196 EBV encodes two abundant non-coding RNAs, the EBV-encoded RNAs (EBERs) 197 EBER1 and EBER2. We tested the ability of the PrimeFlowTM method to detect EBER in 198 an EBV positive, Burkitt lymphoma type I latency cell line, Mutu I [23]. EBER expression 199 was detected in ~45% of Mutu I cells in either untreated or TPA stimulated conditions, 200 with EBER+ cells defined relative to background probe hybridization in the KSHV- and 201 EBV-negative BL41 cell line, a conservative measurement (Fig. 4A). TPA stimulated 202 Mutu I cells showed a modest, 2-fold increase in EBER expression on an individual cell 203 9 9 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint basis, relative to untreated EBER+ cells (Fig. 4B). RESULTS 90 The actin RNAlow population had a 227 fluorescent signal that was only modestly above background fluorescence (defined by 228 the “No probe” sample), suggesting an all-or-none phenomenon in which cells either 229 had no change in actin RNA levels or had pronounced actin RNA degradation. 230 Simultaneous analysis of TMER and actin RNA expression revealed that actin RNAlow 231 cells were frequently TMERhigh, with actinhigh cells frequently TMERnegative at this time 232 (Fig. 5C). 233 To determine whether actin RNA regulation could also be observed during γHV68 234 latency and reactivation, we measured actin RNA levels in A20.γHV68 cells. Parental, 235 virus-negative A20 cells and A20.γHV68 cells had relatively comparable actin RNA 236 levels by qRT-PCR, in both untreated and stimulated cells (Fig. 5D). Given that host 237 shutoff is expected to primarily occur in rare, reactivating cells, we measured actin RNA 238 degradation relative to TMER expression by the PrimeFlowTM method. Untreated 239 A20.γHV68 cultures had no discernable population of TMER+ actin RNAlow events, 240 whereas stimulated cultures were characterized by a rare population of TMERhigh actin 241 RNAlow cells (Fig. 5E). We further compared actin RNA expression between TMERmid 242 and TMERhigh cells, in untreated versus stimulated cultures using our previously defined 243 subpopulations (Fig. 2). While TMERmid cells from either untreated or stimulated 244 cultures were predominantly actin RNA+, TMERhigh cells from stimulated cultures 245 showed a significant increased frequency of actin RNAlow events (Fig. 5F-G). These 246 studies reveal actin RNA as a sensitive indicator of virus-induced host shutoff, and 247 demonstrate this as an all-or-none phenomenon that can be readily queried at the 248 i l ll l l 249 actin RNAhigh and actin RNAlow cells (Fig. 5B). The actin RNAlow population had a 227 fluorescent signal that was only modestly above background fluorescence (defined by 228 the “No probe” sample), suggesting an all-or-none phenomenon in which cells either 229 had no change in actin RNA levels or had pronounced actin RNA degradation. 230 To determine whether actin RNA regulation could also be observed during γHV68 234 latency and reactivation, we measured actin RNA levels in A20.γHV68 cells. Parental, 235 virus-negative A20 cells and A20.γHV68 cells had relatively comparable actin RNA 236 levels by qRT-PCR, in both untreated and stimulated cells (Fig. 5D). RESULTS 90 CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint actin RNAhigh and actin RNAlow cells (Fig. 5B). The actin RNAlow population had a 227 fluorescent signal that was only modestly above background fluorescence (defined by 228 the “No probe” sample), suggesting an all-or-none phenomenon in which cells either 229 had no change in actin RNA levels or had pronounced actin RNA degradation. 230 Simultaneous analysis of TMER and actin RNA expression revealed that actin RNAlow 231 cells were frequently TMERhigh, with actinhigh cells frequently TMERnegative at this time 232 (Fig. 5C). 233 To determine whether actin RNA regulation could also be observed during γHV68 234 latency and reactivation, we measured actin RNA levels in A20.γHV68 cells. Parental, 235 virus-negative A20 cells and A20.γHV68 cells had relatively comparable actin RNA 236 levels by qRT-PCR, in both untreated and stimulated cells (Fig. 5D). Given that host 237 shutoff is expected to primarily occur in rare, reactivating cells, we measured actin RNA 238 degradation relative to TMER expression by the PrimeFlowTM method. Untreated 239 A20.γHV68 cultures had no discernable population of TMER+ actin RNAlow events, 240 whereas stimulated cultures were characterized by a rare population of TMERhigh actin 241 RNAlow cells (Fig. 5E). We further compared actin RNA expression between TMERmid 242 and TMERhigh cells, in untreated versus stimulated cultures using our previously defined 243 subpopulations (Fig. 2). While TMERmid cells from either untreated or stimulated 244 cultures were predominantly actin RNA+, TMERhigh cells from stimulated cultures 245 showed a significant increased frequency of actin RNAlow events (Fig. 5F-G). These 246 studies reveal actin RNA as a sensitive indicator of virus-induced host shutoff, and 247 demonstrate this as an all-or-none phenomenon that can be readily queried at the 248 single-cell level. 249 actin RNAhigh and actin RNAlow cells (Fig. 5B). RESULTS 90 207 expression in Mutu I cells appears to be constitutive, with stimulation under these 205 conditions resulting in minimal consequences on either the frequency or per-cell 206 expression of the EBERs. 207 To extend these findings, we further analyzed EBER expression in a panel of 208 LCLs and during in vitro infection of primary human B cells. EBER expression was 209 readily detected in LCL cultures, with EBER+ cells also found in a subset of human 210 primary B cells following in vitro EBV infection (Supplemental Fig. 2, 3). During EBV 211 infection of human primary B cells, EBER+ B cells had an increased cell size and 212 granularity relative to EBER- B cells, with increased expression of both CD69 and ac 213 mRNA, an activated cell phenotype (Supplemental Fig. 3D). These data demonstrat 214 cell to cell variation in EBER expression and suggest EBER expression as a potentia 215 discriminator to investigate variability during EBV infection. 216 217 Single-cell analysis of actin mRNA degradation as a readout of virus-induced host 218 shutoff. 219 Many herpesviruses, including γHV68, EBV and KSHV, induce host shutoff 220 during lytic replication and reactivation from latency, a process characterized by 221 dramatic decreases in host mRNAs [24, 25]. Consistent with published reports [24], 222 qRT-PCR analysis of a cellular housekeeping gene, β-actin (Actb), showed reduced 223 actin mRNA in γHV68 lytically-infected fibroblasts by 18 hours pi (Fig. 5A). While moc 224 infected samples had a uniformly positive population of actin RNAhigh cells detectable 225 PrimeFlowTM, γHV68-infected fibroblast cultures demonstrated a bimodal distribution 226 To extend these findings, we further analyzed EBER expression in a panel of 208 LCLs and during in vitro infection of primary human B cells. EBER expression was 209 readily detected in LCL cultures, with EBER+ cells also found in a subset of human 210 primary B cells following in vitro EBV infection (Supplemental Fig. 2, 3). During EBV 211 infection of human primary B cells, EBER+ B cells had an increased cell size and 212 granularity relative to EBER- B cells, with increased expression of both CD69 and actin 213 mRNA, an activated cell phenotype (Supplemental Fig. 3D). These data demonstrate 214 cell to cell variation in EBER expression and suggest EBER expression as a potential 215 discriminator to investigate variability during EBV infection. 216 10 . RESULTS 90 Given that host 237 shutoff is expected to primarily occur in rare, reactivating cells, we measured actin RNA 238 degradation relative to TMER expression by the PrimeFlowTM method. Untreated 239 A20.γHV68 cultures had no discernable population of TMER+ actin RNAlow events, 240 whereas stimulated cultures were characterized by a rare population of TMERhigh actin 241 RNAlow cells (Fig. 5E). We further compared actin RNA expression between TMERmid 242 and TMERhigh cells, in untreated versus stimulated cultures using our previously defined 243 subpopulations (Fig. 2). While TMERmid cells from either untreated or stimulated 244 cultures were predominantly actin RNA+, TMERhigh cells from stimulated cultures 245 showed a significant increased frequency of actin RNAlow events (Fig. 5F-G). These 246 studies reveal actin RNA as a sensitive indicator of virus-induced host shutoff, and 247 demonstrate this as an all-or-none phenomenon that can be readily queried at the 248 single-cell level. 249 11 11 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint 250 Heterogeneity of gene expression during de novo lytic replication. 251 Next, we revisited our analysis of gene expression during de novo lytic infection 252 of fibroblasts, to examine co-expression relationships between viral ncRNA (TMERs), 253 viral mRNA (the γHV68 ORF73), viral protein (RCA protein) and cellular actin mRNA 254 degradation [19, 24]. Mouse 3T12 fibroblasts were infected with a multiplicity of infection 255 (MOI=5 plaque forming units of virus/cell), harvested 16 hpi and then subjected to the 256 PrimeFlowTM method. 257 250 Heterogeneity of gene expression during de novo lytic replication. 251 Next, we revisited our analysis of gene expression during de novo lytic infection 252 of fibroblasts, to examine co-expression relationships between viral ncRNA (TMERs), 253 viral mRNA (the γHV68 ORF73), viral protein (RCA protein) and cellular actin mRNA 254 degradation [19, 24]. RESULTS 90 Mouse 3T12 fibroblasts were infected with a multiplicity of infection 255 (MOI=5 plaque forming units of virus/cell), harvested 16 hpi and then subjected to the 256 PrimeFlowTM method. 257 250 To enable an unbiased, automated analysis of gene expression profiles in γHV68 258 lytically infected cells relative to mock infected cells, data were subjected to the 259 automated clustering algorithm X-shift [26], to identify potential subpopulations of cells 260 with heterogeneous gene expression in these cultures. By sampling 1,000 cells from 261 multiple mock- and virus-infected cultures, the X-shift algorithm consistently identified 7 262 major clusters of cells (Fig. 6A) defined by varying gene expression patterns. While 263 some of the clusters were exclusively found in mock-infected cultures, virus-infected 264 cultures contained three broad types of cell clusters: 1) cells, with no detectable 265 expression of either the TMERs or ORF73 and normal actin RNA, 2) fully infected cells, 266 with robust expression of the TMERs, ORF73, actin RNA downregulation and frequent 267 expression of the RCA protein, and 3) intermediate populations characterized by 268 variable expression of the TMERs and ORF73 (Fig. 6A). 269 To validate these findings using a more conventional method, we compared 270 TMER and ORF73 RNA co-expression on a biaxial plot. By comparing mock-infected, 271 WT-infected and TMER-TKO-infected cultures, this analysis revealed five populations of 272 To validate these findings using a more conventional method, we compared 270 TMER and ORF73 RNA co-expression on a biaxial plot. By comparing mock-infected, 271 WT-infected and TMER-TKO-infected cultures this analysis revealed five populations of 272 WT-infected and TMER-TKO-infected cultures, this analysis revealed five populations of 272 12 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint gene expression (Fig. RESULTS 90 ; https://doi.org/10.1101/482042 doi: bioRxiv preprint discernable expression of either early or late genes (Supplemental Fig. 4A). These data 296 emphasize that a subset of 3T12 fibroblasts subjected to infection with an MOI=5 and 297 harvested at 16 hpi have negligible signs of viral gene expression using this method. 298 This is further illustrated by comparing mock and γHV68 infected cultures for TMER, 299 ORF73, ORF64 and ORF18 expression which demonstrates a subset of cells with 300 minimal expression across each of these viral genes (Supplemental Fig. 4B). One 301 potential explanation for this could be that TMER- ORF73- cells are not infected. To test 302 this, we sort purified cells based on TMER and ORF73 expression, followed by 303 quantitation of viral DNA. TMERhigh ORF73high cells and TMER+ 73- cells had 304 comparable levels of viral DNA, which were approximately ten-fold higher than viral 305 DNA levels in TMER- 73- cells (Supplemental Fig. 4C). Despite this moderate difference 306 in viral DNA, however, viral DNA levels in TMER- 73- cells were still significantly above 307 background signal present in mock infected cultures (Supplemental Fig. 4C). Another 308 potential explanation for this limited gene expression in TMER- 73- cells is that perhaps 309 they are in a distinct phase of the cell cycle relative to other cells. We found that there 310 was no sizable impact of cell cycle stage on whether cells were TMER- ORF73- or 311 TMERhigh ORF73high (Supplemental Fig. 4D). These data identify unexpected 312 heterogeneity during in vitro lytic infection, with TMERhigh ORF73high cells characterized 313 by robust viral transcription, TMERlow ORF73low cells characterized by a lower frequency 314 of cells expressing early and late genes, and TMER- ORF73- cells containing viral DNA 315 with little to no detectable viral gene expression. 316 Given the heterogeneous patterns of RNA and protein expression among 317 discernable expression of either early or late genes (Supplemental Fig. 4A). These data 296 emphasize that a subset of 3T12 fibroblasts subjected to infection with an MOI=5 and 297 harvested at 16 hpi have negligible signs of viral gene expression using this method. 298 This is further illustrated by comparing mock and γHV68 infected cultures for TMER, 299 ORF73, ORF64 and ORF18 expression which demonstrates a subset of cells with 300 minimal expression across each of these viral genes (Supplemental Fig. 4B). RESULTS 90 6B), including cells with: 1) no detectable expression of either 273 viral RNA (TMER- ORF73-), 2) TMER+ ORF73- cells (bottom right quadrant), 3) TMER- 274 ORF73+ cells (upper left quadrant), 4) TMERlow ORF73low cells (lower left edge of the 275 upper right quadrant), and 5) TMERhigh ORF73high cells (upper right quadrant). The 276 definition of TMER positive events was defined based on background fluorescent levels 277 observed in TMER-TKO infected cultures (Fig. 6B). These 5 populations were each 278 assigned a unique color for subsequent analysis (Fig. 6C). 279 We compared cellular phenotype and gene expression across these 5 280 populations. Analysis of TMERs, ORF73, actin RNA, RCA protein, cell size (forward 281 scatter), and granularity (side scatter) revealed multiple types of viral gene expression. 282 TMER- ORF73- cells (in black) had no evidence of viral gene expression, with no 283 detectable viral protein (RCA) or actin downregulation (Fig. 6D-E). Cells with low 284 expression of either the TMERs and/or ORF73 contained viral RNAs, but had minimal 285 expression of either viral protein or actin downregulation (Fig. 6D-E). In stark contrast, 286 cells that were TMERhigh ORF73high (in red, Fig. 6D-E) had multiple characteristics of 287 progressive virus infection including a prominent fraction of cells that expressed RCA 288 and/or had actin RNA downregulation. Further, TMERhigh ORF73high cells were 289 consistently smaller in cell size (defined by forward scatter, FSC) and higher in 290 granularity (defined by side scatter, SSC), a feature that was unique to this phenotype 291 (Fig. 6D-E). 292 TMERhigh ORF73high also demonstrated robust expression of an early gene 293 TMERhigh ORF73high also demonstrated robust expression of an early gene 293 (ORF64) and a late gene (ORF18) (Supplemental Fig. 4A). While some TMERlow 294 ORF73low cells expressed early and late genes, cells defined as TMER- 73- had no 295 13 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. RESULTS 90 One 301 potential explanation for this could be that TMER- ORF73- cells are not infected. To test 302 this, we sort purified cells based on TMER and ORF73 expression, followed by 303 quantitation of viral DNA. TMERhigh ORF73high cells and TMER+ 73- cells had 304 comparable levels of viral DNA, which were approximately ten-fold higher than viral 305 DNA levels in TMER- 73- cells (Supplemental Fig. 4C). Despite this moderate difference 306 in viral DNA, however, viral DNA levels in TMER- 73- cells were still significantly above 307 background signal present in mock infected cultures (Supplemental Fig. 4C). Another 308 potential explanation for this limited gene expression in TMER- 73- cells is that perhaps 309 they are in a distinct phase of the cell cycle relative to other cells. We found that there 310 was no sizable impact of cell cycle stage on whether cells were TMER- ORF73- or 311 TMERhigh ORF73high (Supplemental Fig. 4D). These data identify unexpected 312 heterogeneity during in vitro lytic infection, with TMERhigh ORF73high cells characterized 313 by robust viral transcription, TMERlow ORF73low cells characterized by a lower frequency 314 of cells expressing early and late genes, and TMER- ORF73- cells containing viral DNA 315 with little to no detectable viral gene expression. 316 Given the heterogeneous patterns of RNA and protein expression among 317 Given the heterogeneous patterns of RNA and protein expression among 317 lytically-infected cells, we next queried TMER subcellular localization as a function of 318 14 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint viral gene expression using imaging flow cytometry. While the majority of TMER+ cells 319 had a primarily nuclear TMER localization (defined by DAPI co-localization, as in [27]), 320 the frequency of cells with nuclear TMER localization was highest among TMER+ 321 ORF73- cells and lowest among TMER+ ORF73+ RCA+ cells (Fig. 7A-B). RESULTS 90 CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint gating of actin RNA versus RCA protein expression (Fig. 7D). In total, these data 342 indicate heterogeneous progression of lytic replication in vitro. While some cells have 343 robust viral mRNA and protein expression, additional cell subsets are characterized by 344 limited or divergent gene expression. 345 346 Lytic cycle viral gene expression is influenced by target cell type, changes over time, 347 and is modulated by conditions that alter lytic replication. 348 Our previous studies of γHV68 lytic replication used 3T12 fibroblasts, an 349 immortalized cell line. Mouse embryonic fibroblasts (MEFs) are primary cells that are 350 highly permissive for γHV68 infection, with 5 to 10-fold greater sensitivity to virus 351 infection than 3T12 fibroblasts [29]. We therefore compared viral gene expression 352 between 3T12 fibroblasts and MEFs, using MEFs derived either from wild-type or 353 IFNAR1 KO mice. All cells were infected with WT γHV68 (MOI=5) and analyzed at 16 354 hpi. Both WT and IFNAR KO MEFs showed enhanced viral gene expression relative to 355 infected 3T12 cells at 16 hpi, with infected MEF cultures characterized by an increased 356 frequency of events that expressed ORF73, TMERs, RCA and were actinlow (Fig. 8A). 357 Infected MEFs also had an increased frequency of ORF73+TMER+ cells and a 358 decreased frequency of ORF73-TMER- cells relative to 3T12 cells (Fig. 8B). When we 359 analyzed the cellular distribution of viral gene expression across all 16 possible 360 combinations of viral gene expression, resulting from different combinations of ORF73, 361 TMER, RCA and actinlow phenotypes, infected MEF cultures compared to infected 3T12 362 cultures had: i) fewer ORF73-TMER- cells, ii) an increased frequency of 363 Lytic cycle viral gene expression is influenced by target cell type, changes over time, 347 and is modulated by conditions that alter lytic replication. RESULTS 90 These data 322 suggest that the TMERs can be localized in either the nucleus or cytoplasm during 323 γHV68 lytic replication, and that this localization is not strictly a function of magnitude of 324 gene expression. 325 gene expression. 325 Finally, we used tSNE, a dimensionality reduction algorithm, to better delineate 326 the relationship between TMER, ORF73, RCA protein and actin downregulation across 327 populations defined by variable TMER and ORF73 expression. In tSNE-based 328 visualization, events are subjected to dimensionality reduction, with all events plotted 329 according to the composite parameters tSNE1 and tSNE2. In each tSNE-based plot, 330 values from individual cells are depicted as individual dots on the plot. Cells with similar 331 expression profiles are visually clustered together using this algorithm (e.g. [28]). 332 Consistent with our histogram analysis (Fig. 6D-E), cells that were negative for TMER 333 and ORF73 and cells that expressed either TMERs or ORF73 were relatively uniform in 334 gene expression (Fig. 7C). In contrast, TMERhigh ORF73high cells expressed a wider 335 array of phenotypes, including both a predominant fraction of cells that were actin 336 RNAlow RCA+, and a distinct group of cells that were actin RNA+ RCA- (Fig. 7C). 337 Notably, RCA expression and actin degradation were inversely correlated, with very few 338 cells that expressed RCA also high for actin RNA. Actin RNA+ populations among 339 TMERhigh ORF73high cells were associated with larger cell size (Supplemental Fig. 5). 340 The diversity of phenotypes among TMERhigh ORF73high cells was confirmed by biaxial 341 Finally, we used tSNE, a dimensionality reduction algorithm, to better delineate 326 the relationship between TMER, ORF73, RCA protein and actin downregulation across 327 populations defined by variable TMER and ORF73 expression. In tSNE-based 328 Finally, we used tSNE, a dimensionality reduction algorithm, to better delineate 326 the relationship between TMER, ORF73, RCA protein and actin downregulation across 327 populations defined by variable TMER and ORF73 expression. In tSNE-based 328 visualization, events are subjected to dimensionality reduction, with all events plotted 329 according to the composite parameters tSNE1 and tSNE2. In each tSNE-based plot, 330 values from individual cells are depicted as individual dots on the plot. Cells with similar 331 expression profiles are visually clustered together using this algorithm (e.g. [28]). 332 15 . RESULTS 90 369 The increased viral gene expression observed in MEFs raised the possibility that 370 viral gene expression could be further modulated in 3T12 cells, either as a function of 371 time, multiplicity of infection or viral genotype. We therefore compared viral gene 372 expression in 3T12 fibroblasts, comparing: i) WT γHV68 infection with 5 PFU/cell, ii) C- 373 RTA γHV68 (a recombinant virus engineered to overexpress Rta, the immediate early 374 viral transactivator [30]) infection with 5 PFU/cell, and iii) WT infection using 100 375 PFU/cell. Cells were harvested at 6 and 16 hpi. When comparing WT infection at MOI=5 376 between 6 and 16 hpi, ORF73 expression peaked at 6 hpi with decreased expression 377 by 16 hpi, in contrast to TMER, RCA and actinlow phenotypes which were infrequent at 6 378 hpi and increased by 16 hpi (Fig. 8D). 3T12 cells subjected to WT infection at an 379 MOI=100 showed an increased expression of ORF73, TMER and RCA by 6 hpi, above 380 WT infection with an MOI=5. By 16 hpi, cultures infected with C-RTA and WT MOI=100 381 had increased frequencies of TMER+ and ORF73+TMER+ events relative to WT MOI=5 382 cultures (Fig. 8E). 383 viral gene expression (i.e. ORF73+TMER+RCA+actinlow) (Fig. 8C). Viral gene 365 expression distribution between WT and IFNAR1 KO MEFs were generally comparable, 366 suggesting little to no contribution of type I IFN receptor mediated signaling as a 367 regulator of viral gene expression in MEFs. These data demonstrate that the diversity 368 and relative abundance of viral gene expression can be influenced by target cell type. 369 The increased viral gene expression observed in MEFs raised the possibility that 370 viral gene expression could be further modulated in 3T12 cells, either as a function of 371 time, multiplicity of infection or viral genotype. We therefore compared viral gene 372 expression in 3T12 fibroblasts, comparing: i) WT γHV68 infection with 5 PFU/cell, ii) C- 373 RTA γHV68 (a recombinant virus engineered to overexpress Rta, the immediate early 374 viral transactivator [30]) infection with 5 PFU/cell, and iii) WT infection using 100 375 PFU/cell. Cells were harvested at 6 and 16 hpi. RESULTS 90 348 Our previous studies of γHV68 lytic replication used 3T12 fibroblasts, an 349 immortalized cell line. Mouse embryonic fibroblasts (MEFs) are primary cells that are 350 highly permissive for γHV68 infection, with 5 to 10-fold greater sensitivity to virus 351 infection than 3T12 fibroblasts [29]. We therefore compared viral gene expression 352 between 3T12 fibroblasts and MEFs, using MEFs derived either from wild-type or 353 IFNAR1 KO mice. All cells were infected with WT γHV68 (MOI=5) and analyzed at 16 354 hpi. Both WT and IFNAR KO MEFs showed enhanced viral gene expression relative to 355 infected 3T12 cells at 16 hpi, with infected MEF cultures characterized by an increased 356 frequency of events that expressed ORF73, TMERs, RCA and were actinlow (Fig. 8A). 357 Infected MEFs also had an increased frequency of ORF73+TMER+ cells and a 358 decreased frequency of ORF73-TMER- cells relative to 3T12 cells (Fig. 8B). When we 359 analyzed the cellular distribution of viral gene expression across all 16 possible 360 combinations of viral gene expression, resulting from different combinations of ORF73, 361 TMER, RCA and actinlow phenotypes, infected MEF cultures compared to infected 3T12 362 cultures had: i) fewer ORF73-TMER- cells, ii) an increased frequency of 363 ORF73+TMER+actinlow cells, iii) with the most frequent subset defined as cells with full 364 ORF73+TMER+actinlow cells, iii) with the most frequent subset defined as cells with full 364 16 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint viral gene expression (i.e. ORF73+TMER+RCA+actinlow) (Fig. 8C). Viral gene 365 expression distribution between WT and IFNAR1 KO MEFs were generally comparable, 366 suggesting little to no contribution of type I IFN receptor mediated signaling as a 367 regulator of viral gene expression in MEFs. These data demonstrate that the diversity 368 and relative abundance of viral gene expression can be influenced by target cell type. RESULTS 90 When comparing WT infection at MOI=5 376 between 6 and 16 hpi, ORF73 expression peaked at 6 hpi with decreased expression 377 by 16 hpi, in contrast to TMER, RCA and actinlow phenotypes which were infrequent at 6 378 hpi and increased by 16 hpi (Fig. 8D). 3T12 cells subjected to WT infection at an 379 MOI=100 showed an increased expression of ORF73, TMER and RCA by 6 hpi, above 380 WT infection with an MOI=5. By 16 hpi, cultures infected with C-RTA and WT MOI=100 381 had increased frequencies of TMER+ and ORF73+TMER+ events relative to WT MOI=5 382 cultures (Fig. 8E). 383 The diversity and progression of viral gene expression was further illustrated by 384 analyzing the frequency of all 16 different combinations of possible gene expression 385 between these different virus conditions. While WT MOI=5 cultures showed a time- 386 dependent switch from ORF73+ events to TMER+, ORF73+TMER+, and 387 17 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint ORF73+TMER+RCA+actinlow events, the frequency of each of these populations was 388 typically below 10% of cultures (Fig. 8F). In contrast, cultures infected with C-RTA had 389 limited viral gene expression at 6 hpi, but showed enhanced progression to both 390 TMER+RCA+actinlow and ORF73+TMER+RCA+actinlow phenotypes relative to WT 391 infection MOI=5 (Fig. 8F). Cultures infected with WT virus at an MOI=100 showed rapid 392 induction of multiple phenotypes, including a sizable portion of cultures which were 393 ORF73+RCA+ and ORF73+TMER+RCA+ by 6 hpi, both populations which retained 394 actin RNA expression. By 16 hpi, cultures infected with WT virus at an MOI=100 had 395 sizable frequencies of TMER+RCA+actinlow and ORF73+TMER+RCA+actinlow events 396 (Fig. 8F). RESULTS 90 While C-RTA and MOI=100 infected cultures showed enhanced viral gene 397 expression, WT cultures treated with phosphonoacetic acid (PAA), an inhibitor of viral 398 DNA synthesis, showed impaired expression of TMER with a complete absence of 399 TMERhigh events, negligible ORF73 expression and a pronounced inability to induce an 400 actinlow phenotype (Supplemental Fig. 6). These data demonstrate diversity of viral gene 401 expression that can occur during lytic cycle and provide direct demonstration that these 402 different gene expression profiles can be experimentally manipulated by either 403 enhancing or restraining lytic replication. 404 18 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint DISCUSSION 406 444 frequency of ncRNA expressing cells and changes in expression within individual cells. 429 This has been particularly insightful for the identification of rare PAN RNA+ cells in 430 untreated BCBL-1 cells and a TMERhigh subpopulation of cells in reactivating 431 A20.γHV68 cells. Integrating this method with cell sorting will afford future opportunities 432 to investigate unique properties of these rare cell populations. 433 frequency of ncRNA expressing cells and changes in expression within individual cells. 429 This has been particularly insightful for the identification of rare PAN RNA+ cells in 430 untreated BCBL-1 cells and a TMERhigh subpopulation of cells in reactivating 431 A20.γHV68 cells. Integrating this method with cell sorting will afford future opportunities 432 to investigate unique properties of these rare cell populations. 433 Among the viral ncRNAs measured, in-depth analysis of TMER expression 434 during γHV68 infection has revealed new insights into infection. In the context of 435 latency, the TMERs are constitutively expressed in many, but not all, latently infected 436 cells using the A20.γHV68 model. Further, stimulating these cells to undergo 437 reactivation has a minimal effect on the frequency of cells expressing intermediate 438 levels of TMERs (i.e. TMERmid cells), instead resulting in the appearance of a minor 439 population of TMERhigh cells. Notably, TMERhigh cells show additional features of lytic 440 cycle progression, including actin RNA degradation and RCA protein expression. Why 441 only some latently infected cells show the TMERhigh phenotype, and what regulates the 442 inducible expression of the RNA pol III-transcribed TMERs remain important questions 443 raised by this analysis. 444 Among the viral ncRNAs measured, in-depth analysis of TMER expression 434 during γHV68 infection has revealed new insights into infection. In the context of 435 latency, the TMERs are constitutively expressed in many, but not all, latently infected 436 cells using the A20.γHV68 model. Further, stimulating these cells to undergo 437 Of the γHVs studied here, only γHV68 has a robust in vitro lytic replication 445 system. Our studies on γHV68 lytic replication revealed multiple unanticipated results. 446 First, our analysis identified heterogeneity of viral gene expression, stratified by 447 differential viral gene expression of the TMERs and ORF73. DISCUSSION 406 DISCUSSION 406 Herpesvirus gene expression has been historically analyzed in bulk cell 407 populations. These studies have provided an essential cornerstone to understanding 408 the transcriptional and translational capacity of the herpesviruses. Despite this, recent 409 studies on cellular and viral transcription from other systems have emphasized a high 410 degree of cell-to-cell variation in gene expression [6-11, 13, 14], something we have 411 further investigated here. By applying the PrimeFlowTM methodology to measure 412 endogenous viral gene expression across multiple gammaherpesviruses, and multiple 413 stages of infection, we have gained critical new insights into the inter-relationships of 414 gene expression at the single-cell level. 415 A primary focus of the current study has been to analyze expression of γHV 416 ncRNAs. Although the TMERs, EBERs and PAN RNA all represent abundant γHV 417 ncRNAs, these ncRNAs are transcribed by distinct mechanisms: KSHV PAN is a highly- 418 inducible, RNA pol II-transcribed ncRNA [20], in contrast to the RNA pol III-transcribed 419 TMERs and EBERs [15, 31]. This differential regulation was mirrored in the expression 420 patterns we observed. Whereas TMERs and EBERs were detected in a large fraction of 421 latently infected cells, PAN RNA was expressed in a low frequency of latently infected 422 cells, with prominent induction following cell stimulation and the induction of 423 reactivation. The viral ncRNAs were efficiently detected, as might be predicted due to 424 their abundance. The viral ORF73 encodes a transcription factor that is expressed at a 425 far lower level and are also efficiently detected, demonstrating that rare mRNAs can be 426 measured coincidently with abundant RNAs and with proteins, with no modifications 427 required. A unique advantage of our current approach is the ability to measure the 428 19 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. DISCUSSION 406 ; https://doi.org/10.1101/482042 doi: bioRxiv preprint frequency of ncRNA expressing cells and changes in expression within individual cells. 429 This has been particularly insightful for the identification of rare PAN RNA+ cells in 430 untreated BCBL-1 cells and a TMERhigh subpopulation of cells in reactivating 431 A20.γHV68 cells. Integrating this method with cell sorting will afford future opportunities 432 to investigate unique properties of these rare cell populations. 433 Among the viral ncRNAs measured, in-depth analysis of TMER expression 434 during γHV68 infection has revealed new insights into infection. In the context of 435 latency, the TMERs are constitutively expressed in many, but not all, latently infected 436 cells using the A20.γHV68 model. Further, stimulating these cells to undergo 437 reactivation has a minimal effect on the frequency of cells expressing intermediate 438 levels of TMERs (i.e. TMERmid cells), instead resulting in the appearance of a minor 439 population of TMERhigh cells. Notably, TMERhigh cells show additional features of lytic 440 cycle progression, including actin RNA degradation and RCA protein expression. Why 441 only some latently infected cells show the TMERhigh phenotype, and what regulates the 442 inducible expression of the RNA pol III-transcribed TMERs remain important questions 443 raised by this analysis. 444 frequency of ncRNA expressing cells and changes in expression within individual cells. 429 This has been particularly insightful for the identification of rare PAN RNA+ cells in 430 untreated BCBL-1 cells and a TMERhigh subpopulation of cells in reactivating 431 A20.γHV68 cells. Integrating this method with cell sorting will afford future opportunities 432 to investigate unique properties of these rare cell populations. 433 Among the viral ncRNAs measured, in-depth analysis of TMER expression 434 during γHV68 infection has revealed new insights into infection. In the context of 435 latency, the TMERs are constitutively expressed in many, but not all, latently infected 436 cells using the A20.γHV68 model. Further, stimulating these cells to undergo 437 reactivation has a minimal effect on the frequency of cells expressing intermediate 438 levels of TMERs (i.e. TMERmid cells), instead resulting in the appearance of a minor 439 population of TMERhigh cells. Notably, TMERhigh cells show additional features of lytic 440 cycle progression, including actin RNA degradation and RCA protein expression. Why 441 only some latently infected cells show the TMERhigh phenotype, and what regulates the 442 inducible expression of the RNA pol III-transcribed TMERs remain important questions 443 raised by this analysis. DISCUSSION 406 Strikingly, in cultures 448 subjected to infection with 5 PFU per cell, conditions generally considered to induce 449 synchronous lytic replication, there were many cells with limited viral gene expression, 450 expressing low levels of either the TMERs and/or ORF73, but lacking additional signs of 451 20 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint virus gene expression (i.e. actin RNA degradation or RCA protein expression). This 452 analysis further identified a reproducible subset of cells with negligible viral gene 453 expression, defined by a TMER- ORF73- phenotype. Curiously, these cells contain viral 454 DNA but did not have further evidence of viral gene expression. Whether these cells 455 represent an abortive state of infection, or if viral genes are expressed at levels below 456 our current limit of detection remains to be determined. Our studies further revealed that 457 only some viral RNA+ cells showed full progression of virus infection characterized by 458 robust viral gene expression, defined as TMERhigh ORF73high RCA+ actin RNAlow. 459 Though progression to full viral gene expression was enhanced by infection of primary 460 fibroblasts, infection with the C-RTA virus, or a significantly higher MOI, even in these 461 conditions there remained heterogeneity in viral gene expression. Whether 462 heterogeneity of viral gene expression persists when cells manifest cytopathic effect, a 463 process which occurs after 16 hpi, is not yet known. While there is precedence that 464 reactivation from latency in KSHV infection can be asynchronous [9], this heterogeneity 465 of viral gene expression during in vitro lytic replication was unanticipated and suggests 466 that lytic infection under these reductionist conditions is either asynchronous, abortive, 467 or inefficient. DISCUSSION 406 It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint interferon receptor mediated signaling based on relatively comparable gene expression 474 profiles in wild-type and IFNAR1 KO MEFs. 475 This method allows multiplexed analysis of single-cell gene expression, to both 476 directly measure viral RNAs and downstream consequences of gene expression 477 including viral protein production and host RNA degradation, secondary to protein 478 translation. This approach has notable advantages to conventional analyses of gene 479 expression: 1) it can measure endogenous viral gene expression (both mRNA and 480 ncRNA) in the absence of recombinant viruses or marker genes, and 2) it can rapidly 481 analyze gene and protein expression inter-relationships, across millions of cells, 482 providing unique complementary strengths to other single-cell methodologies (e.g. 483 single-cell RNA-seq). In the future, this method can be further integrated with additional 484 antibody-based reagents, to simultaneously query post-translational modifications (e.g. 485 protein phosphorylation) as a function of cell cycle stage. It is also notable that through 486 the use of imaging flow cytometry, it is possible to interrogate subcellular RNA and 487 protein localization throughout distinct stages of virus infection, studies revealing single 488 cell variability in subcellular RNA localization. We anticipate that this approach will have 489 widespread utility, from addressing fundamental questions about herpesvirus gene 490 expression using reductionist approaches, to a better delineation of replication or 491 reactivation defects in viral mutants, to the in vivo identification of viral gene expression 492 in primary infected samples. 493 interferon receptor mediated signaling based on relatively comparable gene expression 474 profiles in wild-type and IFNAR1 KO MEFs. 475 DISCUSSION 406 This heterogeneity of gene expression raises important questions 468 regarding the universality of the prototypical cascade of immediate early, early and late 469 gene expression that is widely accepted in the herpesvirus field and suggests additional 470 levels of complexity that may be obscured by bulk cell analysis. The molecular 471 mechanisms that are responsible for this heterogeneity still remain to be elucidated but 472 appear to be independent of cell cycle stage with little to no contribution of type I 473 virus gene expression (i.e. actin RNA degradation or RCA protein expression). This 452 analysis further identified a reproducible subset of cells with negligible viral gene 453 expression, defined by a TMER- ORF73- phenotype. Curiously, these cells contain viral 454 DNA but did not have further evidence of viral gene expression. Whether these cells 455 represent an abortive state of infection, or if viral genes are expressed at levels below 456 our current limit of detection remains to be determined. Our studies further revealed that 457 only some viral RNA+ cells showed full progression of virus infection characterized by 458 robust viral gene expression, defined as TMERhigh ORF73high RCA+ actin RNAlow. 459 Though progression to full viral gene expression was enhanced by infection of primary 460 fibroblasts, infection with the C-RTA virus, or a significantly higher MOI, even in these 461 conditions there remained heterogeneity in viral gene expression. Whether 462 heterogeneity of viral gene expression persists when cells manifest cytopathic effect, a 463 process which occurs after 16 hpi, is not yet known. While there is precedence that 464 reactivation from latency in KSHV infection can be asynchronous [9], this heterogeneity 465 of viral gene expression during in vitro lytic replication was unanticipated and suggests 466 that lytic infection under these reductionist conditions is either asynchronous, abortive, 467 or inefficient. This heterogeneity of gene expression raises important questions 468 21 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. interferon receptor mediated signaling based on relatively comparable gene expression 474 profiles in wild-type and IFNAR1 KO MEFs. 475 interferon receptor mediated signaling based on relatively comparable gene expression 474 profiles in wild-type and IFNAR1 KO MEFs. 475 This method allows multiplexed analysis of single-cell gene expression, to both 476 directly measure viral RNAs and downstream consequences of gene expression 477 including viral protein production and host RNA degradation, secondary to protein 478 translation. This approach has notable advantages to conventional analyses of gene 479 expression: 1) it can measure endogenous viral gene expression (both mRNA and 480 ncRNA) in the absence of recombinant viruses or marker genes, and 2) it can rapidly 481 analyze gene and protein expression inter-relationships, across millions of cells, 482 providing unique complementary strengths to other single-cell methodologies (e.g. 483 single-cell RNA-seq). In the future, this method can be further integrated with additional 484 antibody-based reagents, to simultaneously query post-translational modifications (e.g. 485 protein phosphorylation) as a function of cell cycle stage. It is also notable that through 486 the use of imaging flow cytometry, it is possible to interrogate subcellular RNA and 487 protein localization throughout distinct stages of virus infection, studies revealing single 488 cell variability in subcellular RNA localization. We anticipate that this approach will have 489 widespread utility, from addressing fundamental questions about herpesvirus gene 490 expression using reductionist approaches, to a better delineation of replication or 491 reactivation defects in viral mutants, to the in vivo identification of viral gene expression 492 in primary infected samples. 493 single-cell RNA-seq). In the future, this method can be further integrated with additional 484 antibody-based reagents, to simultaneously query post-translational modifications (e.g. 485 protein phosphorylation) as a function of cell cycle stage. It is also notable that through 486 the use of imaging flow cytometry, it is possible to interrogate subcellular RNA and 487 protein localization throughout distinct stages of virus infection, studies revealing single 488 cell variability in subcellular RNA localization. We anticipate that this approach will have 489 widespread utility, from addressing fundamental questions about herpesvirus gene 490 expression using reductionist approaches, to a better delineation of replication or 491 reactivation defects in viral mutants, to the in vivo identification of viral gene expression 492 in primary infected samples. 493 The approach presented here provides a powerful complementary method to 494 other single cell methods, affording the opportunity to query a diverse set of 495 experimental manipulations in a relatively rapid manner. It is worthwhile to note, 496 22 . interferon receptor mediated signaling based on relatively comparable gene expression 474 profiles in wild-type and IFNAR1 KO MEFs. 475 CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint however, some important considerations with this approach. First, these studies rely on 497 fluorescent measurements of probe hybridization using a flow cytometer. Though this 498 approach detects a range of viral and host RNAs, sensitivity of this method is influenced 499 by target RNA expression level, fluorophores used for the analysis and controls to 500 define probe specificity and limit of detection. Ideally, comparisons can be strengthened 501 by comparing isogenic conditions (e.g. comparing WT virus with a genetically deficient 502 virus, as done with the TMER deficient virus). In cases where non-isogenic conditions 503 are used (e.g. comparing across cell lines), variable background fluorescence may limit 504 the sensitivity of this method. For new users of this technology, we strongly suggest the 505 use of multiple controls, including a full-minus-one (i.e. FMO) control to accurately 506 define background and signal to noise ratio for targets of interest. We consider 507 multiparameter flow cytometry, as well as mass cytometry [32, 33], two increasingly 508 useful technologies to afford new insight into heterogeneity of virus infection and gene 509 expression that provide a complementary approach to other single cell technologies. 510 In total, these studies demonstrate the power of single-cell analysis of 511 herpesvirus gene expression. Our data emphasize the heterogeneity of γHV gene 512 expression at the single-cell level, even in conditions considered to result in uniform 513 infection. The factors that underlie this heterogeneity are currently unknown, but could 514 reflect either asynchronous or inefficient infection in many infected cells (e.g. in the 515 context of lytic infection). The existence of specific infected cell subsets, based on 516 heterogeneous gene expression, may identify new susceptibilities and points for 517 intervention during the course of virus infection. MATERIALS AND METHODS 520 MATERIALS AND METHODS 520 MATERIALS AND METHODS 520 Viruses and tissue culture. γHV68 viruses were derived from the γHV68 strain WUMS 521 (ATCC VR-1465) [34], using either bacterial artificial chromosome-derived wild-type 522 (WT) γHV68 or γHV68.TMER-Total KnockOut (TMER-TKO) [16], or virus derived by 523 homologous recombination, γHV68.C-RTA [30] . Virus stocks were passaged, grown, 524 and titered as previously described [16]. Mouse 3T12 fibroblasts (ATCC CCL-164) were 525 infected with a multiplicity of infection (MOI) of either 5 or 100 plaque forming units/cell, 526 analyzed 6-18 hpi. Primary mouse embryonic fibroblasts isolated from C57BL/6 mice 527 (B6 MEFs) or from B6 IFNAR1 KO mice (IFNAR KO MEFs, kindly provided by Dr. 528 Thomas E. Morrison at the University of Colorado) were cultured in DMEM with 10% 529 FBS, 1% Penicillin/Streptomycin and then either mock- or virus-infected (MOI=5) 530 harvested at 16 hpi. For experiments using PAA, PAA (Sigma-Aldrich, catalog #284270) 531 was added at a concentration of 200 μg/mL at time of virus infection and left on cultures 532 until the time of harvest. The parental, virus-negative A20 B cell lymphoma cell line was 533 obtained from ATCC (ATCC® TIB-208™) and cultured in RPMI 1640 with 10% FBS, 1% 534 Penicillin/Streptomycin, L-glutamine and 50 µM β-mercaptoethanol (ME). γHV68 535 infected, and hygromycin selected A20.γHV68 (HE2.1) B cells [17] were obtained from 536 Dr. Sam Speck (Emory University) and cultured in RPMI 1640 with 10% FBS, 1% 537 Penicillin/Streptomycin + L-glutamine, 50 µM βME and 300 μg/mL Hygromycin B. A20 538 and A20.γHV68 B cells were treated with vehicle (untreated) or stimulated with 12-O- 539 tetradecanolphrobol-13-actate (TPA) 20 ng/ml (Sigma) (in DMSO) harvested 24 hr later. 540 BCBL-1 B cells, a body-cavity based B cell lymphoma cell line that is latently infected 541 with KSHV (HHV-8), were obtained from the NIH AIDS reagent program (catalog # 542 Viruses and tissue culture. γHV68 viruses were derived from the γHV68 strain WUMS 521 (ATCC VR-1465) [34], using either bacterial artificial chromosome-derived wild-type 522 (WT) γHV68 or γHV68.TMER-Total KnockOut (TMER-TKO) [16], or virus derived by 523 homologous recombination, γHV68.C-RTA [30] . Virus stocks were passaged, grown, 524 and titered as previously described [16]. Mouse 3T12 fibroblasts (ATCC CCL-164) were 525 infected with a multiplicity of infection (MOI) of either 5 or 100 plaque forming units/cell, 526 analyzed 6-18 hpi. interferon receptor mediated signaling based on relatively comparable gene expression 474 profiles in wild-type and IFNAR1 KO MEFs. 475 γHV68 viruses were derived from the γHV68 strain WUMS 521 (ATCC VR-1465) [34], using either bacterial artificial chromosome-derived wild-type 522 (WT) γHV68 or γHV68.TMER-Total KnockOut (TMER-TKO) [16], or virus derived by 523 homologous recombination, γHV68.C-RTA [30] . Virus stocks were passaged, grown, 524 and titered as previously described [16]. Mouse 3T12 fibroblasts (ATCC CCL-164) were 525 infected with a multiplicity of infection (MOI) of either 5 or 100 plaque forming units/cell, 526 analyzed 6-18 hpi. Primary mouse embryonic fibroblasts isolated from C57BL/6 mice 527 (B6 MEFs) or from B6 IFNAR1 KO mice (IFNAR KO MEFs, kindly provided by Dr. 528 Thomas E. Morrison at the University of Colorado) were cultured in DMEM with 10% 529 FBS, 1% Penicillin/Streptomycin and then either mock- or virus-infected (MOI=5) 530 harvested at 16 hpi. For experiments using PAA, PAA (Sigma-Aldrich, catalog #284270) 531 was added at a concentration of 200 μg/mL at time of virus infection and left on cultures 532 until the time of harvest. The parental, virus-negative A20 B cell lymphoma cell line was 533 obtained from ATCC (ATCC® TIB-208™) and cultured in RPMI 1640 with 10% FBS, 1% 534 Penicillin/Streptomycin, L-glutamine and 50 µM β-mercaptoethanol (ME). γHV68 535 infected, and hygromycin selected A20.γHV68 (HE2.1) B cells [17] were obtained from 536 Dr. Sam Speck (Emory University) and cultured in RPMI 1640 with 10% FBS, 1% 537 Penicillin/Streptomycin + L-glutamine, 50 µM βME and 300 μg/mL Hygromycin B. A20 538 and A20.γHV68 B cells were treated with vehicle (untreated) or stimulated with 12-O- 539 tetradecanolphrobol-13-actate (TPA) 20 ng/ml (Sigma) (in DMSO) harvested 24 hr later. 540 BCBL-1 B cells, a body-cavity based B cell lymphoma cell line that is latently infected 541 with KSHV (HHV-8), were obtained from the NIH AIDS reagent program (catalog # 542 interferon receptor mediated signaling based on relatively comparable gene expression 474 profiles in wild-type and IFNAR1 KO MEFs. 475 Whether this variation arises from viral 518 or cellular heterogeneity is a fundamental question for future research. 519 however, some important considerations with this approach. First, these studies rely on 497 fluorescent measurements of probe hybridization using a flow cytometer. Though this 498 approach detects a range of viral and host RNAs, sensitivity of this method is influenced 499 by target RNA expression level, fluorophores used for the analysis and controls to 500 define probe specificity and limit of detection. Ideally, comparisons can be strengthened 501 by comparing isogenic conditions (e.g. comparing WT virus with a genetically deficient 502 virus, as done with the TMER deficient virus). In cases where non-isogenic conditions 503 are used (e.g. comparing across cell lines), variable background fluorescence may limit 504 the sensitivity of this method. For new users of this technology, we strongly suggest the 505 use of multiple controls, including a full-minus-one (i.e. FMO) control to accurately 506 define background and signal to noise ratio for targets of interest. We consider 507 multiparameter flow cytometry, as well as mass cytometry [32, 33], two increasingly 508 useful technologies to afford new insight into heterogeneity of virus infection and gene 509 expression that provide a complementary approach to other single cell technologies. 510 In total, these studies demonstrate the power of single-cell analysis of 511 herpesvirus gene expression. Our data emphasize the heterogeneity of γHV gene 512 expression at the single-cell level, even in conditions considered to result in uniform 513 infection. The factors that underlie this heterogeneity are currently unknown, but could 514 23 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint MATERIALS AND METHODS 520 Viruses and tissue culture. MATERIALS AND METHODS 520 Primary mouse embryonic fibroblasts isolated from C57BL/6 mice 527 (B6 MEFs) or from B6 IFNAR1 KO mice (IFNAR KO MEFs, kindly provided by Dr. 528 24 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint 3233). BCBL-1 cells were cultured in RPMI containing 20% FBS, 1% 543 Penicillin/Streptomycin with L-glutamine, 1% HEPES and 50 µM βME. BCBL-1 B cells 544 were treated with vehicle (untreated) or stimulated with 20 ng/ml TPA (in DMSO) and 545 Sodium Butyrate (NaB) 0.3 mM (Calbiochem) (in water) and then harvested 72 hr later. 546 BL41 B cells (negative for KSHV and EBV), were cultured in RPMI with 10% FBS, 1% 547 Penicillin/Streptomycin with L-glutamine, and 50 µM βME. Mutu I cells, an EBV-infected, 548 type I latency Burkitt’s lymphoma cell line [23] were obtained from Dr. Shannon Kenney 549 (University of Wisconsin), and cultured in RPMI with 10% FBS, 1% 550 3233). BCBL-1 cells were cultured in RPMI containing 20% FBS, 1% 543 Penicillin/Streptomycin with L-glutamine, 1% HEPES and 50 µM βME. BCBL-1 B cells 544 were treated with vehicle (untreated) or stimulated with 20 ng/ml TPA (in DMSO) and 545 Sodium Butyrate (NaB) 0.3 mM (Calbiochem) (in water) and then harvested 72 hr later. 546 BL41 B cells (negative for KSHV and EBV), were cultured in RPMI with 10% FBS, 1% 547 Penicillin/Streptomycin with L-glutamine, and 50 µM βME. Mutu I cells, an EBV-infected, 548 type I latency Burkitt’s lymphoma cell line [23] were obtained from Dr. Shannon Kenney 549 (University of Wisconsin), and cultured in RPMI with 10% FBS, 1% 550 Penicillin/Streptomycin and L-glutamine. Mutu I or BL41 B cells were either treated with 551 vehicle (DMSO) or stimulated with 20 ng/ml TPA (in DMSO) and then harvested 48 hr 552 later. EBV-immortalized B-lymphoblastoid cell lines (LCLs) were cultured in RPMI, 10% 553 FBS, 1% Penicillin/Streptomycin with L-glutamine. MATERIALS AND METHODS 520 LCL3, LCL9 and LCL209 BM were 554 generated from Kenyan samples as previously described [35, 36]. 555 556 EBV infection of primary human B cells. Peripheral blood was obtained from consenting 557 healthy adult donors and layered over Ficoll-Paque to isolate peripheral blood 558 mononuclear cells (PBMCs). B cells were isolated from PBMCs through negative 559 enrichment using EasySep Human B cell isolation kit (Stem Cell Technologies) 560 following manufacturer’s protocol. B cells were plated at 1x106 cells per mL in RPMI, 561 10% FBS, 1% Penicillin/Streptomycin with L-glutamine, then infected with EBV at a MOI 562 of 10 genome copies per cell or mock infected. 5 days post infection cells were 563 harvested for flow cytometry analysis. EBV virus stocks were generated from the EBV+ 564 cell line B95.8 which was reactivated with TPA (50:50 EtOH:Acetone) and Sodium 565 (University of Wisconsin), and cultured in RPMI with 10% FBS, 1% 0 Penicillin/Streptomycin and L-glutamine. Mutu I or BL41 B cells were either treated with 551 vehicle (DMSO) or stimulated with 20 ng/ml TPA (in DMSO) and then harvested 48 hr 552 later. EBV-immortalized B-lymphoblastoid cell lines (LCLs) were cultured in RPMI, 10% 553 FBS, 1% Penicillin/Streptomycin with L-glutamine. LCL3, LCL9 and LCL209 BM were 554 generated from Kenyan samples as previously described [35, 36]. 555 25 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint Butyrate (NaB) for 5 days. Supernatant was collected and centrifuged at 4,000xg for 10 566 min then passed over a 0.7 micron filter. The supernatant was then ultra-centrifuged at 567 16,000x g for 90 mins and resuspended in 1/200th the initial volume using RPMI, 10% 568 FBS, 1% Penicillin/Streptomycin with L-glutamine. Viral stocks were quantified following 569 DNase treatment with qPCR analysis of the EBV BALF 5 gene done as previously 570 described [37]. 571 572 Flow cytometric analysis. MATERIALS AND METHODS 520 Cells were harvested at the indicated time points and 573 processed for flow cytometry using the PrimeFlowTM RNA Assay (Thermo Fisher). 574 Mouse cells were incubated with an Fc receptor blocking antibody (2.4G2) for 10 min 575 and then fixed with 2% PFA (Fisher), washed with PBS (Life Technology). Cells were 576 stained with a rabbit antibody against the γHV68 ORF4 protein, regulator of complement 577 activation (RCA) [19], labeled with Zenon R-phycoerythrin rabbit IgG label reagent (Life 578 Technologies) following manufacturer’s protocol. Human B cells were incubated in 579 human Fc receptor blocking antibody then stained with Zombie Aqua fixable viability 580 dye (1:500 dilution) (BioLegend), according to manufacturer’s protocol. Primary human 581 B cells were subsequently stained with anti-CD19-FITC (Clone HIB19, dilution 1:25), 582 and anti-CD69-PE antibody (clone FN50, dilution 1:20). Samples were subjected to the 583 PrimeFlowTM RNA Assay following manufacturer’s protocols, using viral and host target 584 probes conjugated to fluorescent molecules (Table S1). DAPI (BioLegend) was used on 585 a subset of samples following manufacturer’s protocol, prior to PrimeFlowTM probe 586 hybridization. Flow cytometric analysis was done on LSR II (BD Biosciences), Fortessa 587 (BD Bi i ) d ZE (Bi R d) fl i h i l b d 88 Butyrate (NaB) for 5 days. Supernatant was collected and centrifuged at 4,000xg for 10 566 min then passed over a 0.7 micron filter. The supernatant was then ultra-centrifuged at 567 16,000x g for 90 mins and resuspended in 1/200th the initial volume using RPMI, 10% 568 FBS, 1% Penicillin/Streptomycin with L-glutamine. Viral stocks were quantified following 569 DNase treatment with qPCR analysis of the EBV BALF 5 gene done as previously 570 described [37]. 571 Flow cytometric analysis. Cells were harvested at the indicated time points and 573 processed for flow cytometry using the PrimeFlowTM RNA Assay (Thermo Fisher). 574 Mouse cells were incubated with an Fc receptor blocking antibody (2.4G2) for 10 min 575 and then fixed with 2% PFA (Fisher), washed with PBS (Life Technology). Cells were 576 stained with a rabbit antibody against the γHV68 ORF4 protein, regulator of complement 577 activation (RCA) [19], labeled with Zenon R-phycoerythrin rabbit IgG label reagent (Life 578 Technologies) following manufacturer’s protocol. Human B cells were incubated in 579 human Fc receptor blocking antibody then stained with Zombie Aqua fixable viability 580 dye (1:500 dilution) (BioLegend), according to manufacturer’s protocol. MATERIALS AND METHODS 520 Primary human 581 B cells were subsequently stained with anti-CD19-FITC (Clone HIB19, dilution 1:25), 582 and anti-CD69-PE antibody (clone FN50, dilution 1:20). Samples were subjected to the 583 PrimeFlowTM RNA Assay following manufacturer’s protocols, using viral and host target 584 probes conjugated to fluorescent molecules (Table S1). DAPI (BioLegend) was used on 585 a subset of samples following manufacturer’s protocol, prior to PrimeFlowTM probe 586 hybridization. Flow cytometric analysis was done on LSR II (BD Biosciences), Fortessa 587 (BD Biosciences), and ZE5 (Bio-Rad) flow cytometers, with compensation values based 588 26 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint on antibody-stained beads (BD Biosciences) and cross-validated using cell samples 589 stained with individual antibody conjugates, with compensation modified as needed 590 post-collection using FlowJo. 591 on antibody-stained beads (BD Biosciences) and cross-validated using cell samples 589 stained with individual antibody conjugates, with compensation modified as needed 590 post-collection using FlowJo. 591 592 Flow cytometric analysis utilizing barcoding. Cells were harvested at the indicated time 593 points and processed for flow cytometry using the PrimeFlowTM RNA Assay (Thermo 594 Fisher). Cells were incubated with an Fc receptor blocking antibody (2.4G2), then fixed 595 and permeabilized using reagents from the PrimeFlowTM RNA Assay (Fixation Buffer 1 596 and Permeabilization Buffer). Samples from different experimental conditions were 597 fluorescently barcoded, with cells treated with either: no fluorescent dye, Ghost DyeTM 598 Violet 450 or Ghost DyeTM Violet 510 (Tonbo Biosciences, 1:200 dilution) according to 599 manufacturer’s protocol. After washing, the three labeled samples were pooled together 600 into a single sample and stained with Zenon-labeled polyclonal rabbit antisera against 601 viral RCA as desribed above. Cells were fixed to cross-link antibody stain with Fixation 602 Buffer 2 from PrimeFlowTM RNA Assay, then subjected to the PrimeFlowTM RNA Assay 603 following manufacturer’s protocols. MATERIALS AND METHODS 520 For the analysis of barcoded samples, singlet cells 604 from barcoded samples were analyzed for fluorescence of either Ghost DyeTM Violet 605 450 or Ghost DyeTM Violet 510, to identify the three input populations: cells negative for 606 Ghost 450 and Ghost 510, cells singly positive for Ghost 450, and cell singly positive for 607 Ghost 510. To ensure that no artifacts were introduced due to assignment of one 608 barcode to a specific experimental condition barcodes used for each experimental 609 Flow cytometric analysis utilizing barcoding. Cells were harvested at the indicated time 593 points and processed for flow cytometry using the PrimeFlowTM RNA Assay (Thermo 594 Fisher). Cells were incubated with an Fc receptor blocking antibody (2.4G2), then fixed 595 and permeabilized using reagents from the PrimeFlowTM RNA Assay (Fixation Buffer 1 596 and Permeabilization Buffer). Samples from different experimental conditions were 597 fluorescently barcoded, with cells treated with either: no fluorescent dye, Ghost DyeTM 598 Violet 450 or Ghost DyeTM Violet 510 (Tonbo Biosciences, 1:200 dilution) according to 599 manufacturer’s protocol. After washing, the three labeled samples were pooled together 600 into a single sample and stained with Zenon-labeled polyclonal rabbit antisera against 601 viral RCA as desribed above. Cells were fixed to cross-link antibody stain with Fixation 602 Buffer 2 from PrimeFlowTM RNA Assay, then subjected to the PrimeFlowTM RNA Assay 603 following manufacturer’s protocols. For the analysis of barcoded samples, singlet cells 604 from barcoded samples were analyzed for fluorescence of either Ghost DyeTM Violet 605 450 or Ghost DyeTM Violet 510, to identify the three input populations: cells negative for 606 Ghost 450 and Ghost 510, cells singly positive for Ghost 450, and cell singly positive for 607 Ghost 510. To ensure that no artifacts were introduced due to assignment of one 608 barcode to a specific experimental condition, barcodes used for each experimental 609 condition were shuffled across independent samples. 610 611 27 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. MATERIALS AND METHODS 520 CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available un The copyright holder for this preprint (which was n this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint DAPI content to identify cells in the G0/G1 phase, S phase or G2/M phase of the cell 635 cycle. 636 637 Cell purification and DNA quantitation based on viral gene expression. Mouse 638 fibroblasts (3T12) were infected with WT γHV68 (MOI=5), harvested and processed for 639 PrimeFlowTM at 16 hpi, followed by cell sorting using a BD FACSAria to purify cells 640 based on relative TMER and ORF73 expression. Post-sort purity checks on sorted 641 populations indicated that cell purities were 100% for TMER-ORF73- cells, 94.1% for 642 TMER+ ORF73- and 98% for TMER+ORF73+ cells. DNA was isolated from samples 643 using the DNeasy Blood and Tissue Kit (Qiagen), with an overnight proteinase K 644 incubation followed by heat inactivation (95°C for 10 min). DNA was precipitated using 645 ammonium acetate alcohol. 40 ng of DNA per sample was subjected to qPCR analysis 646 using LightCycler 480 Probe Master-Mix kit (Roche) and primer sets for γHV68 gB and 647 host NFAT5 (Table S3). gB standard curve was generated using a gB plasmid dilution 648 series ranging from 1010 to 101 copies diluted in background DNA, with a limit of 649 detection (LOD) of 100 copies [38]. Host NFAT5 standard curve was generated using 650 HEK293 cells, with 2x105 cell equivalents subjected to serial 10-fold dilutions in 651 background, salmon sperm DNA, from 105 to 101 copies (LOD=10 copies). Quantitation 652 of viral gB copy number was standardized relative to input material using the formula 653 (gB copy number / NFAT5 copy number) / 2, based on the assumption that cells have 2 654 Cell purification and DNA quantitation based on viral gene expression. MATERIALS AND METHODS 520 It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint Imaging flow cytometry. Cells were treated as described above then harvested, and split 612 into two aliquots: one for conventional flow cytometry, and one for imaging flow 613 cytometry, acquired on an Amnis ImageStream®X Mark II imaging flow cytometer 614 (MilliporeSigma) with a 60X objective and low flow rate/high sensitivity using INSPIRE® 615 software. Brightfield (BF) and side scatter (SSC) images were illuminated by LED light 616 and a 785nm laser respectively. Fluorescent probes were excited off 405nm, 488nm, 617 and 642nm lasers with the power adjusted properly to avoid intensity saturation of the 618 camera. Single color controls for compensation were acquired by keeping the same 619 acquisition setting for samples, with the difference of turning the BF LED light and 620 785nm (SSC) laser off. 621 The acquired data were analyzed using IDEAS® software (MilliporeSigma). 622 Single cells that were in focus were defined as a population with a high “gradient RMS” 623 value, an intermediate “Area” value, and a medium to high “Aspect ratio” value for 624 subsequent analysis. Positive and negative events for each fluorescent marker were 625 determined using the “Intensity” feature. TMER nuclear localization was quantified using 626 “Similarity” feature, the log-transformed Pearson’s correlation coefficient by analyzing 627 the pixel values of two image pairs [27]. The degree of nuclear localization of TMER 628 was measured by correlating the pixel intensity of two images with the same spatial 629 registry. The paired TMER and DAPI images were quantified by measuring the 630 “Similarity Score” which cells with high similarity scores display high TMER nuclear 631 localization with similar image pairs. By contrast cells with low similarity scores show 632 low TMER nuclear localization with dissimilar image pairs. Cell cycle stage analysis was 633 performed on data obtained from the imaging flow cytometer, stratifying cells based on 634 28 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . MATERIALS AND METHODS 520 CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint KSHV genome quantification. BCBL-1 or BL41 cells were plated at 7.5e5 cells/well in a 657 6 well plate with 20 ng/ml TPA and 0.3 mM NaB or vehicle only (DMSO and H2O). Cells 658 and supernatant were harvested at 72 hrs post-treatment, hard-spun for 30 min at 4° C 659 and DNA was isolated using the DNeasy Blood and Tissue kit following manufacturer’s 660 protocol, except for sample digestion for 1 hour instead of 10 min. 100 ng of DNA per 661 sample was used for qPCR analysis via SYBR green detection using KSHV ORF50 662 primers (5' -TCC GGC GGA TAT ACC GTC AC- 3' and 5'- GGT GCA GCT GGT ACA 663 GTG TG-3') [39]. qPCR was analyzed using relative quantification normalized against 664 unit mass calculation, ratio = EdeltaCt (Real-Time PCR Application Guide, Bio-Rad 665 Laboratories Inc. 2006). 666 667 RNA and qRT-PCR. RNA was isolated using Trizol (Life Technologies) per 668 manufacturer’s protocol and re-suspended in DEPC treated water. 3 μg of RNA was 669 treated with DNase 1 (Promega) for 2 hours at 37°C, heat inactivated for 10 min at 670 65°C. 500 ng of RNA was then subjected to reverse transcription using SuperScript II 671 (Life Technologies) following manufacturer’s protocol for gene specific, oligo(dT), or 672 random primers (Life Technologies). Quantitative PCR (qPCR) was performed using iQ 673 SYBR Green super mix (Bio-Rad) follow manufacturer’s protocol using host and viral 674 primer sets (Table S2) or using QuantiTech Primer Assay (Qiagen) for 18s (Hs- 675 RRN18S_1_SG). qPCR conditions: 3 min at 95°C, amplification cycles for 40 cycles of 676 15 sec at 95°C, annealing/ extension at temperature for specific primer set for 1 min 677 ending with a melt curve which started at 50°C or 55°C to 95°C increasing 0.5°C for 678 0:05 sec. MATERIALS AND METHODS 520 Mouse 638 fibroblasts (3T12) were infected with WT γHV68 (MOI=5), harvested and processed for 639 PrimeFlowTM at 16 hpi, followed by cell sorting using a BD FACSAria to purify cells 640 based on relative TMER and ORF73 expression. Post-sort purity checks on sorted 641 populations indicated that cell purities were 100% for TMER-ORF73- cells, 94.1% for 642 TMER+ ORF73- and 98% for TMER+ORF73+ cells. DNA was isolated from samples 643 using the DNeasy Blood and Tissue Kit (Qiagen), with an overnight proteinase K 644 incubation followed by heat inactivation (95°C for 10 min). DNA was precipitated using 645 ammonium acetate alcohol. 40 ng of DNA per sample was subjected to qPCR analysis 646 using LightCycler 480 Probe Master-Mix kit (Roche) and primer sets for γHV68 gB and 647 host NFAT5 (Table S3). gB standard curve was generated using a gB plasmid dilution 648 series ranging from 1010 to 101 copies diluted in background DNA, with a limit of 649 detection (LOD) of 100 copies [38]. Host NFAT5 standard curve was generated using 650 HEK293 cells, with 2x105 cell equivalents subjected to serial 10-fold dilutions in 651 background, salmon sperm DNA, from 105 to 101 copies (LOD=10 copies). Quantitation 652 of viral gB copy number was standardized relative to input material using the formula 653 (gB copy number / NFAT5 copy number) / 2, based on the assumption that cells have 2 654 Cell purification and DNA quantitation based on viral gene expression. Mouse 638 fibroblasts (3T12) were infected with WT γHV68 (MOI=5), harvested and processed for 639 PrimeFlowTM at 16 hpi, followed by cell sorting using a BD FACSAria to purify cells 640 based on relative TMER and ORF73 expression. Post-sort purity checks on sorted 641 populations indicated that cell purities were 100% for TMER-ORF73- cells, 94.1% for 642 TMER+ ORF73- and 98% for TMER+ORF73+ cells. DNA was isolated from samples 643 using the DNeasy Blood and Tissue Kit (Qiagen), with an overnight proteinase K 644 incubation followed by heat inactivation (95°C for 10 min). DNA was precipitated using 645 ammonium acetate alcohol. 40 ng of DNA per sample was subjected to qPCR analysis 646 using LightCycler 480 Probe Master-Mix kit (Roche) and primer sets for γHV68 gB and 647 host NFAT5 (Table S3). gB standard curve was generated using a gB plasmid dilution 648 656 29 . MATERIALS AND METHODS 520 CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint each sample together and doing a 1:3 serial dilution. 75 ng of cDNA of the unknown 680 samples was loaded per qPCR reaction/primer set, with reactions run on a Bio-Rad 384 681 CFX LightCycler and data analyzed using Bio-Rad CFX manager software. Data 682 analysis was done using the 1:3 standard curve as the control Ct value to calculate the 683 delta ct, and the Pfaffl equation was used to define the fold difference between the gene 684 of interest and 18s (reference gene) [40]. qPCR products were analyzed by melt curve 685 analysis, with all reactions having a prominent, uniform product. In the case of primers 686 with an aberrant melt curve product (e.g. that arose at late cycles), products were 687 clearly a different product as defined by melt curve analysis. 688 689 Software and Statistical analysis. All flow cytometry data were analyzed in FlowJo 690 (version 8.8.7, 10.5.0, and 10.5.3), with flow cytometry data shown either as histogram 691 overlays or pseudo-color dot plots (with or without smoothing), showing outliers (low or 692 high resolution) on log10 scales. Statistical analysis and graphing were done in 693 GraphPad Prism (Version 6.0d and 7.0d). Statistical significance was tested by 694 unpaired t test (when comparing two conditions) or by one-way ANOVA (when 695 comparing three or more samples) subjected to multiple corrections tests using 696 recommended settings in Prism. X-shift analysis: For automated mapping of flow 697 cytometry data using X-shift, data were obtained from compensated flow cytometry files, 698 exported from FlowJo, using singlets that were live (defined by sequential gating on 699 single cells by FSC-H vs. FSC-W and SSC-H vs. SSC- W, that were DAPI bright vs. 700 SSC-A). These events were imported into the Java based program VorteX 701 (http://web.stanford.edu/~samusik/vortex/) [26]. Four parameters [TMER (AlexaFluor 702 each sample together and doing a 1:3 serial dilution. 75 ng of cDNA of the unknown 680 samples was loaded per qPCR reaction/primer set, with reactions run on a Bio-Rad 384 681 CFX LightCycler and data analyzed using Bio-Rad CFX manager software. MATERIALS AND METHODS 520 A standard curve for each primer set was generated by pooling a portion of 679 KSHV genome quantification. BCBL-1 or BL41 cells were plated at 7.5e5 cells/well in a 657 6 well plate with 20 ng/ml TPA and 0.3 mM NaB or vehicle only (DMSO and H2O). Cells 658 and supernatant were harvested at 72 hrs post-treatment, hard-spun for 30 min at 4° C 659 and DNA was isolated using the DNeasy Blood and Tissue kit following manufacturer’s 660 protocol, except for sample digestion for 1 hour instead of 10 min. 100 ng of DNA per 661 sample was used for qPCR analysis via SYBR green detection using KSHV ORF50 662 primers (5' -TCC GGC GGA TAT ACC GTC AC- 3' and 5'- GGT GCA GCT GGT ACA 663 GTG TG-3') [39]. qPCR was analyzed using relative quantification normalized against 664 unit mass calculation, ratio = EdeltaCt (Real-Time PCR Application Guide, Bio-Rad 665 Laboratories Inc. 2006). 666 RNA and qRT-PCR. RNA was isolated using Trizol (Life Technologies) per 668 manufacturer’s protocol and re-suspended in DEPC treated water. 3 μg of RNA was 669 treated with DNase 1 (Promega) for 2 hours at 37°C, heat inactivated for 10 min at 670 65°C. 500 ng of RNA was then subjected to reverse transcription using SuperScript II 671 (Life Technologies) following manufacturer’s protocol for gene specific, oligo(dT), or 672 random primers (Life Technologies). Quantitative PCR (qPCR) was performed using iQ 673 SYBR Green super mix (Bio-Rad) follow manufacturer’s protocol using host and viral 674 primer sets (Table S2) or using QuantiTech Primer Assay (Qiagen) for 18s (Hs- 675 RRN18S_1_SG). qPCR conditions: 3 min at 95°C, amplification cycles for 40 cycles of 676 15 sec at 95°C, annealing/ extension at temperature for specific primer set for 1 min 677 ending with a melt curve which started at 50°C or 55°C to 95°C increasing 0.5°C for 678 0:05 sec. A standard curve for each primer set was generated by pooling a portion of 679 30 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . MATERIALS AND METHODS 520 Data 682 analysis was done using the 1:3 standard curve as the control Ct value to calculate the 683 delta ct, and the Pfaffl equation was used to define the fold difference between the gene 684 of interest and 18s (reference gene) [40]. qPCR products were analyzed by melt curve 685 analysis, with all reactions having a prominent, uniform product. In the case of primers 686 with an aberrant melt curve product (e.g. that arose at late cycles), products were 687 clearly a different product as defined by melt curve analysis. 688 680 Software and Statistical analysis. All flow cytometry data were analyzed in FlowJo 690 (version 8.8.7, 10.5.0, and 10.5.3), with flow cytometry data shown either as histogram 691 overlays or pseudo-color dot plots (with or without smoothing), showing outliers (low or 692 high resolution) on log10 scales. Statistical analysis and graphing were done in 693 GraphPad Prism (Version 6.0d and 7.0d). Statistical significance was tested by 694 unpaired t test (when comparing two conditions) or by one-way ANOVA (when 695 comparing three or more samples) subjected to multiple corrections tests using 696 recommended settings in Prism. X-shift analysis: For automated mapping of flow 697 cytometry data using X-shift, data were obtained from compensated flow cytometry files, 698 exported from FlowJo, using singlets that were live (defined by sequential gating on 699 single cells by FSC-H vs. FSC-W and SSC-H vs. SSC- W, that were DAPI bright vs. 700 SSC-A). These events were imported into the Java based program VorteX 701 (http://web.stanford.edu/~samusik/vortex/) [26]. Four parameters [TMER (AlexaFluor 702 SSC-A). These events were imported into the Java based program VorteX 701 (http://web.stanford.edu/~samusik/vortex/) [26]. Four parameters [TMER (AlexaFluor 702 31 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint (AF) 488), RCA (PE), ORF73 (AF647), and Actin (AF750)] were selected for clustering 703 analysis using the X-shift algorithm. MATERIALS AND METHODS 520 The following settings were used when importing 704 the data set into VorteX: i) Numerical transformation: arcsinh(x/f), f=150, ii) noise 705 threshold: apply noise threshold of 1.0 (automatic and recommended setting), iii) feature 706 rescaling: none, and iv) normalization: none, v) a Euclidean noise filter was used with a 707 Minimal Euclidean length of the profile of 1.0, and vi) an import max of 1,000 rows from 708 each file after filtering was selected. The following settings were used when preparing 709 the data set for clustering analysis: i) distance measure: angular distance, ii) clustering 710 algorithm: X-shift (gradient assignment), iii) density estimate: N nearest neighbors (fast), 711 iv) number of neighbors for density estimate (K): from 150 to 5, with 30 steps, and v) 712 number of neighbors for mode finding (N): determine automatically. After the cluster 713 analysis was completed, all results were selected and the K value that corresponded 714 with optimal clustering (the elbow point) was calculated, in this case K= 50. All clusters 715 (seven clusters total) for the optimal K value were selected and a Force-Directed Layout 716 was created. The maximum number of events sampled from each cluster was 20, and 717 the number of nearest neighbors was 10. All settings used for this analysis were 718 automated or explicitly recommended (https://github.com/nolanlab/vortex/wiki). Force- 719 Directed layouts in Fig. 6A were saved as graphml files from VorteX, opened in the 720 application Gephi v 0.9.1, and colored by different variables (Cluster ID, experimental 721 group, Actin mRNA, RCA, ORF73, and TMERs respectively) in Adobe Illustrator CC 722 2017. Full details on use of the X-shfit algorithm and analysis pipeline can be found in 723 [41]. tSNE analysis: Gated events for each of the six identified populations were 724 exported from FlowJo, and then imported into Cytobank (www.cytobank.org) for 725 (AF) 488), RCA (PE), ORF73 (AF647), and Actin (AF750)] were selected for clustering 703 analysis using the X-shift algorithm. The following settings were used when importing 704 the data set into VorteX: i) Numerical transformation: arcsinh(x/f), f=150, ii) noise 705 threshold: apply noise threshold of 1.0 (automatic and recommended setting), iii) feature 706 rescaling: none, and iv) normalization: none, v) a Euclidean noise filter was used with a 707 Minimal Euclidean length of the profile of 1.0, and vi) an import max of 1,000 rows from 708 each file after filtering was selected. MATERIALS AND METHODS 520 The following settings were used when preparing 709 the data set for clustering analysis: i) distance measure: angular distance, ii) clustering 710 algorithm: X-shift (gradient assignment), iii) density estimate: N nearest neighbors (fast), 711 iv) number of neighbors for density estimate (K): from 150 to 5, with 30 steps, and v) 712 number of neighbors for mode finding (N): determine automatically. After the cluster 713 analysis was completed, all results were selected and the K value that corresponded 714 with optimal clustering (the elbow point) was calculated, in this case K= 50. All clusters 715 (seven clusters total) for the optimal K value were selected and a Force-Directed Layout 716 was created. The maximum number of events sampled from each cluster was 20, and 717 the number of nearest neighbors was 10. All settings used for this analysis were 718 automated or explicitly recommended (https://github.com/nolanlab/vortex/wiki). Force- 719 Directed layouts in Fig. 6A were saved as graphml files from VorteX, opened in the 720 application Gephi v 0.9.1, and colored by different variables (Cluster ID, experimental 721 group, Actin mRNA, RCA, ORF73, and TMERs respectively) in Adobe Illustrator CC 722 2017. Full details on use of the X-shfit algorithm and analysis pipeline can be found in 723 [41] tSNE analysis: Gated events for each of the six identified populations were 724 32 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint analysis using the viSNE algorithm. Each file was used for a separate viSNE analysis 726 (six total runs), where all available events were selected for clustering (202,669, 727 128,028, 29,096, 5,610, 8,956, 35,850 respectively) and four parameters were selected 728 for clustering (Actin mRNA, RCA, ORF73, and TMERs). The resulting tSNE plots were 729 colored according to expression using the "rainbow" color option, with individual events 730 shown using the stacked dot option. MATERIALS AND METHODS 520 and L.F.V.D., and by an American 747 Heart Association National Scientist Development grant (#13SDG14510023), a 748 Colorado CTSI Novel methods development grant, and funding from the University of 749 Colorado Dept. of Anesthesiology to E.T.C.. The Colorado CTSI is supported by 750 NIH/NCATS Colorado CTSA Grant Number UL1 TR002535. Contents are the authors’ 751 sole responsibility and do not necessarily represent official NIH views. The funders had 752 no role in study design, data collection and analysis, decision to publish, or preparation 753 of the manuscript. 754 755 MATERIALS AND METHODS 520 The channel range was user-defined for each 731 marker according to the range in expression established in Fig. 6E. 732 733 analysis using the viSNE algorithm. Each file was used for a separate viSNE analysis 726 (six total runs), where all available events were selected for clustering (202,669, 727 128,028, 29,096, 5,610, 8,956, 35,850 respectively) and four parameters were selected 728 for clustering (Actin mRNA, RCA, ORF73, and TMERs). The resulting tSNE plots were 729 colored according to expression using the "rainbow" color option, with individual events 730 shown using the stacked dot option. The channel range was user-defined for each 731 marker according to the range in expression established in Fig. 6E. 732 733 analysis using the viSNE algorithm. Each file was used for a separate viSNE analysis 726 (six total runs), where all available events were selected for clustering (202,669, 727 33 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint ACKNOWLEDGMENTS 734 The authors would like to acknowledge insightful comments made by members of the 735 Clambey and van Dyk laboratories, technical assistance by Eva Medina, pilot studies 736 establishing the feasibility of fluorescent barcoding by Shannon Rudolph, support of flow 737 cytometry services through ClinImmune, the Dept. of Immunology & Microbiology, and 738 the University of Colorado Cancer Center, and expert technical guidance from Matt 739 Cato and Dr. Nori Ueno (Thermo Fisher). T.C. and B.A. are employees of EMD Millipore 740 and Luminex respectively and have a potential conflict of interest. E.T.C. was a 741 recipient of the 2016 North America Affymetrix Single-cell Grant Recipient, for studies 742 unrelated to this manuscript. 743 744 FUNDING 745 This research was funded by National Institutes of Health grants R01CA103632 and 746 R01CA168558 to L.F.V.D., R21AI134084 to E.T.C. ACKNOWLEDGMENTS 734 Coleman, Rosemary Rochford, Benjamin 763 Alderete, Tim Chang, Linda F. van Dyk, Eric T. Clambey 764 Project administration: Eric T. Clambey, Linda F. van Dyk 765 Resources: Lauren M. Oko, Rosemary Rochford, Benjamin Alderete, Tim Chang 766 Software: Abigail K. Kimball 767 Supervision: Eric T. Clambey, Linda F. van Dyk 768 Validation: Lauren M. Oko, Eric T. Clambey 769 Visualization: Lauren M. Oko, Abigail K. Kimball, Rachael E. Kaspar, Benjamin 770 Alderete, Tim Chang, Eric T. Clambey 771 Writing – original draft: Eric T. Clambey, Linda F. van Dyk 772 Writing – review & editing: Lauren M. Oko, Linda F. van Dyk, Eric T. Clambey 773 ACKNOWLEDGMENTS 734 The authors would like to acknowledge insightful comments made by members of the 735 Clambey and van Dyk laboratories, technical assistance by Eva Medina, pilot studies 736 establishing the feasibility of fluorescent barcoding by Shannon Rudolph, support of flow 737 cytometry services through ClinImmune, the Dept. of Immunology & Microbiology, and 738 the University of Colorado Cancer Center, and expert technical guidance from Matt 739 Cato and Dr. Nori Ueno (Thermo Fisher). T.C. and B.A. are employees of EMD Millipore 740 and Luminex respectively and have a potential conflict of interest. E.T.C. was a 741 recipient of the 2016 North America Affymetrix Single-cell Grant Recipient, for studies 742 unrelated to this manuscript. 743 FUNDING 745 This research was funded by National Institutes of Health grants R01CA103632 and 746 R01CA168558 to L.F.V.D., R21AI134084 to E.T.C. and L.F.V.D., and by an American 747 Heart Association National Scientist Development grant (#13SDG14510023), a 748 Colorado CTSI Novel methods development grant, and funding from the University of 749 Colorado Dept. of Anesthesiology to E.T.C.. The Colorado CTSI is supported by 750 NIH/NCATS Colorado CTSA Grant Number UL1 TR002535. Contents are the authors’ 751 sole responsibility and do not necessarily represent official NIH views. The funders had 752 no role in study design, data collection and analysis, decision to publish, or preparation 753 of the manuscript. 754 755 34 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint AUTHOR CONTRIBUTIONS 756 Conceptualization: Eric T. Clambey, Linda F. van Dyk 757 Data curation: Lauren M. Oko, Eric T. Clambey 758 Formal analysis: Lauren M. Oko, Abigail K. Kimball, Rachael E. Kaspar, Benjamin 759 Alderete, Tim Chang, Eric T. Clambey 760 Funding acquisition: Eric T. Clambey, Linda F. van Dyk 761 Investigation: Lauren M. Oko, Ashley N. Knox, Carrie B. Coleman 762 Methodology: Lauren M. Oko, Carrie B. AUTHOR CONTRIBUTIONS 756 Conceptualization: Eric T. Clambey, Linda F. van Dyk 757 Data curation: Lauren M. Oko, Eric T. Clambey 758 Formal analysis: Lauren M. Oko, Abigail K. Kimball, Rachael E. Kaspar, Benjamin 759 Alderete, Tim Chang, Eric T. Clambey 760 Funding acquisition: Eric T. Clambey, Linda F. van Dyk 761 Investigation: Lauren M. Oko, Ashley N. Knox, Carrie B. Coleman 762 Methodology: Lauren M. Oko, Carrie B. Coleman, Rosemary Rochf 763 Alderete, Tim Chang, Linda F. van Dyk, Eric T. Clambey 764 Alderete, Tim Chang, Linda F. van Dyk, Eric T. Clambey 764 Project administration: Eric T. Clambey, Linda F. van Dyk 765 Supervision: Eric T. Clambey, Linda F. van Dyk 768 Supervision: Eric T. Clambey, Linda F. van Dyk 768 Validation: Lauren M. Oko, Eric T. Clambey 769 Validation: Lauren M. Oko, Eric T. Clambey 769 Visualization: Lauren M. Oko, Abigail K. Kimball, Rachael E. Kaspar, Benjamin 770 Alderete Tim Chang Eric T Clambey 771 Visualization: Lauren M. Oko, Abigail K. Kimball, Rachael E. Kaspar, Benjamin 770 Alderete, Tim Chang, Eric T. Clambey 771 Writing – original draft: Eric T. Clambey, Linda F. van Dyk 772 Writing – review & editing: Lauren M. Oko, Linda F. van Dyk, Eric T. Clambey 773 774 774 35 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . 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It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint 2014/03/29. doi: 10.1128/JVI.00219-14. PubMed PMID: 24672028; PubMed Central 934 PMCID: PMCPMC4093851. 935 40. Pfaffl MW. A new mathematical model for relative quantification in real-time RT- 936 PCR. Nucleic Acids Res. 2001;29(9):e45. PubMed PMID: 11328886; PubMed Central 937 PMCID: PMCPMC55695. 938 41. Kimball AK, Oko LM, Bullock BL, Nemenoff RA, van Dyk LF, Clambey ET. A 939 Beginner's Guide to Analyzing and Visualizing Mass Cytometry Data. J Immunol. 940 2018;200(1):3-22. Epub 2017/12/20. doi: 10.4049/jimmunol.1701494. PubMed PMID: 941 29255085; PubMed Central PMCID: PMCPMC5765874. 942 943 944 Figure 1. RNA-Flow cytometry using the PrimeFlowTM method affords robust and 947 sensitive analysis of endogenous γHV genes at the single-cell level. Viral RNA 948 analysis in γHV68-infected fibroblasts by qRT-PCR (A, C) or by flow cytometric analysis 949 using PrimeFlowTM at 16 hpi (B, D, E, F). Samples were either mock, TMER-TKO, or 950 WT γHV68-infected, with infections done using 5 plaque forming units/cell. qRT-PCR 951 standardized to 18s RNA, at the indicated times. All flow cytometric events gated on a 952 generous FSC x SSC gate, followed by singlet discrimination. PrimeFlowTM analysis 953 quantified probe fluorescence for (B) TMERs, (D) ORF73, or (E) ORF18, relative to 954 side-scatter area (SSC-A). Probe fluorescence is indicated, with all probes detected 955 using either AlexaFluor (AF) 647 or 488 conjugates. Data representative of 2 956 independent experiments, each done with biological replicates. 957 958 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint 2014/03/29. doi: 10.1128/JVI.00219-14. PubMed PMID: 24672028; PubMed Central 934 PMCID: PMCPMC4093851. 935 40. Pfaffl MW. A new mathematical model for relative quantification in real-time RT- 936 PCR. Nucleic Acids Res. 2001;29(9):e45. PubMed PMID: 11328886; PubMed Central 937 PMCID: PMCPMC55695. 938 944 43 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint FIGURE LEGENDS 945 946 Figure 1. RNA-Flow cytometry using the PrimeFlowTM method affords robust and 947 sensitive analysis of endogenous γHV genes at the single-cell level. Viral RNA 948 analysis in γHV68-infected fibroblasts by qRT-PCR (A, C) or by flow cytometric analysis 949 using PrimeFlowTM at 16 hpi (B, D, E, F). Samples were either mock, TMER-TKO, or 950 WT γHV68-infected, with infections done using 5 plaque forming units/cell. qRT-PCR 951 standardized to 18s RNA, at the indicated times. All flow cytometric events gated on a 952 generous FSC x SSC gate, followed by singlet discrimination. PrimeFlowTM analysis 953 quantified probe fluorescence for (B) TMERs, (D) ORF73, or (E) ORF18, relative to 954 side-scatter area (SSC-A). Probe fluorescence is indicated, with all probes detected 955 using either AlexaFluor (AF) 647 or 488 conjugates. Data representative of 2 956 independent experiments, each done with biological replicates. 957 958 Figure 2. Heterogeneous gene expression in a γHV68 latently infected B cell line. 959 Viral, host RNA analysis during γHV68 latency and reactivation in A20.γHV68 (HE2.1) 960 cells by qRT-PCR (A) and flow cytometric analysis using PrimeFlowTM (B-G), comparing 961 untreated or TPA-stimulated samples at 24 hrs post-treatment. Analysis includes A20, 962 virus-negative cells and A20.γHV68 cells. (A) qRT-PCR analysis of TMER-5 expression 963 relative to 18s RNA in A20 and A20.γHV68 (HE2.1) cells, untreated or stimulated with 964 TPA for 24 hrs. (B) PrimeFlowTM detection of TMER expression in A20.γHV68 (HE2.1) 965 cells, comparing either samples that were unstained (solid gray) or stained for the 966 TMERs (open black line). (C) Analysis of TMER expression in multiple conditions, 967 44 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint comparing untreated and stimulated A20 and A20.γHV68 cells, with gates defining the 968 frequency of events that expressed either intermediate (mid) or high levels of TMERs. 969 Data depict lymphocytes that were singlets, defined by sequential removal of doublets. 970 (D) Quantification of the frequency of TMERhigh cells in stimulated A20.γHV68 cells. (E) 971 Histogram overlays of HygroGFP and RCA protein expression in A20.γHV68 cells 972 comparing TMERmid cells from untreated cultures (top, gray), TMERmid cells from TPA- 973 stimulated cultures (middle, blue), with TMERhigh cells from stimulated cultures (bottom, 974 red). (F) Quantification of the frequencies of HygroGFP+ (left) and RCA protein+ (right) 975 cells as a function of TMER expression and treatment condition. (G) Flow cytometric 976 analysis on an imaging flow cytometer, with each row showing an individual cell and 977 representative images of brightfield (BF), RCA protein (RCA), DAPI, and TMER 978 localization in TMERmid cells from untreated cultures (left) or TMERhigh RCA+ cells from 979 stimulated (right) A20.γHV68 cells. Data are from two independent experiments, with 980 biological replicates within each experiment for all A20.γHV68 cultures. Figure 2. Heterogeneous gene expression in a γHV68 latently infected B cell line. 959 Graphs depict 981 the mean ± SEM, with each symbol identifying data from a single replicate. Statistical 982 analysis was done using an unpaired t test (D) or one-way ANOVA, subjected to 983 Tukey’s multiple comparison test (A, F), with statistically significant differences as 984 indicated, * p<0.05, **** p<0.0001. 985 986 comparing untreated and stimulated A20 and A20.γHV68 cells, with gates defining the 968 frequency of events that expressed either intermediate (mid) or high levels of TMERs. 969 Data depict lymphocytes that were singlets, defined by sequential removal of doublets. 970 (D) Quantification of the frequency of TMERhigh cells in stimulated A20.γHV68 cells. (E) 971 Histogram overlays of HygroGFP and RCA protein expression in A20.γHV68 cells 972 comparing TMERmid cells from untreated cultures (top, gray), TMERmid cells from TPA- 973 stimulated cultures (middle, blue), with TMERhigh cells from stimulated cultures (bottom, 974 red). (F) Quantification of the frequencies of HygroGFP+ (left) and RCA protein+ (right) 975 cells as a function of TMER expression and treatment condition. (G) Flow cytometric 976 analysis on an imaging flow cytometer, with each row showing an individual cell and 977 representative images of brightfield (BF), RCA protein (RCA), DAPI, and TMER 978 localization in TMERmid cells from untreated cultures (left) or TMERhigh RCA+ cells from 979 stimulated (right) A20.γHV68 cells. Data are from two independent experiments, with 980 biological replicates within each experiment for all A20.γHV68 cultures. Graphs depict 981 the mean ± SEM, with each symbol identifying data from a single replicate. Statistical 982 analysis was done using an unpaired t test (D) or one-way ANOVA, subjected to 983 Tukey’s multiple comparison test (A, F), with statistically significant differences as 984 indicated, * p<0.05, **** p<0.0001. 985 986 Figure 3 Single cell analysis of KSHV PAN RNA expression in the BCBL 1 B cell 987 Figure 3. Single-cell analysis of KSHV PAN RNA expression in the BCBL-1 B cell 987 lymphoma cell line. (A) Flow cytometric analysis of PAN RNA expression in multiple 988 conditions, from cells incubated with no probe (left), or cells subjected to hybridization 989 using a probe for PAN RNA, comparing virus-negative BL41 cells (second from left) with 990 45 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint KSHV+ BCBL-1 cells that were either untreated or stimulated (with TPA and sodium 991 butyrate (NaB)) for 72 hours. Data depict lymphocytes that were singlets, defined by 992 sequential removal of doublets. Representative images were defined as samples that 993 were closest to the median frequency. Quantification of (B) the frequency of PAN RNA+ 994 cells and (C) PAN RNA median fluorescence within PAN RNA+ cells, comparing 995 untreated or stimulated BCBL-1 cells. D, E) Flow cytometric analysis of untreated 996 BCBL-1 cells, using histogram overlays, to compare cell size (forward scatter, FSC), 997 granularity (side scatter, SSC) and ORF73 expression in cells that were either PAN 998 RNA negative [-] (blue line) or PAN RNA positive [+] (red line). Data show (D) histogram 999 overlays of these populations with fluorescence quantification provided in panel E. 1000 ORF73 analysis includes samples in which there was no ORF73 probe (i.e. “No Probe”, 1001 in solid gray), to define background fluorescence. (F, G) Flow cytometric analysis of 1002 stimulated BCBL-1 cells, using histogram overlays, to compare cell size (forward 1003 scatter, FSC), granularity (side scatter, SSC) and ORF73 expression in cells that are 1004 either PAN RNA negative [-] (blue) or PAN RNA positive [+] (red). Data show (F) 1005 histogram overlays of these populations with fluorescence quantification provided in 1006 panel G, with ORF73 analysis including a “No Probe” sample (gray) to define 1007 background fluorescence. Figure 3. Single-cell analysis of KSHV PAN RNA expression in the BCBL-1 B cell 987 It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint BL41 control (n = 3), BCBL-1 untreated (n = 6), BCBL-1 stimulated (n = 6). Statistical 1014 analysis was done using an unpaired t test with statistically significant differences as 1015 indicated, * p<0.05, ** p<0.01, **** p<0.0001. 1016 1017 Figure 4. Single-cell analysis of EBV EBER expression in the Mutu I B cell 1018 lymphoma cell line. (A) Flow cytometric analysis using the PrimeFlowTM method, to 1019 quantify EBER expression in multiple conditions, from cells incubated with no probe 1020 (left), or cells subjected to hybridization using a probe for the EBERs, comparing virus- 1021 negative BL41 cells (second from left) with Mutu I EBV+ cells that were either untreated 1022 (with DMSO) or stimulated (with TPA in DMSO) for 48 hours. Data depict lymphocytes 1023 that were singlets, defined by sequential removal of doublets. Representative images 1024 were defined as samples that were closest to the median frequency, with data depicting 1025 mean ± SEM. (B) Quantitation of median EBER fluorescence within EBER+ Mutu I cells 1026 in untreated and stimulated cultures. Horizontal dashed line indicates the background 1027 fluorescent signal from BL41 controls. Data are from two independent experiments, with 1028 biological replicates within each experiment, with total biological replicates as follows: 1029 No Probe (n = 2), BL41 Control untreated (n = 6), Mutu I untreated (n = 6), Mutu I 1030 stimulated (n = 6). Statistical analysis was done using an unpaired t test with statistically 1031 significant differences as indicated, * p<0.05. 1032 1033 BL41 control (n = 3), BCBL-1 untreated (n = 6), BCBL-1 stimulated (n = 6). Statistical 1014 analysis was done using an unpaired t test with statistically significant differences as 1015 indicated, * p<0.05, ** p<0.01, **** p<0.0001. 1016 gure 4. Single-cell analysis of EBV EBER expression in the Mutu I B ce Figure 4. Single cell analysis of EBV EBER expression in the Mutu I B cell 1018 lymphoma cell line. (A) Flow cytometric analysis using the PrimeFlowTM method, to 1019 quantify EBER expression in multiple conditions, from cells incubated with no probe 1020 (left), or cells subjected to hybridization using a probe for the EBERs, comparing virus- 1021 negative BL41 cells (second from left) with Mutu I EBV+ cells that were either untreated 1022 (with DMSO) or stimulated (with TPA in DMSO) for 48 hours. Data depict lymphocytes 1023 that were singlets, defined by sequential removal of doublets. Representative images 1024 were defined as samples that were closest to the median frequency, with data depicting 1025 mean ± SEM. (B) Quantitation of median EBER fluorescence within EBER+ Mutu I cells 1026 in untreated and stimulated cultures. Horizontal dashed line indicates the background 1027 fluorescent signal from BL41 controls. Data are from two independent experiments, with 1028 biological replicates within each experiment, with total biological replicates as follows: 1029 No Probe (n = 2), BL41 Control untreated (n = 6), Mutu I untreated (n = 6), Mutu I 1030 stimulated (n = 6). Statistical analysis was done using an unpaired t test with statistically 1031 significant differences as indicated, * p<0.05. 1032 Figure 3. Single-cell analysis of KSHV PAN RNA expression in the BCBL-1 B cell 987 Due to variable baseline fluorescence values for SSC and 1008 ORF73 between experiments, values were internally standardized to fluorescent 1009 intensities within the PAN RNA negative population for each experiment, with data 1010 depicting mean ± SEM. Symbols in panels B and C indicate values from individual 1011 samples. Data are from two independent experiments, with biological replicates within 1012 each experiment, with total number of biological replicates as follows: No Probe (n = 2), 1013 KSHV+ BCBL-1 cells that were either untreated or stimulated (with TPA and sodium 991 butyrate (NaB)) for 72 hours. Data depict lymphocytes that were singlets, defined by 992 sequential removal of doublets. Representative images were defined as samples that 993 were closest to the median frequency. Quantification of (B) the frequency of PAN RNA+ 994 cells and (C) PAN RNA median fluorescence within PAN RNA+ cells, comparing 995 untreated or stimulated BCBL-1 cells. D, E) Flow cytometric analysis of untreated 996 BCBL-1 cells, using histogram overlays, to compare cell size (forward scatter, FSC), 997 granularity (side scatter, SSC) and ORF73 expression in cells that were either PAN 998 RNA negative [-] (blue line) or PAN RNA positive [+] (red line). Data show (D) histogram 999 overlays of these populations with fluorescence quantification provided in panel E. 1000 ORF73 analysis includes samples in which there was no ORF73 probe (i.e. “No Probe”, 1001 in solid gray), to define background fluorescence. (F, G) Flow cytometric analysis of 1002 stimulated BCBL-1 cells, using histogram overlays, to compare cell size (forward 1003 scatter, FSC), granularity (side scatter, SSC) and ORF73 expression in cells that are 1004 either PAN RNA negative [-] (blue) or PAN RNA positive [+] (red). Data show (F) 1005 histogram overlays of these populations with fluorescence quantification provided in 1006 panel G, with ORF73 analysis including a “No Probe” sample (gray) to define 1007 background fluorescence. Due to variable baseline fluorescence values for SSC and 1008 ORF73 between experiments, values were internally standardized to fluorescent 1009 intensities within the PAN RNA negative population for each experiment, with data 1010 depicting mean ± SEM. Symbols in panels B and C indicate values from individual 1011 samples. Data are from two independent experiments, with biological replicates within 1012 46 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Figure 5. Actin mRNA degradation identifies virally-infected cells experiencing 1034 virus-induced host shutoff. Actin mRNA analysis by qRT-PCR (A,D) or by flow 1035 cytometric analysis using PrimeFlowTM (B,C,E-G), comparing cells with variable 1036 virus-induced host shutoff. Actin mRNA analysis by qRT-PCR (A,D) or by flow 1035 cytometric analysis using PrimeFlowTM (B,C,E-G), comparing cells with variable 1036 47 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint infection status. (A) qRT-PCR analysis of beta-actin (Actb) mRNA expression relative to 1037 18s RNA in mock, WT or TMER-TKO infected 3T12 fibroblasts at 18 hpi. (B) 1038 PrimeFlowTM analysis of actin mRNA in 3T12 fibroblasts, either unstained (“No probe”), 1039 mock-infected or infected with WT γHV68 or TMER-TKO at 18 hpi. (C) PrimeFlowTM 1040 analysis of actin mRNA and TMER expression in WT γHV68 infected fibroblasts at 18 1041 hpi. (D) qRT-PCR analysis of beta-actin (Actb) mRNA expression relative to 18s RNA 1042 in A20, virus-negative cells and A20.γHV68 (HE2.1) cells, untreated or stimulated with 1043 TPA for 24 hrs. (E) PrimeFlowTM analysis of actin mRNA and TMER expression in 1044 untreated and stimulated A20.γHV68 cells, with the frequency of TMERhigh actin 1045 mRNAlow cells indicated, based on the gated events. (F,G) Actin mRNA analysis by 1046 PrimeFlowTM using either (F) histogram overlays or (G) quantifying frequencies, 1047 comparing A20.γHV68 cells that were either untreated or stimulated, further stratified by 1048 whether the cells were TMERmid or TMERhigh (using the gating strategy defined in Fig. 1049 2C). All flow cytometry data depict single cells, defined by sequential removal of 1050 doublets. Data are from two-three independent experiments, with biological replicates 1051 within each experiment. Graphs depict the mean ± SEM, with each symbol identifying 1052 data from a single replicate. Statistical analysis was done using one-way ANOVA, 1053 subjected to Tukey’s multiple comparison test (A, D, G), with statistically significant 1054 differences as indicated, ** p<0.01, *** p<0.001, **** p<0.0001. 1055 Figure 6. Heterogeneous viral gene expression at the single-cell level during lytic 1057 replication. Viral, host RNA flow cytometric analysis in γHV68-infected fibroblasts at 16 1058 hpi defined by the PrimeFlowTM method, comparing (A) X-shift clustering analysis and 1059 48 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint (B-E) biaxial gating analysis for the indicated features. (A) Automated, clustering 1060 analysis using the X-Shift algorithm on 10,000 events total, compiled from mock- and 1061 γHV68-infected fibroblasts at 16 hrs pi (1,000 events randomly imported per sample, 1062 mock infected n=4, γHV68-infected n=6) identifies multiple clusters of cells with 1063 differential gene expression (7 clusters, colored distinctly, “Cluster ID”), with these 1064 clusters then depicted for expression of TMERs, ORF73, Actin mRNA, and RCA. Range 1065 of expression is identified for each parameter. (B) Analysis of TMER and ORF73 co- 1066 expression in mock (left), WT γHV68-infected (middle), and TMER-TKO-infected (right) 1067 samples, with gates depicting populations with different gene expression profiles, 1068 defined relative to mock and TMER-TKO infected samples. (C) Color-coded populations 1069 from WT-infected sample in panel B, with each color indicating a different gene 1070 expression profile. (D) Histogram overlays of the five populations identified in panel C 1071 for the indicated parameters. (E) Quantitation of gene expression among the five 1072 populations identified in panel C, using the same color-coding strategy. Data are from 1073 three independent experiments, with each experiment containing biological replicates. 1074 Flow cytometry data shows single cells that are DNA+ (DAPI+). Statistical significance 1075 tested by one-way ANOVA, comparing the mean of TMERhigh ORF73high cells to all 1076 other means, followed by Dunnett’s multiple testing correction. Significance identified as 1077 *** p<0.001, **** p<0.0001. 1078 1079 Figure 7. Lytic replication is characterized by heterogeneous TMER localization 1080 Figure 7. Lytic replication is characterized by heterogeneous TMER localization 1080 and variable penetrance of actin RNA degradation. Figure 6. Heterogeneous viral gene expression at the single-cell level during lytic 1057 Viral, host RNA flow cytometric 1081 analysis in γHV68-infected fibroblasts at 16 hpi defined by the PrimeFlowTM method. (A) 1082 49 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint The frequency of γHV68-infected fibroblasts with TMERs primarily in the nucleus was 1083 quantified by ImageStream, with data showing the frequency of cells in which 1084 TMER:DAPI colocalization (i.e. similarity score) was >1. (B) Images showing brightfield 1085 (BF), TMER, RCA protein (RCA), DAPI, ORF73 and actin mRNA localization, 1086 comparing cells with nuclear TMER localization (left) versus cytoplasmic TMER 1087 localization (right). (C) Analysis of cell subpopulations stratified by TMER and ORF73 1088 expression (defined in Fig. 6C), subjected to the tSNE dimensionality reduction 1089 algorithm. Data and cell populations are derived from the dataset presented in Fig. 6, 1090 showing all DNA+ (DAPI+) single cells (FSC-A, SSC-A) subjected to the tSNE 1091 algorithm. The tSNE algorithm provides each cell with a unique coordinate according to 1092 its expression of Actin mRNA, RCA, ORF73, and TMERs, displayed on a two- 1093 dimensional plot (tSNE1 versus tSNE2). Visualization grid of tSNE plots, with plots 1094 arranged according to marker expression (rows) relative to phenotype of the cellular 1095 population examined (columns). (D) Biaxial analysis of actin RNA versus RCA protein, 1096 among the five populations identified in Fig. 6. Flow cytometry data shows single cells 1097 that are DNA+ (DAPI+). Data in panels A-B from two independent experiments, in panel 1098 C-D from three independent experiments. 1099 Figure 8. Lytic viral gene expression is influenced by target cell type, changes 1101 over time, is enhanced by conditions which promote lytic replication. Fibroblasts 1102 (3T12 cells or mouse embryonic fibroblasts, MEFs) were infected with WT or C-RTA 1103 γHV68 at 5 or 100 PFU/cell, and harvested at either 6 or 16 hpi as indicated. Figure 6. Heterogeneous viral gene expression at the single-cell level during lytic 1057 (A-C) 1104 Analysis of viral gene expression by PrimeFlowTM in either 3T12 or MEFs following WT 1105 Figure 8. Lytic viral gene expression is influenced by target cell type, changes 1101 over time, is enhanced by conditions which promote lytic replication. Fibroblasts 1102 (3T12 cells or mouse embryonic fibroblasts, MEFs) were infected with WT or C-RTA 1103 γHV68 at 5 or 100 PFU/cell, and harvested at either 6 or 16 hpi as indicated. (A-C) 1104 Analysis of viral gene expression by PrimeFlowTM in either 3T12 or MEFs following WT 1105 50 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint γHV68 infection (MOI=5) and harvested at 16 hpi. (A) Analysis of viral gene expression 1106 by PrimeFlowTM, comparing gene expression by histogram overlays between 3T12, WT 1107 MEFs and IFNAR KO MEFs. (B) The frequency of events stratified based on expression 1108 of ORF73 and TMER, or (C) based on all 16 possible combinations of gene expression 1109 with the frequency of events in each population plotted as a heatmap. Data are from 1110 three independent experiments harvested at the indicated time, subjected to fluorescent 1111 barcoding, pooled together and subjected to PrimeFlowTM analysis in a single analysis. 1112 (D-F) 3T12 fibroblasts were infected with WT or C-RTA γHV68 at 5 or 100 PFU/cell, and 1113 harvested at either 6 or 16 hpi as indicated. (D) Analysis of viral gene expression by 1114 PrimeFlowTM, comparing gene expression by histogram overlays between different virus 1115 infection conditions and times post-infection, as indicated. (E) The frequency of events 1116 stratified based on expression of ORF73 and TMER, or (F) based on all 16 possible 1117 combinations of gene expression with the frequency of events in each population 1118 plotted as a heatmap. Data are from three independent experiments harvested at the 1119 indicated time, subjected to fluorescent barcoding, pooled together and subjected to 1120 PrimeFlowTM analysis in a single analysis. Data depict mean values, with SEM plotted in 1121 panels B, D, and heatmap values colored based on the categorical key provided in the 1122 figure. Flow cytometry data depict single cells, defined by sequential removal of 1123 doublets according to SSC-A x SSC-H and FSC-A x FSC-H, after which files were 1124 subjected to debarcoding to identify each of the independent samples in the analysis. 1125 1126 γHV68 infection (MOI=5) and harvested at 16 hpi. Figure 8. Lytic viral gene expression is influenced by target cell type, changes 1101 over time, is enhanced by conditions which promote lytic replication. Fibroblasts 1102 (3T12 cells or mouse embryonic fibroblasts, MEFs) were infected with WT or C-RTA 1103 γHV68 at 5 or 100 PFU/cell, and harvested at either 6 or 16 hpi as indicated. (A-C) 1104 Analysis of viral gene expression by PrimeFlowTM in either 3T12 or MEFs following WT 1105 (A) Analysis of viral gene expression 1106 by PrimeFlowTM, comparing gene expression by histogram overlays between 3T12, WT 1107 MEFs and IFNAR KO MEFs. (B) The frequency of events stratified based on expression 1108 of ORF73 and TMER, or (C) based on all 16 possible combinations of gene expression 1109 with the frequency of events in each population plotted as a heatmap. Data are from 1110 three independent experiments harvested at the indicated time, subjected to fluorescent 1111 barcoding, pooled together and subjected to PrimeFlowTM analysis in a single analysis. 1112 (D-F) 3T12 fibroblasts were infected with WT or C-RTA γHV68 at 5 or 100 PFU/cell, and 1113 harvested at either 6 or 16 hpi as indicated. (D) Analysis of viral gene expression by 1114 PrimeFlowTM, comparing gene expression by histogram overlays between different virus 1115 infection conditions and times post-infection, as indicated. (E) The frequency of events 1116 stratified based on expression of ORF73 and TMER, or (F) based on all 16 possible 1117 combinations of gene expression with the frequency of events in each population 1118 plotted as a heatmap. Data are from three independent experiments harvested at the 1119 indicated time, subjected to fluorescent barcoding, pooled together and subjected to 1120 PrimeFlowTM analysis in a single analysis. Data depict mean values, with SEM plotted in 1121 panels B, D, and heatmap values colored based on the categorical key provided in the 1122 figure. Flow cytometry data depict single cells, defined by sequential removal of 1123 doublets according to SSC-A x SSC-H and FSC-A x FSC-H, after which files were 1124 subjected to debarcoding to identify each of the independent samples in the analysis. 1125 51 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint SUPPORTING INFORMATION LEGENDS 1128 1129 Supplemental Table 1. Viral and host probes fo 1130 1131 Supplemental Table 2. qPCR primer sets. 1132 1133 Supplemental Table 3. TaqMan PCR probe sets 1134 1135 Supplemental Figure 1. Analysis of KSHV geno 1136 untreated or stimulated. Data depict qPCR analy 1137 quantified using PCR primers for the KSHV gene 5 1138 calculated relative to untreated BCBL-1 cells, using 1139 defined using 100 ng DNA. Figure 8. Lytic viral gene expression is influenced by target cell type, changes 1101 over time, is enhanced by conditions which promote lytic replication. Fibroblasts 1102 (3T12 cells or mouse embryonic fibroblasts, MEFs) were infected with WT or C-RTA 1103 γHV68 at 5 or 100 PFU/cell, and harvested at either 6 or 16 hpi as indicated. (A-C) 1104 Analysis of viral gene expression by PrimeFlowTM in either 3T12 or MEFs following WT 1105 Data compiled from 3 i 1140 cultures, with 3 biological replicates per experimen 1141 a single experiment, with 3 biological replicates. D 1142 individual biological replicates depicted using indiv 1143 the difference between untreated and stimulated s 1144 test, with Welch’s correction, with statistical signific 1145 1146 Supplemental Figure 2. Analysis of EBER expr 1147 of human B cell lines. (A) Comparison of fluores 1148 (EBV+) cells, where cell lines were either incubate 1149 The three populations identified on these plots, ind 1150 SUPPORTING INFORMATION LEGENDS 1128 1129 Supplemental Table 1. Viral and host probes for PrimeFlowTM analysis. 1130 1131 Supplemental Table 2. qPCR primer sets. 1132 1133 Supplemental Table 3. TaqMan PCR probe sets used in this study. 1134 1135 Supplemental Figure 1. Analysis of KSHV genomic DNA in BCBL-1 cells either 1136 untreated or stimulated. Data depict qPCR analysis of KSHV genomic DNA, 1137 quantified using PCR primers for the KSHV gene 50. Fold change in KSHV DNA was 1138 calculated relative to untreated BCBL-1 cells, using ΔCt normalized to mass unit, 1139 defined using 100 ng DNA. Data compiled from 3 independent experiments for BCBL-1 1140 cultures, with 3 biological replicates per experiment. Data from BL-41 cells derived from 1141 a single experiment, with 3 biological replicates. Data depict mean +/- SEM, with 1142 individual biological replicates depicted using individual symbols. Statistical analysis of 1143 the difference between untreated and stimulated samples was done using an unpaired t 1144 test, with Welch’s correction, with statistical significance defined by ***, p<0.001. 1145 1146 Supplemental Figure 2. Analysis of EBER expression by PrimeFlowTM in a panel 1147 of human B cell lines. (A) Comparison of fluorescence in BL41 (EBV-) and Mutu I 1148 (EBV ) ll h ll li i h i b d i h b EBER b 1149 Supplemental Table 2. qPCR primer sets. 1132 Supplemental Table 3. TaqMan PCR probe sets used in this study. 1134 1135 Supplemental Figure 1. Analysis of KSHV genomic DNA in BCBL-1 cells either 1136 untreated or stimulated. Data depict qPCR analysis of KSHV genomic DNA, 1137 quantified using PCR primers for the KSHV gene 50. Fold change in KSHV DNA was 1138 calculated relative to untreated BCBL-1 cells, using ΔCt normalized to mass unit, 1139 defined using 100 ng DNA. Data compiled from 3 independent experiments for BCBL-1 1140 cultures, with 3 biological replicates per experiment. Data from BL-41 cells derived from 1141 a single experiment, with 3 biological replicates. Data depict mean +/- SEM, with 1142 individual biological replicates depicted using individual symbols. Statistical analysis of 1143 the difference between untreated and stimulated samples was done using an unpaired t 1144 test, with Welch’s correction, with statistical significance defined by ***, p<0.001. 1145 1146 Supplemental Figure 2. Analysis of EBER expression by PrimeFlowTM in a panel 1147 of human B cell lines. (A) Comparison of fluorescence in BL41 (EBV-) and Mutu I 1148 (EBV+) cells, where cell lines were either incubated with no probe or an EBER probe. 1149 The three populations identified on these plots, indicated by 3 polygons with black lines, 1150 52 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint correspond to: left, the true negative population; middle, background fluorescence 1151 observed in BL41 cells stained with EBER probes; right, EBER+ events defined by 1152 expression above these two different thresholds. (B) Quantitation of the frequency of 1153 EBER+ events among viable cells across all the cell lines analyzed, with symbols 1154 depicting individual replicate data, bars showing mean ± SEM. (C) Analysis of EBER 1155 expression in three different LCL cultures, as indicated, comparing background 1156 fluorescence (No probe) with fluorescence following EBER probe hybridization. (D) 1157 Gating hierarchy to analyze EBER fluorescence in viable cells. Supplemental Table 3. TaqMan PCR probe sets used in this study. 1134 1135 All flow cytometry plots 1158 show events defined as lymphocytes by forward and side scatter, doublet 1159 discrimination, and viable cells defined by exclusion of a viability dye. Data are from a 1160 single experiment with one to three biological replicates (n=1 Mutu I, n=3 for BL41 and 1161 LCLs). 1162 1163 Supplemental Figure 3. Analysis of EBER expression by PrimeFlowTM in human 1164 primary B cells subjected to in vitro EBV infection. (A) Comparison of EBER 1165 expression in human primary B cells subjected to either mock or EBV infection (10 1166 genome copies/cell) for 5 days. Cells were either incubated with a cocktail of antibodies 1167 and the EBER probe, or with specific exclusion of the EBER probe (i.e. a full minus one 1168 control, No probe). Data depict the frequency of EBER+ events within lymphocytes that 1169 were singlets and CD19+ B cells. Data are from a single experiment. (B) Comparison of 1170 EBER expression in human primary B cells subjected to either mock or EBV infection 1171 (10 genome copies/cell) for 5 days. Data depict the frequency of EBER+ events within 1172 lymphocytes that were viable, singlets, and CD19+ B cells. (C) Quantitation of the 1173 correspond to: left, the true negative population; middle, background fluorescence 1151 observed in BL41 cells stained with EBER probes; right, EBER+ events defined by 1152 expression above these two different thresholds. (B) Quantitation of the frequency of 1153 EBER+ events among viable cells across all the cell lines analyzed, with symbols 1154 depicting individual replicate data, bars showing mean ± SEM. (C) Analysis of EBER 1155 expression in three different LCL cultures, as indicated, comparing background 1156 fluorescence (No probe) with fluorescence following EBER probe hybridization. (D) 1157 Gating hierarchy to analyze EBER fluorescence in viable cells. All flow cytometry plots 1158 show events defined as lymphocytes by forward and side scatter, doublet 1159 discrimination, and viable cells defined by exclusion of a viability dye. Data are from a 1160 single experiment with one to three biological replicates (n=1 Mutu I, n=3 for BL41 and 1161 LCLs). 1162 Supplemental Figure 3. Analysis of EBER expression by PrimeFlowTM in human 1164 primary B cells subjected to in vitro EBV infection. (A) Comparison of EBER 1165 expression in human primary B cells subjected to either mock or EBV infection (10 1166 genome copies/cell) for 5 days. Supplemental Table 3. TaqMan PCR probe sets used in this study. 1134 1135 NIH 3T12 cells were either 1189 mock or virus infected (MOI=5), harvested at 16 hpi, collected in bulk (mock and 1190 infected, no sort) or subject to FACS purification based on expression of TMER and 73 1191 expression to isolate TMER- 73-, TMER+ 73- and TMER high 73 high populations. DNA 1192 was harvested from all samples, subjected to qPCR with primers to the gB gene of 1193 gHV68. DNA content was normalized relative to cellular DNA, defined by parallel 1194 amplification of the NFAT5 gene. Data depict mean ± SEM, with symbols indicating 1195 individual samples Statistical analysis was performed using one-way ANOVA subjected 1196 frequency of EBER+ cells among viable B cells in mock or EBV infected cultures, with 1174 symbols depicting individual replicate data, bars showing mean ± SEM. (D) Comparison 1175 of cell characteristics in EBV infected cells, between EBER- (in black) and EBER+ (in 1176 red) events using histogram overlays, with populations defined in panel B. Each 1177 histogram overlay depicts three biological replicates, comparing expression of the 1178 defined parameter between EBER- and EBER+ populations. 1179 Supplemental Table 3. TaqMan PCR probe sets used in this study. 1134 1135 Cells were either incubated with a cocktail of antibodies 1167 and the EBER probe, or with specific exclusion of the EBER probe (i.e. a full minus one 1168 control, No probe). Data depict the frequency of EBER+ events within lymphocytes that 1169 were singlets and CD19+ B cells. Data are from a single experiment. (B) Comparison of 1170 EBER expression in human primary B cells subjected to either mock or EBV infection 1171 (10 genome copies/cell) for 5 days. Data depict the frequency of EBER+ events within 1172 lymphocytes that were viable, singlets, and CD19+ B cells. (C) Quantitation of the 1173 53 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint frequency of EBER+ cells among viable B cells in mock or EBV infected cultures, with 1174 symbols depicting individual replicate data, bars showing mean ± SEM. (D) Comparison 1175 of cell characteristics in EBV infected cells, between EBER- (in black) and EBER+ (in 1176 red) events using histogram overlays, with populations defined in panel B. Each 1177 histogram overlay depicts three biological replicates, comparing expression of the 1178 defined parameter between EBER- and EBER+ populations. 1179 1180 Supplemental Figure 4. Measurement of viral gene expression heterogeneity by 1181 multiple measures. 3T12 cells were infected with WT γHV68 at an MOI=5 and 1182 analyzed at 16 hpi. (A) Histogram overlays comparing expression of the indicated 1183 parameters between 5 different populations of cells stratified by TMER and ORF73 1184 expression, as defined on the leftmost panel, with data representative of three biological 1185 replicates. (B) Histogram overlays comparing expression of the indicated parameter in 1186 mock (black) or WT γHV68 -infected (red) samples, comparing three biological 1187 replicates with events defined as DAPI+ singlets. (C) Viral DNA as a function of viral 1188 gene expression defined by TMER and 73 expression. Supplemental Figure 4. Measurement of viral gene expression heterogeneity by 1181 Supplemental Figure 4. Measurement of viral gene expression heterogeneity by 1181 multiple measures. 3T12 cells were infected with WT γHV68 at an MOI=5 and 1182 analyzed at 16 hpi. (A) Histogram overlays comparing expression of the indicated 1183 parameters between 5 different populations of cells stratified by TMER and ORF73 1184 expression, as defined on the leftmost panel, with data representative of three biological 1185 replicates. (B) Histogram overlays comparing expression of the indicated parameter in 1186 mock (black) or WT γHV68 -infected (red) samples, comparing three biological 1187 replicates with events defined as DAPI+ singlets. (C) Viral DNA as a function of viral 1188 gene expression defined by TMER and 73 expression. NIH 3T12 cells were either 1189 mock or virus infected (MOI=5), harvested at 16 hpi, collected in bulk (mock and 1190 infected, no sort) or subject to FACS purification based on expression of TMER and 73 1191 expression to isolate TMER- 73-, TMER+ 73- and TMER high 73 high populations. DNA 1192 was harvested from all samples, subjected to qPCR with primers to the gB gene of 1193 gHV68. DNA content was normalized relative to cellular DNA, defined by parallel 1194 amplification of the NFAT5 gene. Data depict mean ± SEM, with symbols indicating 1195 individual samples. Statistical analysis was performed using one-way ANOVA subjected 1196 54 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint to Tukey's multiple comparisons test, comparing viral DNA content between sorted cell 1197 populations, with statistical significance as identified, *** p<0.001. (D) Viral gene 1198 expression during primary lytic replication with gHV68 is relatively comparable between 1199 cells in different stages of the cell cycle stages. Data are from imaging flow cytometry- 1200 based studies, depicting the frequency of cells stratified by expression of TMERs and 1201 ORF73 (as defined in Fig. Supplemental Figure 4. Measurement of viral gene expression heterogeneity by 1181 6D) present among total DAPI+ cells (top row) compared to 1202 cells in either the G0/G1, S or G2/M phase of the cell cycle. Data depict results from 1203 three independent experiments with 8 total replicates, with columns plotting mean ± 1204 SEM. Flow cytometry data depict single cells, defined by sequential removal of 1205 doublets according to SSC-A x SSC-H and FSC-A x FSC-H. 1206 1207 Supplemental Figure 5. tSNE analysis of cell size and granularity as a function of 1208 infection and gene expression status. Data show flow cytometry data using 1209 populations defined in Fig. 6. Data show all DNA+ (DAPI+) single cells (FSC-A, SSC-A) 1210 subjected to the tSNE dimensionality reduction algorithm, depicting relative expression 1211 values for cell size (FSC) and granularity (SSC) in rows relative to the defined cell 1212 populations (columns). The tSNE algorithm provides each cell with a unique coordinate, 1213 displayed on a two-dimensional plot (tSNE1 versus tSNE2), such that FSC and SSC 1214 values within cellular islands can be directly compared to the corresponding cell islands 1215 presented in Fig. 7C. The channel range was locally-defined for each individual and 1216 channel via Cytobank. Flow cytometry data shows single cells that are DNA+ (DAPI+). 1217 Data are from three independent experiments. 1218 to Tukey's multiple comparisons test, comparing viral DNA content between sorted cell 1197 populations, with statistical significance as identified, *** p<0.001. (D) Viral gene 1198 expression during primary lytic replication with gHV68 is relatively comparable between 1199 cells in different stages of the cell cycle stages. Data are from imaging flow cytometry- 1200 based studies, depicting the frequency of cells stratified by expression of TMERs and 1201 ORF73 (as defined in Fig. 6D) present among total DAPI+ cells (top row) compared to 1202 cells in either the G0/G1, S or G2/M phase of the cell cycle. Data depict results from 1203 three independent experiments with 8 total replicates, with columns plotting mean ± 1204 SEM. Flow cytometry data depict single cells, defined by sequential removal of 1205 doublets according to SSC-A x SSC-H and FSC-A x FSC-H. 1206 Supplemental Figure 5. tSNE analysis of cell size and granularity as a function of 1208 Supplemental Figure 5. tSNE analysis of cell size and granularity as a function of 1208 infection and gene expression status. Data show flow cytometry data using 1209 populations defined in Fig. 6. Data show all DNA+ (DAPI+) single cells (FSC-A, SSC-A) 1210 subjected to the tSNE dimensionality reduction algorithm, depicting relative expression 1211 values for cell size (FSC) and granularity (SSC) in rows relative to the defined cell 1212 populations (columns). The tSNE algorithm provides each cell with a unique coordinate, 1213 displayed on a two-dimensional plot (tSNE1 versus tSNE2), such that FSC and SSC 1214 values within cellular islands can be directly compared to the corresponding cell islands 1215 presented in Fig. 7C. The channel range was locally-defined for each individual and 1216 channel via Cytobank. Flow cytometry data shows single cells that are DNA+ (DAPI+). 1217 Data are from three independent experiments. 1218 1219 55 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint Supplemental Figure 6. Phosphonoacetic acid treatment alters viral gene 1220 expression during lytic replication. 3T12 fibroblasts were infected with WT gHV68 1221 (MOI=5), either in the absence of phosphonoacetic acid (no PAA) or incubated with 1222 PAA (200 mg/mL, +PAA), harvested at 18 hpi and subjected to PrimeFlow analysis. 1223 (A,B) Analysis of TMER and ORF73 expression profiles on a biaxial plot (A), with total 1224 data shown in (B). (C,D) Analysis of TMER and Actin mRNA expression profiles on a 1225 biaxial plot (C), with total data shown in (D). Events were gated on cells subjected to 1226 doublet discrimination. Supplemental Figure 5. tSNE analysis of cell size and granularity as a function of 1208 Data depict mean ± SEM from two independent experiments, 1227 with 4 biological replicates per group denoted by individual symbols. Statistical analysis 1228 done using either a two-way ANOVA with Sidak’s multiple correction test (panel B) or an 1229 unpaired t test (panel D), with statistical significance as identified ****, p<0.0001. 1230 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/482042 doi: bioRxiv preprint lemental Figure 6. Phosphonoacetic acid treatment alters viral gene 56 8 12 16 20 10-3 10-2 10-1 100 101 Time (hpi) TMER5/18s RNA (Fold change) Mock WT γHV68 TMER-TKO 8 12 16 20 10-6 10-5 10-4 10-3 10-2 10-1 100 101 Time (hpi) ORF73/18s RNA (Fold change) Mock WT γHV68 TMER-TKO TMERs-AF647 (Virus small ncRNAs) SSC Mock WT γHV68 TMER-TKO 0.1 0.2 79.8 B. A. C. 0.5 70.6 71.3 ORF73-AF647 (Viral mRNA) Mock WT γHV68 TMER-TKO SSC D. E. 0.2 61.0 Mock WT γHV68 SSC ORF18-AF488 (Viral mRNA) 8 12 16 20 10-3 10-2 10-1 100 101 Time (hpi) TMER5/18s RNA (Fold change) Mock WT γHV68 TMER-TKO TMERs-AF647 (Virus small ncRNAs) SSC Mock WT γHV68 TMER-TKO 0.1 0.2 79.8 B. A. A. B. A. 8 12 16 20 10-6 10-5 10-4 10-3 10-2 10-1 100 101 Time (hpi) ORF73/18s RNA (Fold change) Mock WT γHV68 TMER-TKO (Virus small ncRNAs) C. 0.5 70.6 71.3 ORF73-AF647 (Viral mRNA) Mock WT γHV68 TMER-TKO SSC D. C. 8 D. ( ) E. 0.2 61.0 Mock WT γHV68 SSC ORF18-AF488 (Viral mRNA) E. 0.2 61.0 Mock WT γHV68 SSC ORF18-AF488 (Viral mRNA) E. 0.2 61.0 Mock WT γHV68 SSC ORF18-AF488 (Viral mRNA) E. Figure 1 Oko et al BCBL-1 Untreated ORF73 Granularity (SSC) Cell size (FSC) PAN RNA No Probe PAN RNA [-] PAN RNA [+] F. BCBL-1 Stimulated SSC (Granularity) G. BCBL-1 Stimulated Untreated Stimulated 0 1 2 10 15 20 25 30 % of PAN RNA+ **** E. BCBL-1 Untreated C. Untreated Stimulated 1 10 100 1000 10000 PAN Median Fluorescence among PAN+ cells - + 0 20 40 60 500 1000 1500 2000 PAN RNA (Median) PAN RNA: ** - + 0 50 100 150 200 % SSC (relative to PAN RNA [-] events) PAN RNA: - + 0 20000 40000 60000 80000 FSC (Median) PAN RNA: 40000 60000 80000 C (Median) ** 100 150 200 C (relative to NA [-] events) 60 500 1000 1500 2000 NA (Median) **** - + 0 100 200 300 % ORF73 (relative to PAN RNA [-] events) PAN RNA: ** 100 200 300 73 (relative to NA [-] events) * A. B. PAN RNA - AF488 SSC (Granularity) 99.7 0.32 0.99 0.068 98.0 2.02 71.6 28.3 No probe BL41 Control (KSHV- EBV-) Untreated BCBL-1 (KSHV+) Untreated BCBL-1 (KSHV+) Stimulated AF488 ORF73 Cell size (FSC) Granularity (SSC) PAN RNA No Probe PAN RNA [-] PAN RNA [+] D. BCBL-1 Untreated SSC (Granularity) Untreated Stimulated 0 1 2 10 15 20 25 30 % of PAN RNA+ **** E. BCBL-1 Untreated C. Untreated Stimulated 1 10 100 1000 10000 PAN Median Fluorescence among PAN+ cells A. PAN RNA - AF488 SSC (Granularity) 99.7 0.32 0.99 0.068 98.0 2.02 71.6 28.3 No probe BL41 Control (KSHV- EBV-) Untreated BCBL-1 (KSHV+) Untreated BCBL-1 (KSHV+) Stimulated AF488 SSC (Granularity) A. B. Untreated Stimulated 0 1 2 10 15 20 25 30 % of PAN RNA+ **** C. Untreated Stimulated 1 10 100 1000 10000 PAN Median Fluorescence among PAN+ cells Figure 3 Oko et a B. ORF73 Cell size (FSC) Granularity (SSC) PAN RNA No Probe PAN RNA [-] PAN RNA [+] D. BCBL-1 Untreated ORF73 Granularity (SSC) Cell size (FSC) PAN RNA No Probe PAN RNA [-] PAN RNA [+] F. BCBL-1 Stimulated G. BCBL-1 Stimulated Untreated Stimulated 0 1 2 10 15 20 25 30 % of PAN RNA+ **** E. BCBL-1 Untreated C. Figure 1 Oko et al Figure 1 Oko et al E. TMER Mid TMER Mid TMER High HygroGFP RCA protein A20.γHV68, TPA stimulated A20.γHV68, Untreated D. A20 +TPA (Virus-) A20.γHV68 Untreated A20.γHV68 TPA stimulated No probe TMERs-AF647 SSC 0.89 0.001 0.16 0.007 80.6 0.46 51.2 1.82 G. TMERmid Untreated TMERhigh RCA+ TPA stimulated DAPI TMER RCA BF DAPI TMER RCA BF 0 20 40 60 80 100 % HygroGFP+ **** * TMER: Stimulus: Mid Mid High No + + 0 20 40 60 80 100 % RCA protein+ **** TMER: Stimulus: Mid Mid High No + + F. B. AF647 % of max No probe TMER probe A. C. AF647 SSC Untreated + TPA 0.0 0.5 1.0 1.5 2.0 2.5 % TMER high p=0.0147 0.00 0.02 0.04 0.06 0.08 0.10 TMER5/18s RNA (Relative Expression) * A20 A20.γHV68 Stimulus: - + + - B. AF647 % of max No probe TMER probe A. 0.00 0.02 0.04 0.06 0.08 0.10 TMER5/18s RNA (Relative Expression) * A20 A20.γHV68 Stimulus: - + + - B. AF647 % of max No probe TMER probe A. C. Untreated + TPA 0.0 0.5 1.0 1.5 2.0 2.5 % TMER high p=0.0147 0.00 0.02 0.04 0.06 0.08 0.10 TMER5/18s RNA (Relative Expression) * A20 A20.γHV68 Stimulus: - + + - C. Untreated + TPA 0.0 0.5 1.0 1.5 2.0 2.5 % TMER high p=0.0147 C. B. A. D. A20 +TPA (Virus-) A20.γHV68 Untreated A20.γHV68 TPA stimulated No probe TMERs-AF647 SSC 0.89 0.001 0.16 0.007 80.6 0.46 51.2 1.82 AF647 SSC D. E. E. TMER Mid TMER Mid TMER High HygroGFP RCA protein A20.γHV68, TPA stimu A20.γHV68, Untreated TMERs AF647 AF647 G. TMERmid Untreated TMERhigh RCA+ TPA stimulated DAPI TMER RCA BF DAPI TMER RCA BF h G. TMERmid Untreated TMERhigh RCA+ TPA stimulated DAPI TMER RCA BF DAPI TMER RCA BF 0 20 40 60 80 100 % HygroGFP+ **** * TMER: Stimulus: Mid Mid High No + + 0 20 40 60 80 100 % RCA protein+ **** TMER: Stimulus: Mid Mid High No + + F. G. F. TMERhigh RCA+ TPA stimulated Figure 2 Oko et al Figure 2 Oko et al A. B. PAN RNA - AF488 SSC (Granularity) 99.7 0.32 0.99 0.068 98.0 2.02 71.6 28.3 No probe BL41 Control (KSHV- EBV-) Untreated BCBL-1 (KSHV+) Untreated BCBL-1 (KSHV+) Stimulated AF488 ORF73 Cell size (FSC) Granularity (SSC) PAN RNA No Probe PAN RNA [-] PAN RNA [+] D. Figure 1 Oko et al Untreated Stimulated 1 10 100 1000 10000 PAN Median Fluorescence among PAN+ cells - + 0 20 40 60 500 1000 1500 2000 PAN RNA (Median) PAN RNA: ** - + 0 50 100 150 200 % SSC (relative to PAN RNA [-] events) PAN RNA: - + 0 20000 40000 60000 80000 FSC (Median) PAN RNA: - + 0 20000 40000 60000 80000 FSC (Median) PAN RNA: ** - + 0 50 100 150 200 % SSC (relative to PAN RNA [-] events) PAN RNA: - + 0 20 40 60 500 1000 1500 2000 PAN RNA (Median) PAN RNA: **** - + 0 100 200 300 % ORF73 (relative to PAN RNA [-] events) PAN RNA: ** - + 0 100 200 300 % ORF73 (relative to PAN RNA [-] events) PAN RNA: * B. Untreated Stimulated 0 1 2 10 15 20 25 30 % of PAN RNA+ **** C. Untreated Stimulated 1 10 100 1000 10000 PAN Median Fluorescence among PAN+ cells B. C. ORF73 Cell size (FSC) Granularity (SSC) PAN RNA No Probe PAN RNA [-] PAN RNA [+] D. BCBL-1 Untreated Untreated Stimulated 0 1 % Untreated Stimulated 1 PAN M am ORF73 Cell size (FSC) Granularity (SSC) PAN RNA No Probe PAN RNA [-] PAN RNA [+] D. BCBL-1 Untreated D. E. (FSC) (SSC) E. BCBL-1 Untreated - + 0 20 40 60 500 1000 1500 2000 PAN RNA (Median) PAN RNA: ** - + 0 50 100 150 200 % SSC (relative to PAN RNA [-] events) PAN RNA: - + 0 20000 40000 60000 80000 FSC (Median) PAN RNA: - + 0 100 200 300 % ORF73 (relative to PAN RNA [-] events) PAN RNA: ** F. ORF73 Granularity (SSC) Cell size (FSC) PAN RNA No Probe PAN RNA [-] PAN RNA [+] F. BCBL-1 Stimulated G. Figure 3 Oko et al (SSC) (FSC) G. BCBL-1 Stimulated - + 0 20000 40000 60000 80000 FSC (Median) PAN RNA: ** - + 0 50 100 150 200 % SSC (relative to PAN RNA [-] events) PAN RNA: - + 0 20 40 60 500 1000 1500 2000 PAN RNA (Median) PAN RNA: **** - + 0 100 200 300 % ORF73 (relative to PAN RNA [-] events) PAN RNA: * Figure 3 Oko et al A. Figure 1 Oko et al EBERs – AF647 SSC No probe BL41 Control (KSHV- EBV-) Untreated Mutu I (EBV+) Untreated Mutu I (EBV+) Stimulated AF647 B. 0.05 ± 0.02% 0.047 42.1 ± 8.47% 45.1 ± 10.2% Untreated Stimulated 100 1000 10000 EBER Median Fluorescence among EBER+ cells * A. EBERs – AF647 SSC No probe BL41 Control (KSHV- EBV-) Untreated Mutu I (EBV+) Untreated Mutu I (EBV+) Stimulated AF647 B. 0.05 ± 0.02% 0.047 42.1 ± 8.47% 45.1 ± 10.2% eated lated 100 1000 10000 EBER Median Fluorescence among EBER+ cells * BL41 Control (KSHV- EBV-) Untreated A. SSC No probe AF647 0.047 B. A. B. Figure 4 Oko et al Figure 4 Oko et al Mock WT TMER-TKO 0.0 0.5 1.0 1.5 2.0 Fold change Actin/18s (18 hpi, Relative to Mock) *** ** A. B. No probe Mock WT γHV68 Actin mRNA TMER-TKO TMERs-AF488 Actin mRNA-AF750 C. 36.8 2.7 27.3 33.2 D. TMERs-AF647 E. Actin mRNA AF750 A20.γHV68 Untreated A20.γHV68 Stimulated 0.13 2.11 Actin mRNA TMER Mid TMER Mid TMER High A20.γHV68, Stimulated A20.γHV68, Untreated 0 20 40 60 80 100 % Actin mRNA low **** TMER: Stimulus: Mid Mid High No + + F. G. 0.0 0.1 0.2 0.3 0.4 0.5 Actin/18s RNA (Relative Expression) A20 A20.γHV68 Stimulus: - + + - Mock WT TMER-TKO 0.0 0.5 1.0 1.5 2.0 Fold change Actin/18s (18 hpi, Relative to Mock) *** ** A. Mock WT TMER-TKO 0.0 0.5 1.0 1.5 2.0 Fold change Actin/18s (18 hpi, Relative to Mock) *** ** A. B. No probe Mock WT γHV68 Actin mRNA TMER-TKO TMERs-AF488 Actin mRNA-AF750 C. 36.8 2.7 27.3 33.2 TMERs-AF488 Actin mRNA-AF750 C. 36.8 2.7 27.3 33.2 A. C. B. Actin mRNA Actin mRNA D. TMERs-AF647 E. Actin mRNA AF750 A20.γHV68 Untreated A20.γHV68 Stimulated 0.13 2.11 0.0 0.1 0.2 0.3 0.4 0.5 Actin/18s RNA (Relative Expression) A20 A20.γHV68 Stimulus: - + + - TMERs-AF647 E. Actin mRNA AF750 A20.γHV68 Untreated A20.γHV68 Stimulated 0.13 2.11 68 D. E. Actin mRNA TMER Mid TMER Mid TMER High A20.γHV68, Stimulated A20.γHV68, Untreated 0 20 40 60 80 100 % Actin mRNA low **** TMER: Stimulus: Mid Mid High No + + F. G. G. F. Figure 4 Oko et al Figure 5 Oko et al Figure 5 Oko et al γHV68 Infected Mock #1 #2 #3 #4 #5 #6 #7 Cluster ID: TMERs: 0.22 7.58 TMER- 73- TMER+ 73- TMER- 73+ MER low 73 low ER high 73 high 0 500 1000 1500 2000 2500 TMER (Median) **** TMER- 73- TMER+ 73- TMER- 73+ MER low 73 low ER high 73 high 0 1000 2000 3000 4000 ORF73 (Median) **** TMER- 73- TMER+ 73- TMER- 73+ MER low 73 low ER high 73 high 0 50 100 150 200 250 RCA (Median) *** **** TMER- 73- TMER+ 73- TMER- 73+ MER low 73 low ER high 73 high 0 1000 2000 3000 4000 5000 Actin mRNA (Median) **** TMER- 73- TMER+ 73- TMER- 73+ MER low 73 low R high 73 high 0 25 50 75 100 % FSC (relative to TMER- 73- events) **** TMER- 73- TMER+ 73- TMER- 73+ MER low 73 lo R high 0 25 50 75 100 125 150 % SSC (relative to TMER- 73- events) **** A. B. C. D. E. TMERs-AF488 (Virus ncRNAs) Mock WT γHV68 TMER-TKO ORF73-AF647 (Viral mRNA) TMERs-AF488 (Virus ncRNAs) ORF73-AF647 (Viral mRNA) TMERs ORF73 mRNA RCA Protein Actin mRNA Cell size (FSC) Granularity (SSC) ORF73 mRNA: 0.86 7.80 RCA protein: 0.33 5.74 Actin mRNA: 0.00 8.43 γHV68 Infected Mock #1 #2 #3 #4 #5 #6 #7 Cluster ID: TMERs: 0.22 7.58 A. B. TMERs-AF488 (Virus ncRNAs) Mock WT γHV68 TMER-TKO ORF73-AF647 (Viral mRNA) ORF73 mRNA: 0.86 7.80 RCA protein: 0.33 5.74 Actin mRNA: 0.00 8.43 γHV68 Infected Mock #1 #2 #3 #4 #5 #6 #7 Cluster ID: TMERs: 0.22 7.58 A. B ORF73 mRNA: 0.86 7.80 RCA protein: 0.33 5.74 Actin mRNA: 0.00 8.43 A. Figure 4 Oko et al γHV68 Infected #7 0.22 TMER- 73- TMER+ 73- TMER- 73+ TMER low 73 low TMER high 73 high 0 500 1000 1500 2000 2500 TMER (Median) **** TMER- 73- TMER+ 73- TMER- 73+ TMER low 73 low TMER high 73 high 0 1000 2000 3000 4000 ORF73 (Median) **** TMER- 73- TMER+ 73- TMER- 73+ TMER low 73 low TMER high 73 high 0 50 100 150 200 250 RCA (Median) *** **** TMER- 73- TMER+ 73- TMER- 73+ TMER low 73 low TMER high 73 high 0 1000 2000 3000 4000 5000 Actin mRNA (Median) **** TMER- 73- TMER+ 73- TMER- 73+ TMER low 73 low TMER high 73 high 0 25 50 75 100 % FSC (relative to TMER- 73- events) **** TMER- 73- TMER+ 73- TMER- 73+ TMER low 73 low TMER high 73 high 0 25 50 75 100 125 150 % SSC (relative to TMER- 73- events) **** B. C. D. E. TMERs-AF488 (Virus ncRNAs) Mock WT γHV68 TMER-TKO ORF73-AF647 (Viral mRNA) TMERs-AF488 (Virus ncRNAs) ORF73-AF647 (Viral mRNA) TMERs ORF73 mRNA RCA Protein Actin mRNA Cell size (FSC) Granularity (SSC) 0.86 7.80 p ote 0.33 5.74 0.00 8.43 B. TMERs-AF488 (Virus ncRNAs) Mock WT γHV68 TMER-TKO ORF73-AF647 (Viral mRNA) 0.86 B. TMERs-AF488 (Virus ncRNAs) Mock WT γHV68 TMER-TKO ORF73-AF647 (Viral mRNA) 0.86 0.33 0.00 B. D. TMERs ORF73 mRNA RCA Protein Actin mRNA Cell size (FSC) Granularity (SSC) C. TMERs-AF488 (Virus ncRNAs) ORF73-AF647 (Viral mRNA) D. C. E. TMER- 73- TMER+ 73- TMER- 73+ TMER low 73 low TMER high 73 high 0 500 1000 1500 2000 2500 TMER (Median) **** TMER- 73- TMER+ 73- TMER- 73+ TMER low 73 low TMER high 73 high 0 1000 2000 3000 4000 ORF73 (Median) **** TMER- 73- TMER+ 73- TMER- 73+ TMER low 73 low TMER high 73 high 0 50 100 150 200 250 RCA (Median) *** **** TMER- 73- TMER+ 73- TMER- 73+ TMER low 73 low TMER high 73 high 0 1000 2000 3000 4000 5000 Actin mRNA (Median) **** TMER- 73- TMER+ 73- TMER- 73+ TMER low 73 low TMER high 73 high 0 25 50 75 100 % FSC (relative to TMER- 73- events) **** TMER- 73- TMER+ 73- TMER- 73+ TMER low 73 low TMER high 73 high 0 25 50 75 100 125 150 % SSC (relative to TMER- 73- events) **** E. Figure 4 Oko et al Mock, 6 hpi Mock, 16 hpi WT MOI=5, 6 hpi WT MOI=5, 16 hpi C-RTA MOI=5, 6 hpi C-RTA MOI=5, 16 hpi WT MOI=100, 6 hpi WT MOI=100, 16 hpi ORF73 TMER RCA Actin mRNA - - - + - - - - + - - + + - - - - + - + - + - - - - + + - - + - + + - + + + - - + - + + + - + - - + + + - + + - + + + + + + + - 2-3 # Markers of infection: 0 3-4 1-2 0 - 0.99 1.00 - 4.99 5.00 - 9.99 10.00 - 19.99 20.00 - 49.99 50.00 - 100.00 % of events 0 20 40 60 80 100 % of events ORF73- TMER- ORF73+ TMER- ORF73- TMER+ ORF73+ TMER+ WT MOI=5 WT MOI=100 C-RTA MOI=5 6 Virus: Hours pi: 16 6 16 6 16 Mock, 16 hpi 3T12 MOI=5, 16 hpi WT MEF MOI=5, 16 hpi IFNAR KO MEF MOI=5, 16 hpi ORF73 TMER RCA Actin mRNA - - - + - - - - + - - + + - - - - + - + - + - - - - + + - - + - + + - + + + - - + - + + + - + - - + + + - + + - + + + + + + + - 2-3 # Markers of infection: 0 3-4 1-2 0 - 0.99 1.00 - 4.99 5.00 - 9.99 10.00 - 19.99 20.00 - 49.99 50.00 - 100.00 % of events Figure 8 Oko et al Actin mRNA AF750 B. C. Figure 4 Oko et al ( ) ( ) Figure 6 Oko et al DAPI ORF73 RCA BF TMER Actin mRNA DAPI ORF73 RCA BF TMER Nuclear TMERs Cytoplasmic TMERs A. B. C 0 20 40 60 80 100 % Cells with Nuclear TMER Expt 1 Expt 2 TMER: ORF73: RCA: + + + - - +- + + Actin mRNA A. DAPI ORF73 RCA BF TMER Actin mRNA DAPI ORF73 RCA BF TMER Nuclear TMERs Cytoplasmic TMERs B. Actin mRNA B. ORF73 Actin mRNA TMER DAPI RCA BF Cytoplasmic TMERs C. γHV68 Infected TMER- 73- Input #: 202,669 Mock Infected TMER- 73- Input #: 128,028 TMER+ 73- Input #: 29,096 TMER- 73+ Input #: 5,610 TMER low 73 low Input #: 8,956 TMER high 73 high Input #: 35,850 TMERs Gene 73 mRNA Actin mRNA RCA protein 25 0 18 62 12 40 0 30 10 20 50 0 37 12 25 25 0 18 62 12 C. D. TMER- 73- TMER+ 73- TMER- 73+ TMER low 73 low TMER high 73 high γHV68 Infected TMER- 73- Input #: 202,669 Mock Infected TMER- 73- Input #: 128,028 TMER+ 73- Input #: 29,096 TMER- 73+ Input #: 5,610 TMER low 73 low Input #: 8,956 TMER high 73 high Input #: 35,850 TMERs Gene 73 mRNA in C. γHV68 Infected TMER- 73- Input #: 202,669 Mock Infected TMER- 73- Input #: 128,028 TMER+ 73- Input #: 29,096 TMER- 73+ Input #: 5,610 TMER low 73 low Input #: 8,956 TMER high 73 high Input #: 35,850 TMERs Gene 73 mRNA Actin mRNA RCA protein 250 0 187 625 125 400 0 300 100 200 500 0 375 125 250 250 0 187 62.5 125 Figure 7 Ok t l C. D. TMER- 73- TMER+ 73- TMER- 73+ TMER low 73 low TMER high 73 high RCA-PE Actin mRNA AF750 TMER low 73 low Input #: 8,956 TMER high 73 high Input #: 35,850 TMERs Gene 73 mRNA RCA protein Actin mRNA D. TMER- 73- TMER+ 73- TMER- 73+ TMER low 73 low TMER high 73 high RCA-PE Actin mRNA AF750 D. TMER high 73 high Figure 7 Oko et al Figure 7 Oko et al 3T12 cell line WT MEFs IFNAR KO MEFs Cell type Infection Mock Virus Mock Virus Mock Virus A. B. C. Figure 4 Oko et al ORF73 AF647 TMER AF488 RCA PE Actin mRNA AF750 0 20 40 60 80 100 % of events ORF73- TMER- ORF73+ TMER- ORF73- TMER+ ORF73+ TMER+ 3T12 IFNAR KO WT MEFs E. D. Mock WT gHV68, MOI=5 C-RTA gHV68, MOI=5 WT gHV68, MOI=100 Infection ORF73 AF647 TMER AF488 RCA PE Actin mRNA AF750 Time (hpi) 6 16 6 16 6 16 6 16 F. Mock, 6 hpi Mock, 16 hpi WT MOI=5, 6 hpi WT MOI=5, 16 hpi C-RTA MOI=5, 6 hpi C-RTA MOI=5, 16 hpi WT MOI=100, 6 hpi WT MOI=100 16 hpi 2-3 # Markers of infection: 0 3-4 1-2 0 - 0.99 1.00 - 4.99 5.00 - 9.99 10.00 - 19.99 20.00 - 49.99 50.00 - 100.00 % of events 0 20 40 60 80 100 % of events ORF73- TMER- ORF73+ TMER- ORF73- TMER+ ORF73+ TMER+ Mock, 16 hpi 3T12 MOI=5, 16 hpi WT MEF MOI=5, 16 hpi IFNAR KO MEF MOI=5, 16 hpi ORF73 TMER RCA Actin mRNA - - - + - - - - + - - + + - - - - + - + - + - - - - + + - - + - + + - + + + - - + - + + + - + - - + + + - + + - + + + + + + + - 2-3 # Markers of infection: 0 3-4 1-2 0 - 0.99 1.00 - 4.99 5.00 - 9.99 10.00 - 19.99 20.00 - 49.99 50.00 - 100.00 % of events 3T12 cell line WT MEFs IFNAR KO MEFs Cell type Infection Mock Virus Mock Virus Mock Virus A. ORF73 AF647 TMER AF488 RCA PE Actin mRNA AF750 A. WT MEFs IFNAR KO MEFs Mock Virus Mock Virus B. C. ORF73 AF647 TMER AF488 RCA PE Actin mRNA AF750 0 20 40 60 80 100 % of events ORF73- TMER- ORF73+ TMER- ORF73- TMER+ ORF73+ TMER+ 3T12 IFNAR KO WT MEFs E. D. Mock WT gHV68, MOI=5 C-RTA gHV68, MOI=5 WT gHV68, MOI=100 Infection ORF73 AF647 TMER AF488 RCA PE Actin mRNA AF750 Time (hpi) 6 16 6 16 6 16 6 16 F. Figure 4 Oko et al 0 20 40 60 80 100 % of events ORF73- TMER- ORF73+ TMER- ORF73- TMER+ ORF73+ TMER+ 3T12 IFNAR KO WT Mock, 16 hpi 3T12 MOI=5, 16 hpi WT MEF MOI=5, 16 hpi IFNAR KO MEF MOI=5, 16 hpi ORF73 TMER RCA Actin mRNA - - - + - - - - + - - + + - - - - + - + - + - - - - + + - - + - + + - + + + - - + - + + + - + - - + + + - + + - + + + + + + + - 2-3 # Markers of infection: 0 3-4 1-2 0 - 0.99 1.00 - 4.99 5.00 - 9.99 10.00 - 19.99 20.00 - 49.99 50.00 - 100.00 % of events B. 0 20 40 60 80 100 % of events ORF73- TMER- ORF73+ TMER- ORF73- TMER+ ORF73+ TMER+ 3T12 IFNAR KO WT MEFs B. C. D. Mock WT gHV68, MOI=5 C-RTA gHV68, MOI=5 WT gHV68, MOI=100 Infection ORF73 AF647 TMER AF488 RCA PE Actin mRNA AF750 Time (hpi) 6 16 6 16 6 16 6 16 D. Actin mRNA AF750 E. F. 0 20 40 60 80 100 % of events ORF73- TMER- ORF73+ TMER- ORF73- TMER+ ORF73+ TMER+ WT MOI=5 WT MOI=100 C-RTA MOI=5 6 Virus: Hours pi: 16 6 16 6 16 Mock, 6 hpi Mock, 16 hpi WT MOI=5, 6 hpi WT MOI=5, 16 hpi C-RTA MOI=5, 6 hpi C-RTA MOI=5, 16 hpi WT MOI=100, 6 hpi WT MOI=100, 16 hpi ORF73 TMER RCA Actin mRNA - - - + - - - - + - - + + - - - - + - + - + - - - - + + - - + - + + - + + + - - + - + + + - + - - + + + - + + - + + + + + + + - 2-3 # Markers of infection: 0 3-4 1-2 0 - 0.99 1.00 - 4.99 5.00 - 9.99 10.00 - 19.99 20.00 - 49.99 50.00 - 100.00 % of events F. E. Figure 8 Oko et al
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Natural Selection beyond Life? A Workshop Report
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Natural Selection beyond Life? A Workshop Report Sylvain Charlat, André Ariew, Pierrick Bourrat, María Ferreira Ruiz, Thomas Heams, Philippe Huneman, Sandeep Krishna, Michael Lachmann, Nicolas Lartillot, Louis Le Sergeant d’Hendecourt, et al. To cite this version: Sylvain Charlat, André Ariew, Pierrick Bourrat, María Ferreira Ruiz, Thomas Heams, et al.. Natural Selection beyond Life? A Workshop Report. Life, 2021, 11 (10), pp.1051. ￿10.3390/life11101051￿. ￿hal-03418757￿ Distributed under a Creative Commons Attribution 4.0 International License Sylvain Charlat 1,*, André Ariew 2, Pierrick Bourrat 3,4, María Ferreira Ruiz 5, Thomas Heams 6, Philippe Huneman 7, Sandeep Krishna 8, Michael Lachmann 9, Nicolas Lartillot 1, Louis Le Sergeant d’Hendecourt 10, Christophe Malaterre 11, Philippe Nghe 12, Etienne Rajon 1, Olivier Rivoire 13, Matteo Smerlak 14 and Zorana Zeravcic 15 Natural Selection beyond Life? A Workshop Report. Life 2021, 11, 1051. https://doi.org/10.3390/ life11101051 Citation: Charlat, S.; Ariew, A.; Bourrat, P.; Ferreira Ruiz, M.; Heams, T.; Huneman, P.; Krishna, S.; Lachmann, M.; Lartillot, N.; Le Sergeant d'Hendecourt, L.; et al. Natural Selection beyond Life? A Workshop Report. Life 2021, 11, 1051. https://doi.org/10.3390/ life11101051 Academic Editor: David A. Baum Received: 24 August 2021 Accepted: 29 September 2021 Published: 7 October 2021 g 9 Santa Fe Institute, Santa Fe, NM 87501, USA; lachmann@santafe.edu 10 Centre de St-Jérôme, Laboratoire de Physique des Interactions Ioniques et Moléculaires, Aix-Marseille Université, CNRS, UMR 7345, 13013 Marseille, France; ldh@univ-amu.fr 11 Centre de Recherche Interuniversitaire sur la Science et la Technologie (CIRST), Département de Philosophie, Université du Québec à Montréal (UQAM), 455 Boulevard René-Lévesque Est, Montréal, QC H3C 3P8, Canada; malaterre.christophe@uqam.ca p q 12 Laboratoire Biophysique et Evolution, CNRS UMR Chimie Biologie Innovation 8231, ESPCI Paris, p q 12 Laboratoire Biophysique et Evolution, CNRS UMR Chimie Biologie Innovation 8231, ESPCI Paris, Université PSL, 10 Rue Vauquelin, 75005 Paris, France; philippe.nghe@espci.fr Université PSL, 10 Rue Vauquelin, 75005 Paris, France; philippe.nghe@espci.fr 0 Rue Vauquelin, 75005 Paris, France; philippe.nghe@espci.f 13 Center for Interdisciplinary Research in Biology (CIRB), Collège de France, CNRS, INSERM, Université PSL, 75005 Paris, France; olivier.rivoire@college-de-france.fr 13 Center for Interdisciplinary Research in Biology (CIRB), C 75005 Paris, France; olivier.rivoire@college-de-france.fr 75005 Paris, France; olivier.rivoire@college-de-france.fr 14 Max Planck Institute for Mathematics in the Sciences, Inselstrasse 22, 04103 Leipzig, Germany; msmerlak@perimeterinstitute.ca p 15 Gulliver Lab, CNRS UMR 7083, ESPCI Paris, PSL University, 75005 Paris, France; zorana.zeravcic@espci.fr Received: 24 August 2021 Accepted: 29 September 2021 Published: 7 October 2021 * Correspondence: sylvain.charlat@univ-lyon1.fr Abstract: Natural selection is commonly seen not just as an explanation for adaptive evolution, but as the inevitable consequence of “heritable variation in fitness among individuals”. Although it re- mains embedded in biological concepts, such a formalisation makes it tempting to explore whether this precondition may be met not only in life as we know it, but also in other physical systems. This would imply that these systems are subject to natural selection and may perhaps be investigated in a biological framework, where properties are typically examined in light of their putative functions. Here we relate the major questions that were debated during a three-day workshop devoted to discussing whether natural selection may take place in non-living physical systems. HAL Id: hal-03418757 https://univ-lyon1.hal.science/hal-03418757v1 Submitted on 8 Nov 2021 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 Perspective Sylvain Charlat 1,*, André Ariew 2, Pierrick Bourrat 3,4, María Ferreira Ruiz 5, Thomas Heams 6, Philippe Huneman 7, Sandeep Krishna 8, Michael Lachmann 9, Nicolas Lartillot 1, Louis Le Sergeant d’Hendecourt 10, Christophe Malaterre 11, Philippe Nghe 12, Etienne Rajon 1, Olivier Rivoire 13, Matteo Smerlak 14 and Zorana Zeravcic 15 Sylvain Charlat 1,*, André Ariew 2, Pierrick Bourrat 3,4, María Ferreira Ruiz 5, Thomas Heams 6, Philippe Huneman 7, Sandeep Krishna 8, Michael Lachmann 9, Nicolas Lartillot 1, Louis Le Sergeant d’Hendecourt 10, Christophe Malaterre 11, Philippe Nghe 12, Etienne Rajon 1, Olivier Rivoire 13, Matteo Smerlak 14 and Zorana Zeravcic 15 1 Laboratoire de Biométrie et Biologie Évolutive, Université de Lyon, Université Lyon 1, CNRS, UMR 5558, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne, France; nicolas.lartillot@univ-lyon1.fr (N.L.); Etienne.Rajon@univ-lyon1.fr (E.R.) 1 Laboratoire de Biométrie et Biologie Évolutive, Université de Lyon, Université Lyon 1, CNRS, UMR 5558, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne, France; nicolas.lartillot@univ-lyon1.fr (N.L.); Etienne.Rajon@univ-lyon1.fr (E.R.) j y 2 Department of Philosophy, University of Missouri, 438 Strickland Hall, Columbia, MO 65211, USA; ariewa@missouri.edu 3 Department of Philosophy, Macquarie University, Balaclava Road, North Ryde, NSW 2109, Australia; pierrick.bourrat@mq.edu.au 4 Charles Perkins Centre, Department of Philosophy, The University of Sydney, Camperdown, NSW 2006, Australia 5 Department of Philosophy, University of Bielefeld, 33615 Bielefeld, Germany; mariaferreiraruiz@gmail.com p p y y y g 6 INRAE, Domaine de Vilvert Bâtiment 211, 78352 Jouy-en-Josas, France; thomas.heams@agroparistech.fr 6 INRAE, Domaine de Vilvert Bâtiment 211, 78352 Jouy-en-Josas, France; thomas.heams@agroparistech.fr 7 Institut d’Histoire et de Philosophie des Sciences et des Techniques, CNRS (Centre National de la Recherche Scientifique), Université Paris I Sorbonne, 13 Rue du Four, 75006 Paris, France; philippe.huneman@gmail.com 7 Institut d’Histoire et de Philosophie des Sciences et des Techniques, CNRS (Centre National de la Recherche Scientifique), Université Paris I Sorbonne, 13 Rue du Four, 75006 Paris, France; philippe.huneman@gmail.com 8 Simons Centre for the Study of Living Machines, National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560065, India; sandeep@ncbs.res.in Citation: Charlat, S.; Ariew, A.; Bourrat, P.; Ferreira Ruiz, M.; Heams, T.; Huneman, P.; Krishna, S.; Lachmann, M.; Lartillot, N.; Le Sergeant d'Hendecourt, L.; et al. Natural Selection beyond Life? A Workshop Report. Life 2021, 11, 1051. https://doi.org/10.3390/ life11101051 Academic Editor: David A. Baum Received: 24 August 2021 Accepted: 29 September 2021 Published: 7 October 2021 Citation: Charlat, S.; Ariew, A.; Bourrat, P.; Ferreira Ruiz, M.; Heams, T.; Huneman, P.; Krishna, S.; Lachmann, M.; Lartillot, N.; Le Sergeant d'Hendecourt, L.; et al. Natural Selection beyond Life? A Workshop Report. Life 2021, 11, 1051. https://doi.org/10.3390/ life11101051 Citation: Charlat, S.; Ariew, A.; Bourrat, P.; Ferreira Ruiz, M.; Heams, T.; Huneman, P.; Krishna, S.; Lachmann, M.; Lartillot, N.; Le Sergeant d'Hendecourt, L.; et al. 1. Introduction: Why Investigate “Natural Selection Beyond Life”? The principle of natural selection occupies a central role in biology: explaining why living organisms harbour properties apparently fitted to particular functions, and thus denoted as “adaptive”. In doing so, it provides a non-finalistic justification for “functional thinking” [1,2]; a typically biological mode of inquiry where structures, or more generally features, are investigated in light of their observed or putative effects, in interrelations with others, with which they form a functioning “whole”, the organism. Within the stand- ard evolutionary framework, the process of natural selection is commonly conceived as the inevitable consequence of necessary and sufficient preconditions, namely “heritable variation in fitness related traits” [3] (provided it is not overwhelmed by random events). Such a formulation naturally leads one to wonder whether non biological systems may also fulfil these conditions. In turn, such an interrogation constitutes an occasion to revisit whether evolution by natural selection necessarily produces features that can be qualified as functions, that is, whether functional thinking becomes a relevant mode of inquiry whenever the conditions for natural selection are fulfilled. Life-derived objects not typically seen as “living”, such as words, ideas or computer programs, are nevertheless endowed with some kind of self-replicating ability, and thus stand as obvious candidates for “evolution by natural selection” outside of biology. Ac- cordingly, the principle of natural selection made its way into linguistics, cultural evolu- tion and computer sciences [4,5]. The potential relevance of natural selection to physical sciences is a priori less obvious: since living entities are part of the physical world, con- cepts from the physical sciences commonly flow into biology, but the reverse flow would be unusual. Perhaps unusual but not logically impossible insofar as concepts may flow between scientific disciplines without respecting the natural hierarchy of their objects. If the a priori objection that natural selection cannot be relevant to physical sciences is thus ruled out, the question of whether it is remains largely unexplored. This issue was at the centre stage of a workshop held in November 2019, that gath- ered a group of evolutionary biologists, chemists, physicists, and philosophers of science. Here we relate the major questions that were debated in this context. The article is struc- tured as follows. We first briefly describe the research objects of connected fields, from synthetic biology to the origins of life, that we take as starting points for considering nat- ural selection at the edge of biology. Sylvain Charlat 1,*, André Ariew 2, Pierrick Bourrat 3,4, María Ferreira Ruiz 5, Thomas Heams 6, Philippe Huneman 7, Sandeep Krishna 8, Michael Lachmann 9, Nicolas Lartillot 1, Louis Le Sergeant d’Hendecourt 10, Christophe Malaterre 11, Philippe Nghe 12, Etienne Rajon 1, Olivier Rivoire 13, Matteo Smerlak 14 and Zorana Zeravcic 15 Overall, these discussions thus lead us to conjecture that understanding, in physico-chemical terms, how individuality emerges and how it can be recognised, will be essential in the search for instances of evolution by natural selection outside of living systems. Keywords: natural selection; individuality; levels of selection; evolutionary biology; physics; phi- losophy of biology; exobiology; origins of life Sylvain Charlat 1,*, André Ariew 2, Pierrick Bourrat 3,4, María Ferreira Ruiz 5, Thomas Heams 6, Philippe Huneman 7, Sandeep Krishna 8, Michael Lachmann 9, Nicolas Lartillot 1, Louis Le Sergeant d’Hendecourt 10, Christophe Malaterre 11, Philippe Nghe 12, Etienne Rajon 1, Olivier Rivoire 13, Matteo Smerlak 14 and Zorana Zeravcic 15 We start this report with a brief overview of research fields dealing with “life-like” or “proto-biotic” systems, where mimicking evolution by natural selection in test tubes stands as a major objective. We con- tend the challenge may be as much conceptual as technical. Taking the problem from a physical angle, we then discuss the framework of dissipative structures. Although life is viewed in this con- text as a particular case within a larger ensemble of physical phenomena, this approach does not provide general principles from which natural selection can be derived. Turning back to evolution- ary biology, we ask to what extent the most general formulations of the necessary conditions or Abstract: Natural selection is commonly seen not just as an explanation for adaptive evolution, but as the inevitable consequence of “heritable variation in fitness among individuals”. Although it re- mains embedded in biological concepts, such a formalisation makes it tempting to explore whether this precondition may be met not only in life as we know it, but also in other physical systems. This would imply that these systems are subject to natural selection and may perhaps be investigated in a biological framework, where properties are typically examined in light of their putative functions. Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and institu- tional affiliations. Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and con- ditions of the Creative Commons At- tribution (CC BY) license (http://crea- tivecommons.org/licenses/by/4.0/). www.mdpi.com/journal/life Life 2021, 11, 1051. https://doi.org/10.3390/life11101051 Life 2021, 11, 1051 2 of 12 2 of 12 signatures of natural selection may be applicable beyond biology. In our view, such a cross-discipli- nary jump is impeded by reliance on individuality as a central yet implicit and loosely defined con- cept. Overall, these discussions thus lead us to conjecture that understanding, in physico-chemical terms, how individuality emerges and how it can be recognised, will be essential in the search for instances of evolution by natural selection outside of living systems. signatures of natural selection may be applicable beyond biology. In our view, such a cross-discipli- nary jump is impeded by reliance on individuality as a central yet implicit and loosely defined con- cept. 2. Natural Selection in Protobiotic Systems? Although not always stated in such terms, the very possibility of applying the prin- ciple of natural selection to physico-chemical systems is a common theme of several re- search areas lying on the fringe of physical and life sciences: synthetic biology, exobiology, and the origins of life. While pursuing distinct objectives, these fields share a common vast technical and conceptual challenge: bridging the gap between physico-chemical and biological systems, explaining the transition from inanimate to living matter. Darwinian evolution being recognised as an important component of such a transition, many exper- imental and theoretical systems have been designed with the objective of mimicking this process. Such setups originally implemented some form of experimenter-assisted natural selection in the laboratory [6]. This approach led to a now well-established process of “in vitro” or “directed” evolution [7,8]. Yet such settings take for granted recurrent human interventions to handle the core feature of replication, while our focus here is on systems that replicate autonomously and are left to evolve by themselves. Many potential candidates have been designed, falling into partially overlapping broad groups. Some are directly inspired by the template-based replication of nucleic ac- ids seen in extant organisms. Such systems typically follow up from the discovery of nat- urally occurring ribozymes [9,10] and fit in a model where RNA occupies a central role in the emergence of life. The continued search for an RNA-replicase has uncovered increas- ingly powerful ribozymes capable of ligating up to a hundred nucleotides in emulsions, though still short of exhibiting complete self-replication capability [11,12], for a review, see [13]. Other systems take the form of autocatalytic networks and are thus more centred on metabolism as a central feature. Some are based on peptides [14,15] but others involve RNA and remain tightly connected with the RNA-world hypothesis [16–18]. Yet another group of protobiotic replicating systems involves more physical or self-organizing entities such as vesicles, crystals, colloids, or nanotubes [19–21]. Objects from these different cat- egories may also be merged to combine their respective advantages, e.g., [13]. g y g p g g Many of these systems arguably display some form of self-replication. Some varia- tions among replicating entities may also exist but only a limited fraction of them is herit- able. As a result, it remains currently unclear whether such systems should be considered as evolvable by natural selection. 1. Introduction: Why Investigate “Natural Selection Beyond Life”? Experimentally mimicking evolution by natural se- lection in these fields stands as a key objective, that seems hindered not only by technical difficulties, but also by the conceptual challenge of tracking this process, identifying its conditions, and expected outcomes. We then discuss whether natural selection can be sit- uated in the framework of far-from-equilibrium physics, a field that is explicitly aimed at encompassing living systems. Our tentative conclusion is that it cannot, perhaps because natural selection, at least as currently formalised, is at odds with the epistemic status and usage of physical principles. Next, we conversely examine whether some physical systems may be situated in the framework of evolutionary biology and argue that this will require further formalisation of the natural selection principle, to make it portable across disci- plines. In particular, we highlight that natural selection requires “individuals” (i.e., well- identified units) as a precondition although, paradoxically, biological individuals are also Life 2021, 11, 1051 3 of 12 3 of 12 considered as outcomes of this process. We end this report with the conjecture that solving this paradox may be a necessary first step toward the search for natural selection beyond life. This implies understanding how individuality may emerge and perhaps be reinforced in the course of evolution. 3. Natural Selection in the Context of Physical Phenomena Assessing the possibility that natural selection takes place in non-living physical sys- tems first implies positioning living systems, where natural selection is recognized to hap- pen, in the range of physical phenomena. This question has been predominantly exam- ined from a thermodynamic perspective, starting in the 1940s with Schrödinger’s influen- tial book [30]. We discuss more specifically the approach developed in the 1970s by the Brussels school of thermodynamics [31–33], which continues to this date to inspire new works [34,35]. Within this approach, as explained in the following, living systems are viewed as belonging to a larger class of open and far-from-equilibrium systems called dissipative structures, and one seeks a general framework in the form of a variational principle (as defined below), of which natural selection could be a particular case. p p p The term “dissipative structures” designates steady states that display spatial and/or temporal patterns (e.g., inhomogeneous distribution of chemical species, or sustained os- cillations) which typically occur due to an instability from a homogeneous, patternless steady state subject to a small perturbation. Dissipative structures commonly occur in sys- tems which are open (i.e., can exchange matter and energy with the environment), non- linear (in terms of the governing dynamics equations) and far from equilibrium. A canon- ical example arises from the hydrodynamic instability known as the Rayleigh–Bénard convection [36,37]. This instability is observed in a horizontal layer of fluid heated from below. As the temperature difference between the bottom and the top is raised, a thresh- old is reached at which the fluid loses its spatial homogeneity and shows motions orga- nized in patterns. This transition formally corresponds to an instability of the homogene- ous state upon fluctuations (small random variations in density for instance). Depending on the difference of temperatures, the initial and boundary conditions (i.e., spatial and temporal constraints set by the environment), and the protocol followed to raise the tem- perature, one can observe patterns of many different forms, from simple convection cells and rolls to more complicated spatio-temporal structures. Dissipative structures are also found in reaction-diffusion systems in chemistry (Turing patterns, Belousov–Zhabotinsky oscillations), in fluid mechanics (Faraday waves) and in nonlinear optics (light beam mod- ulations). Several biological phenomena have also been studied from the standpoint of dissipative structures, including gene regulation and biological rhythms [38]. 2. Natural Selection in Protobiotic Systems? This may reflect the technical difficulty of designing systems that will effectively display a larger diversity of heritable states. Strikingly, the challenge is also conceptual, to the point that it appears difficult to even imagine theoret- ical systems that would radically differ from the biological paradigm of self-replicating nucleic acids and could yet be considered as evolvable by natural selection [22–24]. Ad- dressing this challenge may require shifting away from a categorical to a continuous per- spective, as previously advocated in the context of the origin of “lifeness” [25–29]: asking, not if these systems are evolvable through natural selection, but to what extent they are, on a quantitative scale that remains to be formalised in cross-disciplinary terms. In the next section, we discuss whether the physical approach to far-from-equilibrium systems may take us closer to that objective. Life 2021, 11, 1051 4 of 12 3. Natural Selection in the Context of Physical Phenomena The over- arching theme in all these examples is that structures can emerge from fluctuations through instabilities when a system is maintained out of equilibrium, for instance by a gradient of temperature or an influx of chemical compounds. From a thermodynamic perspective, living organisms constitute far-from-equilib- rium open systems, but populations of organisms can also be conceived this way, and their evolution through natural selection may then be framed in terms of instability [39]. In particular, introducing a mutant in a previously stable population can be seen as pro- voking an instability in the population dynamics, eventually leading to a new steady state with a completely different structure, that is, a different composition of the population. Furthermore, in numerical simulations of artificial chemistries aiming at modelling the emergence of evolutionary dynamics from elementary physical rules [40,41] one observes instabilities giving rise to various dissipative structures, such as competing catalytic cy- cles. From this standpoint, one may therefore consider biological populations subject to natural selection as particular dissipative structures. A major goal of the Brussels school was to identify a variational principle that gener- ally applies to non-equilibrium steady states, and therefore to dissipative structures. In physics, variational principles take the form of mathematical functions of one or more physical quantities whose minimisation allows one to predict the final state of a system, without reference to its initial conditions or particular dynamics. For example, in equilib- rium thermodynamics, this is achieved by minimizing free energy. Far from equilibrium, however, in systems such as dissipative structures, no such principle has been found [42]. In fact, it is well-established through explicit counter examples that the most likely steady state of a non-equilibrium system can depend on parameters that cannot be estimated Life 2021, 11, 1051 5 of 12 5 of 12 from the immediate vicinity of stable steady states only [43]. Hence, a state-function whose optima (that is, whose minima) indicates which non-equilibrium steady states are favoured cannot be derived. In other words, dissipative structures cannot be generally predicted from summary descriptors of the steady states. A general framework exists for rationalizing a variety of patterns observed in non-equilibrium systems, but it is limited to local stability analyses [42]. In short, one can recognize that a particular steady state is unstable but not generally predict which final state it will reach. From this standpoint, natural selection is not different. 3. Natural Selection in the Context of Physical Phenomena In particular cases, a fitness function can be defined in which the maximum describes the steady state of population dynamics. Many other examples exist, however, where such functions cannot be defined, for example in cases where optimal trait values depend on the composition of the popu- lations, e.g., in systems following dynamics analogous to those of the rock-paper-scissors game [44]. This is unsurprising from the standpoint of general dynamical systems, where no steady state is even generally guaranteed. What non-equilibrium thermodynamics teaches us is that even describing biological systems in a physical framework, as subclasses of dynamical systems (e.g., by invoking physical constraints), would not be sufficient to solve this problem, that is, to understand them using a general variational principle. From the standpoint of its epistemic status and usage, the principle of natural selec- tion is, however, markedly different from variational principles in physics: unlike varia- tional principles in physics, the principle of natural selection is used even in the absence of a precise quantitative description or a well-defined optimum. Trying to subsume natu- ral selection into a variational principle, or reciprocally, may therefore be inappropriate. One may nevertheless wonder if the principle of natural selection, which is so fruitful in biology despite not constituting a valid variational principle from a physical standpoint, can provide comparable insights into physical but non-biological systems. In the follow- ing section, we discuss whether the conditions and signatures of natural selection, as de- picted in the framework of evolutionary biology, can provide hints on what such systems could be. 4. Natural Selection as a Framework We take here two complementary approaches to try and characterise non-living physical systems that may be subject to natural selection. One is to look for the causes of natural selection, its necessary and sufficient conditions. The other is to look for its conse- quences, the patterns it generates. The former may thus be labelled as “causal” or “mech- anistic”, while the latter is more correlative or phenomenological, and will only provide strong evidence for natural selection if it relies on specific and unambiguous signatures. 4.1. The Conditions of Natural Selection If we turn to the notion of “fitness-related traits”, perhaps not encouraging is the acknowledgment that even within biology, reaching a consensual definition of this ex- pression is probably impossible, for fitness is a notoriously slippery term [51–53]. Yet most biologists would probably agree that it relates to survival and/or reproduction, that con- dition one’s own persistence and the number of offspring one may produce. Taking only the “survival” component, the fitness of an object may be regarded as equivalent to its “stability”, a concept that seems readily applicable to any physical entity. Including the “reproduction” component breaks this equivalence. Fitness may then be related to an ex- tended notion of stability that would apply to dynamic structures maintained although they are made of unstable components, that is, to steady states. Yet, proposals to define fitness in physical terms along those lines, including for instance dynamic kinetic stability [34] are subject to the limitations reviewed in Section 3: they cannot provide a general criterion for specifying the steady state that a system will adopt unless their scope of ap- plication is precisely and rigorously circumscribed. The third component of the above-defined Darwinian recipe, individuality, is prob- ably the most central although it is generally kept implicit and thus goes unnoticed: her- itable differences and fitness related traits just cannot be conceived without referring to individuals. Applying the recipe approach to non-biological systems thus requires defin- ing individuality without referring to biological concepts. Clarifying the meaning of this term within biology would probably be a good start, but this task in itself is acknowledged as very challenging [54]. Although central to virtually any biological reasoning, “individ- uals” are perhaps sufficiently evident in many contexts to let biologists live well without defining the underlying concept. Yet many problematic situations can also be found within biology. Vegetative propagation through cutting, or more generally clonal repro- duction, is a situation where many individuals may be seen as a single individual of a higher level [45,51,55]. Obligate symbiotic associations pose similar difficulties. These bor- der-line cases have prompted the development of more rigorous approaches to individu- ality [56–58]. 4.1. The Conditions of Natural Selection Following Lewontin [3] and many subsequent authors, natural selection is often pre- sented as necessarily resulting from heritable differences in fitness-related traits among individuals. This Darwinian recipe provides a starting point for the causal approach, alt- hough, as abstract and general as it may sound to most biologists, it remains very much dependent upon biological concepts. Making it portable across disciplines requires more formal definitions of its components: “heritable differences”, “fitness-related traits” and “individuals”. “Heritable differences”, first, refer to parent-offspring resemblance, and more specif- ically to the fraction of differences seen among individuals that stem from differences among their parents. The concept of inheritance is tightly connected to that of reproduc- tion, although they are not strictly equivalent. Specifically, reproduction may take place without heritable differences if all individuals are identical, but heritable differences, in- sofar as they refer to parents and offspring, imply reproduction. It has been argued that reproduction should not be seen as a necessary ingredient of natural selection [45–48] be- cause mere differences in “survival” among different kinds of entities, if they are persis- tent enough, suffice to induce deterministic changes in their relative abundances over Life 2021, 11, 1051 6 of 12 6 of 12 time. Under this broadened view, natural selection is the biological name for the sorting process taking place, with or without reproduction, in any dynamical systems composed of entities differing in their inherent stability or emergence rates. As discussed in Box 1 using a toy mathematical model (and illustrated in Figure 1), reproduction nevertheless introduces radical changes in the efficiency of this sorting process, opening the possibility of otherwise unlikely cumulative changes. It may thus be recognised that reproduction is a necessary component, if not of natural selection in its most general sense, at least of “cumulative changes through natural selection”, which we take as an equivalent to “evo- lution by natural selection”, and to which we happily restrict our focus. The biological concept of reproduction is equivalent to that of autocatalysis in chemistry [49,50] and can also be related to exponential growth. The latter is essentially a mathematical concept but captures the dynamics of processes involving self-amplification. Yet we note that many physical systems, such as nuclear chain reactions, display exponential dynamics without being reducible to self-amplification of particular entities. In that sense, reproduction and auto-catalysis constitute, not a general equivalent of exponential growth, but rather a par- ticular case. 4.1. The Conditions of Natural Selection In particular, the research fields of “major evolutionary transitions” and “levels of selection” provide us with useful conceptual tools to apprehend this problem in an evolutionary perspective, by describing how new levels of individuality may emerge through increased cooperation and decreased conflict among lower-level entities, turning higher levels into more effective targets of natural selection (e.g., see [56,59–62]). In this framework, individuals are thus depicted as the product of an evolutionary process, which comes as a paradox if individuals are also recognised as an essential ingredient of evolution by natural selection. Life 2021, 11, 1051 7 of 12 Figure 1. Efficiency of a sorting process with or without reproduction. Without reproduction (full bullet points), the fittest entities (in green) dominate the system at equilibrium, but the blue ones still constitute a fifth of the population. In contrast, with reproduction (empty bullet points), the blue entities are just not present at equilibrium if one assumes a finite population size (1000 in these simulations). Figure 1. Efficiency of a sorting process with or without reproduction. Without reproduction (full bullet points), the fittest entities (in green) dominate the system at equilibrium, but the blue ones still constitute a fifth of the population. In contrast, with reproduction (empty bullet points), the blue entities are just not present at equilibrium if one assumes a finite population size (1000 in these simulations). 4.2. The Signatures of Natural Selection When it comes to describing the outcomes of natural selection, one conceptual tool is frequently invoked: the Price equation [63]. This equation formalizes in statistical terms the notion that the change in the mean value of a biological trait across two time points (typically, between two generations) can be partitioned into an “individual-level term” (how much individuals themselves or their offspring have changed) and a “population- level term” (how much the relative abundances of the different classes of individuals have changed). Technically, both terms are defined at the population level, but the first refers to individual changes, hence the terminology used here. The population term captures the outcome of natural selection as a covariance between trait value and abundance, although this covariance may also be inflated by pure chance [64]. The Price equation is most re- nowned for its abstractness (thus its generality) and its robustness to particular assump- tions, making it a versatile tool adapted to a wide diversity of evolutionary questions, from epidemiology to non-genetic inheritance and social or cultural evolution [65–68]. Yet, also because of its abstractness and generality, this equation remains purely descrip- tive and may be judged of limited utility unless it is used in a biologically well-delimited context [69]. While initially derived in reference to genetics and evolutionary biology, the Price equation was also explicitly perceived by its own author as a possible first step toward “a general selection theory” that may be used beyond its original field [70]. In principle, it may indeed apply to any dynamical system where sets of individual entities can be mapped over different time points, for example through parent-offspring relationships, or simply through conserved “identities” in non-reproducing systems. It then formalizes the notion that the average change over time in any property can be partitioned into some individual level changes and some higher-level changes, where the latter is a covariance between the property value and its relative abundance. If, under this framework, the ob- servation of a systematic (that is, non-random) non-zero covariance, for a given property, is to be taken as a signature of natural selection on this property, one may conclude that natural selection is just everywhere, or more specifically, in any system where some form of stability is deterministically associated with some property. Box 1. Mere sorting versus evolution by natural selection. Box 1. Mere sorting versus evolution by natural selection. One way to discuss whether natural selection should be seen as equivalent to mere sorting is to assess the efficiency of a sorting process with or without reproduc- tion. This can be performed with a simple mathematical model (equivalent to those previously used by Earnshaw-Whyte [79] and Bourrat [80]) simulating the dynamics of a system composed of two or more types of entities, only differing in their rates of decay, the equivalent of “survival” in biology. Specifically, let us consider a system of green and blue entities, produced at the same rate but differing in their respective sta- bilities, e.g., with 80% chance of being maintained at each time step for the green kind, and only 40% for the blue kind. We assume no transition between the two types, that is, the colour (and thus the degree of stability) of the entities does not change. ( g y) g Starting from an equal proportion of the two, Figure 1 shows what would then happen in two situations: one without reproduction (small points), where the lost en- tities are replaced with green or blue ones with equal chance, versus one with repro- duction (open circles) where the lost entities are more often replaced by the most abundant type, with a probability that equals its frequency (equivalent to choosing a random entity to reproduce). Without reproduction, green entities dominate the sys- tem at equilibrium, but the blue ones still constitute a fifth of the population. In con- trast, with reproduction, the blue entities are just not present at equilibrium if one as- sumes a finite population size (1000 in these simulations). These radically different dynamics also mean that reproduction increases the chances of cumulative changes, where each new step is facilitated by the very high abundance of the fittest type. In sum, it remains theoretically possible to envisage a process akin to natural selection without reproduction, but reproduction so radically changes the dynamics that it in- troduces the possibility of otherwise very improbable cumulative changes. In that sense, reproduction may be considered an essential ingredient, perhaps not of natural selection in its broadest sense, but of evolution by natural selection, implying cumula- tive changes. Life 2021, 11, 1051 8 of 12 8 of 12 4.2. The Signatures of Natural Selection 4.2. The Signatures of Natural Selection In that sense, using the Price equation to detect natural selection appears as an excessively permissive approach, that fails at limiting our focus on what we defined above as evolution by natural selection, which implies reproduction. p p Another difficulty lies in finding the appropriate level of description. If the individ- ual- versus population-level partitioning is always possible, the equation in itself does not help to define those levels, because of the so-called problem of “cross-level by-products” [56,71]: natural selection acting at one level will also give rise to a non-zero covariance at higher organisational levels, provided an appropriate grouping scheme. Notably, this dif- ficulty may also occur in standard applications of the Price equation, that is, in biological systems, if different levels may be perceived as “individuals”. To account for these cases, some sophistications have been added to produce a “multi-level Price equation”. Yet, even then, any arbitrary choice may reveal elevated covariance terms at higher levels than those where fitness differences effectively take place, so that the choice of the description level requires some other kind of knowledge [56,72]. This problem is obviously inflated when it comes to considering non biological candidates for evolution by natural selection, where intuition is of no help to circumscribe individuals and populations. p p p In principle, multiplying the features to be measured and scanning various grains of description may provide a means to circumvent this problem: if individuals correspond to levels of organisation where selection is indeed effective, they should be identifiable as grains of description where most of the observed change can be attributed to the covari- ance term, and simultaneously so for a diversity of features (that is, in biological terms, for a diversity of “traits”). To our knowledge, such an approach has not been developed yet (though see discussions in [73,74]), perhaps because it implies that the features to be measured must be defined and measurable at any granularity, a constraint that does not Life 2021, 11, 1051 9 of 12 9 of 12 readily fit with the intuition that individuals should display “emergent properties” that is, features that precisely cannot be defined at all levels of description. The information theory may offer a possible way out of this difficulty, by providing a means to “measure” individuality in non-biological terms [75], but the very feasibility of this approach is also questioned [73]. 5. Conclusion: Individuality Beyond Life? Building on the conception that natural selection should follow from some necessary and sufficient conditions, our discussions aimed at exploring the possibility that this pro- cess could take place beyond life as we know it, that is, in other physical systems where these conditions would be met. A short survey of protobiotic systems revealed how much evolvability through natural selection is perceived in this research field as an important yet unattained objective, perhaps because of an excessively categorical scheme, as op- posed to continuous, which we take as evidence that the challenge is not only technical, but also conceptual. Considering the problem from a physical perspective, we discussed the possibility of placing natural selection within the context of dissipative structures. While this framework makes it possible to situate life among other physical phenomena, it has not produced general principles, of which natural selection might have been a par- ticular case, reflecting that current physics does not include a readily usable equivalent to natural selection. Turning to evolutionary biology, we asked whether the conditions for natural selection, or its signatures, were defined with sufficient formalism to be identified outside of their original context; we contend they are not. Most strikingly, the implicit but essential notion of individuality stands as a major conceptual obstacle. How can individuals be recognised without a priori knowledge of the appropriate level of description? How can individuals be at the same time considered as essential ingredients and outcomes of evolu- tion by natural selection? Addressing these paradoxical questions appears as an essential prerequisite for further investigating where and how natural selection may take place. In fact, the question of how individuality may emerge resembles one that has at- tracted much attention in the field of major evolutionary transitions: how and why, in the history of life, have individuals merged into higher organisational levels, such as procar- yotic symbionts into large eucaryotic cells, or clonal cells into multicellular organisms [54,56,59,71]? Yet in the case of the very first emergence of individuality, low level indi- viduals cannot be part of the initial conditions. In other words, a general theory for the emergence of individuality cannot, by definition, rely on individuals. In biology, explain- ing jumps in levels of individuality implies identifying conditions, such as limited spatial diffusion or relatedness, that generate interdependence among the fitness of various enti- ties, setting the stage for the evolution of cooperative traits [77]. 4.2. The Signatures of Natural Selection More generally, we note a connection between this problem and the field of coarse graining, where one aims to determine the optimal levels of description to char- acterise a system [76]. If natural selection is taking place, this objective may be much akin to that of defining individuals and populations. 5. Conclusion: Individuality Beyond Life? This mode of reasoning has recently been applied to very early stages of life evolution [78] and may also help in explaining the very origin of individuality. Yet at that stage it remains unclear how to even model these questions without assuming the existence of some kind of well-delim- ited self-replicators, that is, without starting from individuals of lower levels. Future work will hopefully clarify whether the biological concept of individuality, and the biological principle of natural selection, can be grounded in physico-chemical roots, to perhaps ex- tend the breadth of their applicability. Life 2021, 11, 1051 10 of 12 10 of 12 Author Contributions: This paper is the culmination of discussions that involved all the authors. S.C. initiated the workshop, produced the first draft, and integrated the diverse contributions to the manuscript. P.B., C.M., O.R. and Z.Z. contributed from early writing stages. E.R. performed the simulations behind Figure 1. All authors further contributed to improving the manuscript. Authors other than S.C. are listed in alphabetical order. 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. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We are most grateful to the scientific committee of the “Institut d’Etudes Sci- entifiques de Cargèse” (IESC), the “Mission pour les Initiatives Transverses” (MITI) of the CNRS, and the “Groupement de Recherche” Sapienv (GDR CNRS 3770) for supporting this workshop. Acknowledgments: We are most grateful to the scientific committee of the “Institut d’Etudes Sci- entifiques de Cargèse” (IESC), the “Mission pour les Initiatives Transverses” (MITI) of the CNRS, and the “Groupement de Recherche” Sapienv (GDR CNRS 3770) for supporting this workshop. Conflicts of Interest: The authors declare no conflict of interest. References 1. Neander, K. The Teleological Notion of ‘Function’. Australas. J. Philos. 1991, 69, 454–468. https://doi.org/10.1080/00048409112344881. 2. Millikan, R.G. Language, Thought, and Other Biological Categories; MIT Press: Cambridge, MA, USA, 198 2. Millikan, R.G. Language, Thought, and Other Biological Categories; MIT Pres 2. Millikan, R.G. 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Distanced self-talk increases rational self-interest
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Distanced self‑talk increases rational self‑interest N Izzy Gainsburg1,2,4*, Walter J. Sowden1,3, Brittany Drake5, Warren Herold6 & Ethan Kross1, Does stepping back to evaluate a situation from a distanced perspective lead us to be selfish or fair? This question has been of philosophical interest for centuries, and, more recently, the focus of extensive empirical inquiry. Yet, extant research reveals a puzzle: some studies suggest that adopting a distanced perspective will produce more rationally self-interested behavior, whereas others suggest that it will produce more impartial behavior. Here we adjudicate between these perspectives by testing the effects of adopting a third-person perspective on decision making in a task that pits rational self-interest against impartiality: the dictator game. Aggregating across three experiments (N = 774), participants who used third-person (i.e., distanced) vs. first-person (i.e., immersed) self-talk during the dictator game kept more money for themselves. We discuss these results in light of prior research showing that psychological distance can promote cooperation and fairmindedness and how the effect of psychological distance on moral decision-making may be sensitive to social context. When LeBron James was 25-years old, he had to make an agonizingly difficult decision: whether he should stay with his current team, the Cleveland Cavaliers, or join the Miami Heat. Being from Akron, Ohio and having been drafted by the Cavaliers, James could have chosen the Cavaliers out of loyalty to his home-state and fairness to the franchise that drafted him. By joining the Heat, though, LeBron would have more talented teammates, an organization with a championship pedigree, warmer weather, and no state income tax. In other words, James had to choose what was best for himself and what was best for the many fans that wanted him to stay in Cleveland. James ultimately chose to join the Heat, explaining to an interviewer, “I wanted to do what’s best for LeBron James and to do what makes LeBron James happy.”1. Notably, by using his own name to refer to himself in the above quotation, James thought about himself from a third-person perspective when making this decision. In this case, this linguistic shift co-occurred with a self-interested decision. But to what extent did James’ choice of language actually help promote his rational self-interest? Distanced‑self talk. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ Finally, one study demonstrated that distanced self-talk was associated with increased cooperative behavior in a public goods game, which involves players privately choosing how much money to contribute to a shared pot, with the pot’s sum multiplied and distributed evenly among the ­players29. The present research. Critically, prior research cannot clearly distinguish whether distanced self-talk is more likely to promote rational self-interest or fair-minded impartiality because the two outcomes are not at odds with one another in prior research. For instance, cooperation in a public goods game has the potential to benefit the self; likewise, lying to a police officer to protect a criminal loved one can jeopardize one’s own reputa- tion or innocence.h Thus, we aimed to test whether distanced self-talk increases rational self-interest in a situation where rational self-interest is at odds with fair-minded impartiality: the dictator ­game30. In the dictator game, participants are randomly assigned to the role of “decider” or “recipient.” The decider is allotted a sum of money and can choose how much money to give (if any) to the recipient. Unlike in other economic games (e.g., a public goods game, an ultimatum game), there is no financial incentive to give money to the recipient in the dictator game, and in fact, giving money to the recipient always comes at a financial cost to the self. Thus, the dictator game is a situation where rational self-interest or fair-minded impartiality are not conflated with one another. l In the present research, we conducted three experiments with samples from two populations (university undergraduates and participants from Amazon’s Mechanical Turk) to examine the effect of self-distancing on rational self-interest. In each study, participants played the dictator game as the decider, but were randomly assigned to refer to themselves in either first or third-person language while thinking about their decision (i.e., immersed vs. distanced self-talk9).hi The first experiment (Study 1) recruited university undergraduates in an exploratory study testing the two competing predictions pertaining to the relationship between self-distance and rational self-interest. The second experiment (Study 2) sought to replicate the first study with a sample from a different population (Amazon’s Mechanical Turk). The third experiment (Study 3) was mostly identical, except that we had participants write about their decision while referring to themselves in either first or third-person language. www.nature.com/scientificreports/ linking psychological distance to increased ­deliberation20,21, distanced self-talk could increase rational self-inter- est by promoting more deliberative thinking. Similarly, given theorizing that a third-person, “outside view” can promote more rational decision-making22, distanced self-talk may also promote more rationally self-interested decisions. Supporting this possibility, people making financial decisions for others (versus the self) make more rationally self-interested decisions that con- form to normative models of economic ­behavior23,24. Research also shows that distanced self-talk can increase objectivity in judgments and ­decisions13,14,25,26, albeit in contexts unrelated to economic decisions. Finally, one study found that people use more second-person language during goal-directed (vs. spontaneous) self-talk27, suggesting that psychological distance may foster goal-directed (i.e., rational) behavior. Taken together, distanced self-talk may shift people’s thinking to be more rational, increasing rationally self-interested financial decisions. Why self‑distancing might decrease rational self‑interest. Research from other areas suggests, however, that there are instances in which distanced self-talk may decrease rational self-interest by promoting a sense of fairness or ­impartiality28. This line of reasoning can be found in Adam Smith’s treatise on the self and morality, The Theory of Moral Sentiments. According to Smith, humans are naturally self-centered, but that if we imagine ourselves “at a certain distance…from the place and with the eyes of a third person,” we are able to see “the real littleness of ourselves” and to make a “proper comparison between our own interests and those of other people.” In other words, Smith believed that a distanced view of the self would allow people weigh their interests and those of other people less partially and to behave more fairly. Although research has yet to test the full extent of Smith’s logic, several of the underlying principles have been empirically demonstrated. g y g p p p y For instance, research shows that distanced self-talk promotes impartiality. In one set of studies, self-distanced (vs. self-immersed) reflection was associated with less extreme political ideology and greater willingness to join bipartisan discussion ­groups14. In another set of studies where participants imagined close others having committed crimes, participants using distanced (vs. immersed) self-talk were less likely to lie on behalf of their felonious close others to protect them from punishment, demonstrating that distanced self-talk can promote fair-minded (e.g., honest) and impartial (i.e., nondiscriminatory) ­behavior25. www.nature.com/scientificreports/ We did this so that we could get a window into participants’ thinking, and whether they were thinking about their decision in different ways across conditions. In particular, we coded the degree to which participants were thinking about the self, others, and moral principles (e.g., fairness), given that each of these themes may be tied to how much money participants keep for the self (vs. give to others) in the dictator game. Results focus on data collapsed across all studies given the similar designs and outcomes. Distanced self‑talk increases rational self‑interest N Self-talk is a basic dimension of one’s inner experience, along with other dimensions such as feeling and sensory awareness, and differs from other forms of thought in that it uses language, as opposed to images or unsymbolized ­thinking2. There are also different kinds of self-talk—for instance, some dual-systems models differentiate between spontaneous and goal-directed self-talk3.t yf p g Relevant to the present research, people often talk about the self using their given name, a phenomenon sometimes referred to as illeism4,5 or distanced self-talk6,7. Only recently, however, has research explored the psychological function of this linguistic shift. In particular, research indicates that when people use their own name and other non-first person pronouns (e.g., he/she, you) to refer to the self, they are able to relatively effort- lessly transcend their default, egocentric perspective and see the self from a distanced, third-person ­viewpoint8. More importantly, distanced self-talk can facilitate emotion-regulation9–11 (for reviews ­see7,12) and elements of wise reasoning (e.g., intellectual humility)13,14. Yet, as the LeBron James example indicates, it is possible that this type of distanced self-talk may also have another important function: promoting rational self-interest during decision-making. Why self‑distancing might promote rational self‑interest. Many theories of human judgment and decision-making, from Plato’s writings to contemporary dual-systems models, have suggested that our intui- tions can interfere with rational thinking and behavior. Consistent with such accounts, deliberative (i.e., slow and reflective), compared to intuitive (i.e., fast and automatic), thinking is associated with more rationally self- interested economic decisions that adhere closer to normative models of economic ­behavior15–19. Given research 1Psychology Department, University of Michigan, Ann Arbor, USA. 2Ross School of Business, University of Michigan, Ann Arbor, USA. 3Department of Behavioral Health, Tripler Army Medical Center, Honolulu, USA. 4Harvard Kennedy School, Harvard University, Cambridge, MA 02138, USA. 5University of California, Los Angeles, Los Angeles, USA. 6University of Arkansas, Fayetteville, USA. *email: izzyg@umich.edu | https://doi.org/10.1038/s41598-021-04010-3 Scientific Reports | (2022) 12:511 www.nature.com/scientificreports/ Results All β values represent partially standardized coefficients, consistent with recommendations from Hayes (2017) to use partially standardized coefficients with dichotomous X variables. In a separate model, we ran this ANOVA, but without the condition by study interaction term included—the effect of condition was the same across both studies. Below, we report results for this ANOVA with the interac- tion term for completeness, including estimated marginal means to measure central tendency and their 95% confidence intervals to estimate variance. In a separate model, we ran this ANOVA, but without the condition by study interaction term included—the effect of condition was the same across both studies. Below, we report results for this ANOVA with the interac- tion term for completeness, including estimated marginal means to measure central tendency and their 95% confidence intervals to estimate variance. i Across studies, participants using distanced self-talk to think through their decision kept more for themselves (M = $7.30; 73.0% of the money) than those in the self-immersed condition (M = $6.78; 67.8% of the money), F(1, 768) = 7.83, p = 0.005, d = 0.20. There was also a main effect of study (F(2, 768) = 29.84, p < 0.001, η2 p = 0.072), which captured that MTurk participants kept more money for themselves than those from the subject pool (see in Table 1). The condition x study interaction was not significant, F(2, 768) = 1.83, p = 0.16, η2 p = 0.005.f hi p In addition, we analyzed the effect of distanced self-talk in each individual study. Participants using distanced (vs. immersed) self-talk to think about their decision condition kept significantly more money for themselves in Study 1 and Study 3; there was no difference between conditions in Study 2. See Table 1 for raw means and standard deviations across conditions and independent t-tests for each study.f Finally, given that our observed effect size (d = 0.20) was smaller than what we initially predicted (d = 0.50), we wanted to run a post-hoc power-analyses to estimate how much power we had to observe the effect of interest in the aggregated analysis and in each individual study. Results We tested whether distanced self-talk affects rational self-interest for each individual study and meta-analytically by collapsing data across studies. We first present these results. Following this, we analyze results from the coded essays in Study 3 to examine if the effects of distanced self-talk on rational self-interest could be explained by shifts in how participants thought about the decision. Does distanced self‑talk influence rational self‑interest? We tested whether distanced self-talk influenced rational self-interest using an ANOVA that included “condition” (distanced vs. immersed), “study” (Study 1 vs. Study 2. vs. Study 3) as categorical covariate, and the condition x study interaction. For this ANOVA, the dependent variable for Study 3 was doubled so that it used the same scale as Studies 1 and 2 (i.e., out of $10). https://doi.org/10.1038/s41598-021-04010-3 Scientific Reports | (2022) 12:511 | www.nature.com/scientificreports/ Table 1. Effect of self-distancing on money kept by participants. Immersed M (SD) Distanced M (SD) Inferential statistics Study 1 5.54 [1.80] 6.28 [2.41] t(139) = 2.01, p = .04, d = .35 Study 2 7.38 [2.45] 7.46 [2.34] t(396) = 0.36, p = .72, d = .04 Study 3 7.43 [2.52] 8.17 [2.22] t(233) = 2.27, p = .02, d = .31 Table 1. Effect of self-distancing on money kept by participants. Immersed M (SD) Distanced M (SD) Inferential statistics Study 1 5.54 [1.80] 6.28 [2.41] t(139) = 2.01, p = .04, d = .35 Study 2 7.38 [2.45] 7.46 [2.34] t(396) = 0.36, p = .72, d = .04 Study 3 7.43 [2.52] 8.17 [2.22] t(233) = 2.27, p = .02, d = .31 Table 1. Effect of self-distancing on money kept by participants. Table 1. Effect of self-distancing on money kept by participants. Figure 1. Mediation model from Study 3. Participants using distanced (vs. immersed) self-talk thought less about other people and less about moral principles. Decreases in moral-focus (but not other-focus) mediated the finding that distanced (vs. immersed) self-talk was associated with more money kept for the self. All β values represent partially standardized coefficients, consistent with recommendations from Hayes (2017) to use partially standardized coefficients with dichotomous X variables. Figure 1. Mediation model from Study 3. Participants using distanced (vs. immersed) self-talk thought less about other people and less about moral principles. Decreases in moral-focus (but not other-focus) mediated the finding that distanced (vs. immersed) self-talk was associated with more money kept for the self. General discussion How does adopting an objective, third-person view of the self influence rational self-interest? For basketball superstar LeBron James, his use of distanced self-talk famously coincided with the difficult, but rationally self- interested decision to switch teams during free agency. Prior research, however, offered mixed forecasts as to whether a distanced perspective of the self would promote rational self-interest vs. fair-minded impartiality when the two were at odds with one another. The present research offers insight into this debate by examining how distanced self-talk affects decision-making in a zero-sum game where rational self-interest and impartiality are at odds with one another. Aggregating across three studies, participants using distanced (vs. immersed) self-talk kept more money for themselves in one-shot, anonymous dictator games, suggesting that distanced self-talk promotes rational self-interest. This effect was observed among college students in a laboratory setting, as well as among MTurk participants in an online setting. The aggregated effect size (d = 0.20) is small by conventional standards, but is comparable to the estimated effects from several dictator game meta-analyses, including trait altruism (d = 0.22; 33); gender (d = 0.25; 34); the presence of eye-like stimuli prompting donations (d = 0.18; 35); and stake size (d = 0.15; 36). Theoretical implications. Findings from the present research accord with other research showing that distanced self-talk promotes rational thinking and ­behavior26. The economically rational decision in this con- text—a one-shot, anonymous allocation decision—is to keep money for the self, given that there is no opportu- nity for retaliation, reputation costs/benefits, or reciprocity. Defining the decision to keep money as rational is consistent with normative economic (i.e., maximizing expected utility;37) and evolutionary (acquiring resources that increase biological and reproductive fitness;38) conceptualizations of rational behavior.h gi This broader trend for self-distancing to promote rational thinking and behavior may explain seemingly contradictory findings in other research, in which distanced self-talk was associated with increased money given to others in a different economic game—a public goods ­game29. Unlike the one-shot dictator game used in the present research, a public goods game offers the possibility for cooperation to result in increased gains for the self, meaning that cooperative behavior can be consistent with rational self-interest. General discussion Thus, it is possible that distanced self-talk allows one to behave in the way that is more rational and beneficial for the situation at hand (i.e., keeping money in a one-shot dictator game, cooperating in a public goods game), consistent with the idea that deliberation will only increase selfish decisions when it is the most beneficial ­option39. Future research should more systematically examine how these contextual variables modulate the effect of self-distancing on people’s moral cognition and behavior.hi p p g These findings are consistent with research on construal level theory, i.e., the idea that psychological distance can result in more abstract and decontextualized construals of people, objects, and ­situations40. Prior research on construal level theory has shown that other dimensions of psychological distance (e.g., social distance; temporal distance) and abstract thinking can shift people’s economic decisions to be more in line with their future or desired selves, such as reducing preferences for smaller present rewards over greater future rewards (i.e., temporal discounting)41–43 and rejections of unfair but objectively beneficial financial ­offers44. The fact that self-distance may have the similar effects on rationally self-interested decision-making as other dimensions of psychological distance (e.g., temporal distance; social distance) is consistent with research showing that different dimensions of psychological distance have similar psychological and behavioral ­consequences45.hf The present work also accords with prior work examining the effect of delegation on allocation decisions in the dictator game. When dictators delegate allocation decisions to third parties, they choose third parties that will allocate less money to the recipients and more money to themselves. In other words, these delegates function as a buffer between dictators and recipients, warding off concerns people feel about appearing ­selfish46. In the present research, distanced self-talk may have had a similar effect, shifting the focus from “How much money should I give to the other person?” or “What is the fair decision?” to “What is in the best interest of the person making this decision (i.e., the self)?” Future research should explore whether there are contexts in which differ- ent types of psychological distance (e.g., distanced self-talk vs. making decisions on behalf of others) produce divergent effects in financial decision-making. Limitations and future directions. The present research offered a window into how distanced self-talk affects rational self-interest, but there are several limitations to our work that should be considered when inter- preting the results. Results Using G*Power 31, a post-hoc power analysis (using two- tails, α = 0.05) revealed that we had sufficient power (power = 0.80) to detect the aggregate effect size (d = 0.20) in the aggregated sample using an ANOVA (as was used in the present analyses), and that we were underpowered to detect the this effect in Study 1 (power = 0.22), Study 2 (power = 0.51), and Study 3 (power = 0.33). What mediates the relationship between distanced self‑talk and rational self‑interest? We tested other-focus and moral-focus as simultaneous mediators in a single model (Model 4) using the PROCESS Macro 32; we did not test self-focus as a potential mediator because it was not affected by condition (t(233) = 0.65, p = 0.52, d = 0.09) . Results revealed a significant indirect effect through moral-focus (partially standardized indi- rect effect = 0.11, 95% CI [0.003, 0.236], SE = 0.06, 37.3% of the total effect), but no indirect effect through other- focus (partially standardized indirect effect = 0.04, 95% CI [− 0.01, 0.12], SE = 0.03, 12.7% of the total effect). In addition, the direct effect of condition on rational self-interest was no longer significant after accounting for these two indirect effects (unstandardized effect = 0.37, 95% CI [− 0.21, 0.95], SE = 0.30, p = 0.21). See Fig. 1. Scientific Reports | (2022) 12:511 | https://doi.org/10.1038/s41598-021-04010-3 www.nature.com/scientificreports/ Method d In other words, as you think about how to split up the money, ask yourself things like, “What should [participant’s name] do?’” with the participant’s first name piped into the question from when they entered it earlier. Note that in both conditions, we specifically prompted participants to engage in self-talk (as opposed to other types of thinking) by asking them to think to themselves verbally (the defining feature of self-talk) and providing an example using language, consistent with other research that has used similar instructions to elicit self-talk9,26.f p g g g All participants then chose to keep anywhere from $10 dollars to $0, with the different options representing one-dollar increments in between those two extremes (participants were given this exact dollar amount at the end of the study). Participants in the self-immersed condition chose from options that used “I” (e.g., “I keep $10 and give the recipient $0), whereas participants in the self-distanced condition chose from options that had their name piped into the text (e.g., “[Name] keeps $10 and gives the recipient $0”). Participants kept an average of $5.89 (SD = 2.14) out of a possible $10. p Participants then rated satisfaction with their decision, completed follow-up measure of their emotional state (for descriptive statistics, see Supplementary Table S1) and were funnel-debriefed by the experimenter. Study 2. Participants. Participants from the United States were recruited from Amazon Mechanical Turk (MTurk). We needed 260 participants to detect the observed effect distanced self-talk in Study 1 (Power = 0.80 for d = 0.35 and α = 0.05). We recruited 400 participants to account for potential attrition and the fact that we were sampling from a new population. One participant was excluded for failing to enter their first name and one participant was a research assistant testing the survey, leaving a total of 398 participants (43.5% female; Mage = 34.4; 80.2% white), giving us 94% power to detect our effect size of interest. Procedure. Participants were told that they would be paid a flat fee of $0.50 but could earn an additional $10 depending on how they performed during the experiment. After consenting to participate, participants com- pleted a measure of baseline mood (see Supplementary Table S1 for descriptive statistics) and entered their first name into the survey so that it could be piped into later prompts. Method d Study 1. Participants. Participants were recruited from the University of Michigan Psychology Depart- ment’s Paid Subject Pool. They were told that they could earn up to $10 by participating. We anticipated a moderate effect (d = 0.50) for self-distancing based on the previous research on self-distancing and decision- making9,13,25. We used G*Power31 to determine that we needed 128 participants to have 80% power to detect this effect. We recruited 10% more than our target to account for potential exclusions (N = 145). Four participants were excluded failing our a priori criteria for quality responses (two had taken similar studies; one did not have a gender-matched experimenter; one was not a native English-speaker), leaving a final sample of 141 participants (59.6% female) with 80% power to detect an effect size of d = 0.48. Procedure. Participants arrived at the laboratory at the same time as one other participant of the same gender. An experimenter of the same gender as the two participants told them they would be participating together in the experiment and led them to computers in separate cubicles, where participants completed informed con- sent and read about the tasks they would be doing. Participants then answered questions about their current emotional state (for descriptive statistics, see Supplementary Table S1). All participants entered their first name into the survey so that it could be piped into later prompts for those in the self-distancing condition. Next, par- ticipants were briefed on the rules of the dictator game and told that they and their partner would be assigned to either the role of the decider or the recipient (in reality, all participants were assigned the role of the decider). From there, participants were randomly assigned to either the self-immersed or the self-distanced condition. Participants in the self-immersed condition read: “Some people report thinking about themselves using first person language, so that’s what we’d like you to do as you make your decision. In other words, as you think about how to split up the money, ask yourself things like, ‘What should I do?’”. y y g Participants in the self-distanced condition read: “Some people report thinking about themselves using third person language, so that’s what we’d like you to do as you decide what to do when making your decision. www.nature.com/scientificreports/ Fourth, our research was limited by focusing on a single task. As previously discussed, the effects of self- distancing appear to depend on context, and thus, more work is needed to examine whether different tasks and contexts moderate the effects of self-distancing on people’s moral judgments and decisions.f f Finally, and related to the above point, it is possible that there are individual differences that moderate the effects of distanced self-talk has different effects on rational self-interest. In particular, given that rationality is often defined as people behaving in line with their own goals and ­preferences47, distanced self-talk may have het- erogeneous effects for people with different goals and preferences, with the common thread being the distanced self-talk promoting instrumental rationality. Method d Participants were then briefed on the rules of the dictator game and told that they would be randomly part- nered with another MTurk participant. From there, participants were directed to a screen that had a loading icon and the text, “Please wait while we pair you with another MTurk worker. (Usually less than one minute.).” All participants saw this screen for 17s before the survey automatically advanced to the next page. There, all partici- pants were told they had been assigned to the “Decider” role and reminded of the decision that this role entails. p g y y p nered with another MTurk participant. From there, participants were directed to a screen that had a loading icon and the text, “Please wait while we pair you with another MTurk worker. (Usually less than one minute.).” All participants saw this screen for 17s before the survey automatically advanced to the next page. There, all partici- pants were told they had been assigned to the “Decider” role and reminded of the decision that this role entails. Next, participants were randomly assigned to either the distanced vs. immersed self-talk condition and subsequently chose how much money to keep for themselves. The instructions used, the manipulation, and the choice phase were identical to Study 1. Participants kept an average of $7.42 (SD = 2.39) out of a possible $10. participants saw this screen for 17s before the survey automatically advanced to the next page. There, all partici- pants were told they had been assigned to the “Decider” role and reminded of the decision that this role entails. Next, participants were randomly assigned to either the distanced vs. immersed self-talk condition and subsequently chose how much money to keep for themselves. The instructions used, the manipulation, and the choice phase were identical to Study 1. Participants kept an average of $7.42 (SD = 2.39) out of a possible $10. Next, participants were randomly assigned to either the distanced vs. immersed self-talk condition and subsequently chose how much money to keep for themselves. The instructions used, the manipulation, and the choice phase were identical to Study 1. Participants kept an average of $7.42 (SD = 2.39) out of a possible $10. General discussion g First, our self-talk instructions may have prompted other kinds of inner experience beyond self-talk, such as inner ­seeing2. This would actually be consistent with other research showing that third-person (vs. first person) self-talk results in people visualizing the self from a more distanced ­perspective9. Thus, it is possible that our first-person vs. third-person manipulation also changed other dimensions of inner experience, and these changes may have also had effects on participants’ dictator game decisions. f Second, our research is limited in that it examined experimentally-induced self-talk, as opposed to sponta- neous self-talk. For instance, typical spontaneous self-talk is not prompted by others or written down (as it was in Study 3). Although future research could test the effects of distanced (vs. immersed) spontaneous self-talk on rational self-interest, prior research suggests similar effects would emerge. For instance, prior research on spontaneous visual self-distancing shows that, like experientially induced visual self-distancing manipulations, it carries similar emotion regulation benefits (e.g., less rumination).hf i Third, our individual studies were underpowered to detect the effect size that we observed in the meta- analysis. Therefore, we encourage readers to interpret the results from individual studies with caution, and to instead treat the meta-analysis as the best estimate of the effect of distanced self-talk on rational self-interest. Scientific Reports | (2022) 12:511 | https://doi.org/10.1038/s41598-021-04010-3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ intended for exploratory analyses (these were not explored in the present research; see supplementary materials for all scales), and demographics. intended for exploratory analyses (these were not explored in the present research; see supplementary materials for all scales), and demographics. Study 3. Participants. We recruited 296 participants from the United States from MTurk. This gave us 85% power for detecting the effect observed in Study 1. Participants in Study 3 were instructed to write about their decision using the language associated with the condition they were placed in (i.e., first-person or third-person language). There were 61 participants who were excluded for failing to do this (all in the distanced self-talk con- dition). This left a final sample of 235 (40.8% female; ­Mage = 34.64; 74.0% white), giving us 74% power to detect the effect observed in Study 1. Procedure. The procedure in Study 3 was the same in Study 2 except for two differences. First, participants were told that they would be paid a flat fee of $0.50 but could earn an additional $5 depending on how they per- formed during the experiment (they could earn additional $10 in Study 2). This affected the choice set available to participants (they could choose to keep $5, $4, $3, $2.50, $2, $1, or $0). Participants kept an average of $3.86 (SD = 1.22) out of a possible $5. Second, participants wrote about their decision using distanced vs. immersed self-talk (rather than merely thinking in those terms, as in Study 2). Even though participants wrote down their thoughts, we treated these thoughts as self-talk given that they were self-directed and mediated by language, consistent with other research that has analyzed written responses using self-talk ­frameworks9,26,48. Measures. Two condition-blind coders read all essays and coded them for five variables: Self-focus, Other focus, Moral focus, Altruism focus, and Emotionality. “Self focus” was defined as, “To what degree does the participant focus their attention on themselves?” (Cronbach’s α = 0.68). “Other-focus” was defined as, “To what degree does the participant focus their attention on the recipient?” (Cronbach’s α = 0.81). “Moral focus” was defined as, “To what degree does the participant focus on moral considerations (e.g., fairness, equality, rights, right/wrong, ‘shoulds’)?” (Cronbach’s α = 0.84). “Altruism focus” was defined as, “To what degree does the participant focus on altruism, explicitly? References References 1. 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Method d p y p p g ( ) p Participants rated satisfaction with their decision, a follow-up measure of mood akin to the measure of baseline mood (see Supplementary Table S1 for descriptive statistics), a series of personality scales that were Scientific Reports | (2022) 12:511 | https://doi.org/10.1038/s41598-021-04010-3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ By altruism, we aren’t referring to fairness or equality, but rather the specific motivation to help or benefit another person” (Cronbach’s α = 0.59). “Emotionality” was defined as “To what degree to they express or refer to their own emotions? This can be explicit (them writing about their feelings) or something you perceive in their writing (their emotions leaking out more subtly through their writing)” (Cronbach’s α = 0.87). g g y g g Each of these variables was coded on a 0 (the concept was not at all mentioned) to 3 (the concept was a major theme in the essay) scale. Descriptive statistics and correlations between all five variables are in Supplementary Tables S2 and S3. We review findings pertaining to “Altruism focus” and “Emotionality” in the supplement, because interrater reliability for “altruism focus” was low (Cronbach’s α = 0.59) and because the authors agreed that the “emotionality” code was an insufficient measure of the emotions that participants were feeling regarding their decision. We proceeded to test whether the first three coded variables mediated the effect of the self-talk manipulation on money kept. Ethics declarations. All research protocols was approved by the University of Michigan Institutional Review Board—Health Sciences and Behavioral Sciences and performed in accordance with their guidelines and APA ethical standards. Across studies, we obtained informed consent from all participants. 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(Metaphysics Research Lab, Stanford University, 2020). p , ( ) 47. Kolodny, N. & Brunero, J. Instrumental Rationality. in The Stanford Encyclopedia of Philosophy (ed. Zalta, E. N.) (Metaphysics Research Lab, Stanford University, 2020). , y, ) 48. Latinjak, A. T. Goal-directed, spontaneous, and stimulus-independent thoughts and mindwandering in a competitive context. Sport Psychol. 32, 51–59 (2018). , y, ) 48. Latinjak, A. T. Goal-directed, spontaneous, and stimulus-independent thoughts and mindwandering in a competitive context. Sport Psychol. 32, 51–59 (2018). Author contributions I.G. and E.K. wrote the main manuscript. I.G. performed the data analysis and contributed to study design. B.D. contributed to study design. W.S., W.H., and E.K. contributed to project conception, study design, data analysis, and writing. References Exploring Solomon’s Paradox: Self-distancing eliminates the self-other asymmetry in wise reasoning about close relationships in younger and older adults. 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T., Zourbanos, N., López-Ros, V. & Hatzigeorgiadis, A. Goal-directed and undirected self-talk: Exploring a new perspective for the study of athletes’ self talk Psychol Sport Exerc 15 548 558 (2014) e study of athletes’ self-talk. Psychol. Sport Exerc. 15, 548–558 (20 y y p 28. Snow, N. E. & Narvaez, D. Self, Motivation, and Virtue: Innovative Interdisciplinary Research. (Routledge, 2019). 29. Grossmann, I., Brienza, J. P. & Bobocel, D. R. Competing interests h g The authors declare no competing interests. 7 Vol.:(0123 022) 12:511 | https://doi.org/10.1038/s41598-021-04010-3 The authors declare no competing interests. Additional information Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​021-​04010-3. Correspondence and requests for materials should be addressed to I.G. Reprints and permissions information is available at www.nature.com/reprints. h p g Additional information Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​021-​04010-3. Correspondence and requests for materials should be addressed to I.G. Reprints and permissions information is available at www.nature.com/reprints. Additional information Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​021-​04010-3. Correspondence and requests for materials should be addressed to I.G. Reprints and permissions information is available at www.nature.com/reprints. Additional information Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​021-​04010-3. Additional information Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​021-​04010-3. Correspondence and requests for materials should be addressed to I.G. Scientific Reports | (2022) 12:511 | https://doi.org/10.1038/s41598-021-04010-3 www.nature.com/scientificreports/ 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. 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. 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Thoracoscopic T2-T3 versus T4 sympathectomy for primary palmar hyperhidrosis in children and adolescents: a randomized comparative study
Annals of Pediatric Surgery /Annals of Pediatric Surgery
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* Correspondence: ismaelelhalaby@gmail.com Presented at the 2020 Pan African Pediatric Surgical Association PAPSA e- Annual Scientific Congress on 9-10 October, 2020 Tanta University Hospital, Faculty of Medicine, Tanta University, Tanta, Egypt © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Thoracoscopic T2-T3 versus T4 sympathectomy for primary palmar hyperhidrosis in children and adolescents: a randomized comparative study lhalaby* , Mohamed Awad Mansour, Ahmed Mohamed Ismail Tawfik and Hisham Fayad Aly Ismael Essam Elhalaby* , Mohamed Awad Mansour, Ahmed Mohamed Ismail Tawfik and Hisham Fayad Aly Annals of Pediatric Surgery Annals of Pediatric Surgery Elhalaby et al. Annals of Pediatric Surgery (2021) 17:26 https://doi.org/10.1186/s43159-021-00092-w Elhalaby et al. Annals of Pediatric Surgery https://doi.org/10.1186/s43159-021-00092-w Open Access Abstract Background: Controversy exists regarding the appropriate level of sympathectomy for primary palmar hyperhidrosis (PH) as different levels are associated with variable postoperative patients’ satisfaction and potential complications. The aim of this study was to investigate the safety, efficacy, and outcome of T2-T3 thoracoscopic sympathectomy (TS) versus T4 TS in this age group. Results: This prospective study included 32 patients (17 males and 15 females) with primary PH treated at the department of pediatric surgery, in our institution during the period from February 2019 to February 2020. Inclusion criteria included moderate and/or severe degrees of PH not responding to conservative measures. The patients were divided randomly into two groups: group I treated by T2-T3 TS and group II underwent only T4 TS. All patients were evaluated regarding operative details and postoperative outcome. Follow-up ranged from 6 to 24 months. Group I included 18 patients (14 operated on both sides and 4 operated on one side), and group II included 14 patients (11 operated on both sides and 3 operated on one side). The ages ranged between 5 and 18 years (mean 14.25 ± 3.14 years). The difference in mean age among both groups (14.5 versus 13.9 years) was not statistically significant. The mean operative time was significantly longer in group I (22.4 versus 17.2 min, p value 0.046). The hospital stay (1 day) was similar for both groups. Postoperative compensatory hyperhidrosis (CH) was more frequent in group I (n=7, 50% versus n=5, 45.5%), but the difference was not statistically significant. Postoperative over dryness occurred in 5 patients in group I (28.6%) and temporary Horner’s syndrome in one patient (7.14%). No over dryness or Horner’s syndrome occurred in any patient in group II. The QOL score has improved in both groups; the degree of improvements was better in group II. q g p y g Postoperative over dryness occurred in 5 patients in group I (28.6%) and temporary Horner’s syndrome in one patient (7.14%). No over dryness or Horner’s syndrome occurred in any patient in group II. The QOL score has improved in both groups; the degree of improvements was better in group II. Conclusion: Both T2-T3 TS and T4 TS are effective in treating primary palmar hyperhidrosis in children and adolescents. T4 TS is preferred than T2-3 TS due to less frequent postoperative complications and better patients’ satisfaction. rds: Hyperhidrosis, Thoracic sympathectomy, Children, Adolescents, VATS, Compensatory sweating Surgical technique The procedure was performed under general anesthesia with single-lumen endotracheal intubation. Video-assisted thoracoscopic surgery was used for all patients. Patients were placed in a semi-Fowler’s pos- ition with abduction of both arms, the back lifted to 45° from the horizontal level, and the patient was then tilted to the opposite side with the side of pro- cedures being up to help deflate the lung and im- prove the field. Primary hyperhidrosis is an underrated problem in children and adolescents. Several systemic and topical treatment modalities have been used for the treat- ment of hyperhidrosis. Topical treatment includes as- tringents, iontophoresis, and botulinum toxin. Systemic treatment consists of the administration of anticholinergic drugs and psychotherapy. Despite the availability of such modalities, their use has limita- tions and side effects, and they are not always effect- ive especially in severe cases [2]. Two 5-mm ports were inserted in the fifth inter- costal space at mid-axillary line and fourth intercos- tal space at anterior axillary line for the camera and working ports respectively (Fig. 1). Sympathetic chain was interrupted by electrocautery with elec- trical hook; this was done on either side of the chain which was then freed and hooked from the under- surface before transection (Fig. 2). The transection range was extended about 1–2 cm laterally along the surface of the upper border of the corresponding rib in order to interrupt the potential bypass nerve fi- bers to decrease the possibility of recurrence. Sym- pathectomy was always done on one side starting with the dominant hand, and operation on the op- posite side was conducted after a period of 3–5 months. Thoracoscopic sympathectomy (TS) has been advo- cated in severe cases or after failure of non-surgical methods. Previously published studies have showed that TS is feasible in young children and adolescents. Contro- versy still exists regarding the most appropriate level of sympathectomy. The different levels are associated with different outcomes especially in terms of postoperative complications and patients’ satisfaction. The aim of this study was to investigate the safety, effi- cacy, and outcome of T2-T3 versus T4 TS in this age group. Elhalaby et al. Annals of Pediatric Surgery (2021) 17:26 Page 2 of 8 Elhalaby et al. Annals of Pediatric Surgery (2021) 17:26 Page 2 of 8 Background Patients were randomly divided by closed envelopes into 2 groups: group I, treated by T2-T3 TS; group II, treated by T4 TS. Hyperhidrosis is defined as excessive perspiration be- yond physiological needs [1]. Primary palmar hyperhi- drosis (PH) is part of a triad of excessive sweating affecting the hands, feet, and axillae. The major com- plaints are bouts of significant amounts of perspiration of the palms, causing severe psychological, social, and occupational inconvenience. Methods All patients remained at the hospital overnight. Dis- charge was allowed on the first postoperative day after exclusion of pneumothorax or other complication. This prospective randomized comparative study was conducted at the pediatric and vascular surgery units, in our institution during the period from February 2019 to February 2020. A total of 32 patients (children and ado- lescents) presented with PH with a total of 57 proce- dures were performed. Patients were followed up at outpatient clinic once weekly for the first month and once every month after that for at least 3 months. The study design was approved by the institutional re- search ethical committee. Informed consent was taken from all patients/parents after detailed description of the procedure, any other available alternative treatment, or potential complications. Fig. 1 Port sites at fifth intercostal space at mid-axillary line for the camera (C) and fourth intercostal space at anterior axillary line for working port (W) Inclusion criteria included children and adolescents with primary PH after failure of conservative treatment and patients with severe primary PH. Exclusion criteria were the following: (1) patients with mild symptoms, who can benefit from conservative man- agement; their sweating can be described and noticed as only slight wetting and dampness of the hands not grossly intervening with patients’ activities; (2) refusal of the patient or the parents of thoracoscopic approach; (3) major chest deformities; (4) previous open chest or heart surgery due to expected pleural adhesions; (5) age older than 18 years. Fig. 1 Port sites at fifth intercostal space at mid-axillary line for the camera (C) and fourth intercostal space at anterior axillary line for working port (W) Elhalaby et al. Annals of Pediatric Surgery (2021) 17:26 Page 3 of 8 Fig. 2 Both sides of the sympathetic chain were cauterized using electric hook; the chain was then freed and hooked from the undersurface before transection Fig. 2 Both sides of the sympathetic chain were cauterized using electric hook; the chain was then freed and hooked from the undersurface before transection he sympathetic chain were cauterized using electric hook; the chain was then freed and hooked from the undersurface Fig. 2 Both sides of the sympathetic chain were cauterized using electric hook; the chain was then freed and hooked from the undersurface before transection Results The current study included 32 patients (17 males and 15 females). Their ages ranged from 5 to 18 years with a mean age of 14.25 ± 3.14. The mean age of patients at time of surgery was almost equal in both groups (14.5 versus 13.9 years) The patients’ age at the onset of mani- festation varied from 4 to 12 years (mean value 6.31 ± 1.97 years). Sixteen of 32 patients (50%) presented at the age of 5–6 years. Intraoperative and postoperative results Patients in group I and group II were compared as regards to operative time, any intraoperative complica- tions, and hospital stay (Table 2). Preoperative severity of symptoms Preoperative severity of symptoms Thirteen patients (40.6%) had moderate degree, 7 in group I versus 6 in group II. The sweating was described and noticed as significant wetting, needing frequent hand wiping, and affecting the patients’ activities and manual work. Nineteen patients (59.4%), 11 in group I and 8 in group II, had severe degree of hyperhidrosis. Sweating was described and noticed to be excessive, even reaching to dripping from the hands, very frequent and routine wiping of the hands with marked affection of the patient’s daily activities. For each domain, a set of five questions was provided. Points for each question range from 1 to 5 for the first three domains; 1 is very poor and 5 is excellent. For the last domain, points are scored as 1 (do not know), 2 (strongly agree), 3 (agree), 4 (disagree), and 5 (strongly disagree). The maximum score to be achieved is 100, scores below 33 signify a poor QOL, scores from 34 to 66 sig- nify a fair QOL, and scores from 67 to 100 indicate good QOL. Assessment of the quality of life were satisfied with their primary surgery and were not enthusiastic to proceed for surgical intervention for the other side; the other 2 patients were lost to follow-up after 3 months of surgery. Patients/parents were asked to fill out a questionnaire before surgery with some details added during the post- operative period. This questionnaire was adopted from the questionnaire proposed by Laje et al. [3] to evaluate objectively the results of TS in the treatment of PH. The score assesses 4 domains: functional aspects, social as- pects, interaction with others, and performance under stressful circumstances (Table 1). Group I In a warm room When you are worried Taking an exam Speaking in public Other situation Total score (5–25) Do not know 1 Strongly agree 2 Agree 3 Disagree 4 Strongly disagree 5 Interaction with others People notice my excessive sweeting People avoid me because of my excessive sweeting I frequently avoid social interaction I am overall less confident I have a trouble maintaining relationship Table 1 Questionnaire for assessment of the quality of life (QOL) Writing Manual work Grasping objects Using a touch screen Leisure Total score (5–25) Very poor 1 Poor 2 Good 3 Very good 4 Excellent 5 Social area: How do you rate your QOL when doing the following activities? Shaking hands Holding hands Sociability with friends Playing sports Other activities Total score (5–25) Very poor 1 Poor 2 Good 3 Very good 4 Excellent 5 Stressful circumstances: How would you rate your QOL in these situations? In a warm room When you are worried Taking an exam Speaking in public Other situation Total score (5–25) Do not know 1 Strongly agree 2 Agree 3 Disagree 4 Strongly disagree 5 Interaction with others People notice my excessive sweeting People avoid me because of my excessive sweeting I frequently avoid social interaction I am overall less confident I have a trouble maintaining relationship Table 2 Operative time and immediate results Group I (n = 18) (32 procedures) Group II (n = 14) (25 procedures) Significance Operative time for each procedure (minutes) range (mean ± SD) 30–40 (22.4 ± 5.8) 25–37 (17.2 ± 5.7) 0.046* Intraoperative complications No No ---- Duration of hospital stay 1 day 1 day ---- *Significant as p value < 0.05 Very poor 1 Poor 2 Social area: How do you rate your QOL when doing the following activities? Shaking hands Holding hands Sociability with friends Playing sports Other activities Total score (5–25) Very poor 1 Poor Stressful circumstances: How would you rate your QOL in these situations? Group I Fourteen patients had bilateral T2-T3 TS, 7 of them de- veloped compensatory sweating (50%) ranging from mild to moderate degrees, yet all of them were satisfied. Five patients of this subgroup developed postoperative over dryness, three in one hand and 2 in both hands. Four out of those 5 patients were not bothered by such dry- ness, while one patient requested prescription of sooth- ing agents. One patient sustained transient Horner’s syndrome postoperatively (7.14%) and resolved spontan- eously after few weeks; four patients had unilateral T2- T3 TS. One of them developed postoperative over dry- ness. No compensatory sweating was noticed in any of these 4 unilateral T2-T3 TS patients (Table 3) Group I included 18 patients treated by T2-T3 TS (14 operated bilaterally and 4 on one side). A total of 32 procedures were done in this group. Group II included 14 patients who underwent T4 TS (11 operated bilat- erally and 3 on one side). A total of 25 procedures were done in the second group. Seven out of the total 32 (21.89%) patients underwent unilateral sympathectomy. Five out of the 7 patients Elhalaby et al. Annals of Pediatric Surgery (2021) 17:26 Page 4 of 8 Table 1 Questionnaire for assessment of the quality of life (QOL) Very poor 1 Poor 2 Good 3 Very good 4 Excellent 5 Functional area: How do you rate your QOL performing these tasks? Writing Manual work Grasping objects Using a touch screen Leisure Total score (5–25) Very poor 1 Poor 2 Good 3 Very good 4 Excellent 5 Social area: How do you rate your QOL when doing the following activities? Shaking hands Holding hands Sociability with friends Playing sports Other activities Total score (5–25) Very poor 1 Poor 2 Good 3 Very good 4 Excellent 5 Stressful circumstances: How would you rate your QOL in these situations? Group II Group II Eleven patients had had bilateral T4 TS, 5 of them (45.4%) developed compensatory sweating ranging from mild to severe degrees, all of them were satisfied with the exception of one case that complained of the degree of the compensatory sweating resulting in some discom- fort, yet the patient did not regret having the procedure. None of the eleven patients has developed postoperative over dryness. On the contrary, four of the patients expe- rienced slight moisture of one side (36.3%) not causing a problem for any of these patients. Three patients had unilateral T4 TS, one of them had slight moistness of hand, yet he was satisfied with the result. No compensa- tory sweating was noticed in any of these 3 patients (Table 3). The overall subjective evaluation of QOL was fairly concomitant with the objective evaluation using detailed QOL score. Postoperatively, patients were again asked to evaluate their QOL subjectively on a score ranging from 1 to 5 during the clinic follow-up visits: In group I, 12 patients (66.6 %) rated their QOL as excellent, and 5 patients (27.7%) rated very good. One patient was lost to follow- up after 3 months of the first operation. In group II, 13 patients (%) rated their QOL as excellent (92.8%). One patient was lost to follow-up after 3 months of the first operation. A repeated postoperative questionnaire 3 months after the last surgery showed a marked change in the patients’ QOL and functionality. In group I, 12 patients scored above 67 (good score); 5 patients scored between 34 and 66 (fair). One patient was lost to follow-up after 3 months of the first operation. Change of QOL Preoperative overall subjective assessment according to a score ranging from 1 to 5 showed that in group I, 12 patients of the 18 patients (66.6%) rated their QOL as very poor, 4 (22.2%) poor, and 1 (5.5%) good, and one patient (5.5%) rated very good. None of them rated excellent. In group II, 10 patients of the 14 pa- tients (71.4%) rated their QOL as very poor, 3 (21.4%) poor, and 1 (7.1%) good. None of them rated very good or excellent. In group II, 13 patients scored above 67 (good). One patient was lost to follow-up after 3 months of the first operation (Table 4). Group I In a warm room When you are worried Taking an exam Speaking in public Other situation Total score (5–25) Table 2 Operative time and immediate results Group I (n = 18) (32 procedures) Group II (n = 14) (25 procedures) Significance Operative time for each procedure (minutes) range (mean ± SD) 30–40 (22.4 ± 5.8) 25–37 (17.2 ± 5.7) 0.046* Intraoperative complications No No ---- Duration of hospital stay 1 day 1 day ---- *Significant as p value < 0.05 Elhalaby et al. Annals of Pediatric Surgery (2021) 17:26 Page 5 of 8 Table 3 Postoperative outcome Table 3 Postoperative outcome Group I (18 patients, 32 procedures) Group II (14 patients, 25 procedures) Significance Bilateral (n= 14) Unilateral (n= 4) Bilateral (n= 11) Unilateral (n= 3) Postoperative over dryness 5 (28.6%) 1 0 0 - Compensatory sweating 7 (50%) 0 5 (45.4%) 0 0.570 Temporary Horner’s syndrome 1 (7.14%) 0 0 0 - Recurrence of significant hand sweating 0 0 0 0 0 *Significant as p value < 0.05 between 34 and 66 which is fair; none of the patients scored 67 or above (Table 4). Discussion Hyperhidrosis (especially PH) significantly affects the so- cial life and functional aspects of the affected individuals. Many scoring systems have been proposed for evaluation of the condition and the response to provided treatment [3]. Some children have gross problems at their schools with many of them being bullied by their colleagues, schoolmates, and, even worse, their teachers. An objective validation of the patients/parents assess- ment using the QOL questionnaire has revealed that in group I, 11 patients had scores < 33 which is poor, and 7 patients had score between 34 and 66 (fair). None of the patients scored 67 or above. In group II, 9 patients had scores below 33 (poor), and 5 patients had score Table 4 Change of QOL Group I (18 patients) Group II (14 patients) Preoperative QOL score Postoperative QOL score Preoperative QOL score Postoperative QOL score Good 0 12 0 13 Fair 7 5 5 0 Poor 11 0 9 0 Total number of patients 18 17 (plus one patient lost to follow-up) 14 13 (plus one patient lost to follow-up) Page 6 of 8 Page 6 of 8 Page 6 of 8 Elhalaby et al. Annals of Pediatric Surgery (2021) 17:26 Elhalaby et al. Annals of Pediatric Surgery (2021) 17:26 T3 sympathectomy. A high satisfaction rate (94.2%) was noted in the T4 TS group in his study [13]. Hyperhidrosis has also major psychological effects over both patients and their parents. Many of them suffer from significantly lower self-esteem and self-confidence and sometimes force them to self-isolation and negative thoughts. Despite the good results regarding resolution of PH and cessation of sweating, TS has its potential side ef- fects. Postoperative compensatory sweating or over dry- ness of hands occur in some cases. The incidence and severity of these side effects vary significantly in different published series [14, 15]. These postoperative unfavor- able consequences are the main focus of current research. The appropriate time for surgical intervention has re- ceived some attention in recent years. Some studies have showed that the earlier this condition is addressed, the better is the outcome [4]. Steiner et al. Discussion included 325 pa- tients, 116 were children (8–14 years of age, median = 13), and 209 were adolescents and adults (15–41 years of age, median =18); they concluded that the children who underwent sympathectomies benefited of the effects the procedure on their daily lives [4] and that the inci- dence of side effects of the procedure (e.g., compensa- tory sweating) was significantly lower than that occurred in older adolescents and adults who underwent the same procedure (69.8% vs. 88.5%); in addition, satisfaction rates were also higher 92.2% vs. 80.7% [4]. Compensatory sweating after TS is a common side ef- fect. In the current study, postoperative compensatory sweating developed in both groups (50% versus 45.4%). The incidence of this complication varies widely throughout the literature. Zacherl et al. [14] reported an incidence of 69% in a series of 352 patients. In another study by Fredman et al., 90% of patients had compensa- tory sweating postoperatively [15]. This variability is probably due to the differences in surgical technique and classification of compensatory sweating. One of the topics that needs to be studied and researched is the mechanism of development of CH, since this is not fully understood till now. One of the interesting findings in our study is the onset of occurrence of compensatory sweating as it did not develop in any of the cases after operating on one side and only developed after operating on both sides of the patient. The group of patients who underwent unilateral sympathectomy did not develop any compensatory sweating. Despite the development of compensatory sweating in almost half of the patients, all of them were satisfied with the exception of one case, yet the patient did not regret having the procedure. The overall incidence of compensatory sweating in this study is much less than that in many other series. The impact of sequential bilateral TS rather than simultaneous bilat- eral TS on development of compensatory sweating was highlighted in a study conducted by Youssef et al. where sequential sympathectomy was found to be a more opti- mal technique for reduction of compensatory sweating [16]. Confirming this finding needs further studies and prospective controlled randomized clinical trials. In the majority of published TS procedures, a biportal approach was used with the first port for the scope and the second as a working port. We have successfully used the same approach in all procedures. Discussion There was no need for introducing a third port. Some of the studies how- ever are in favor of introducing a second working port especially in recurrent cases or when adhesions are ex- pected [5]. T2 ganglion has been viewed traditionally as the key pathway for the hands [6–8]. T2 interruption has been found to cause dry hands and denervation of the face. Drott suggested that turning off head and face sweating may be the main trigger for compensatory sweating on the trunk [9]. Schmidt et al. found that avoiding T2 gan- glion may limit compensatory sweating [10]. The most appropriate segment to be interrupted for the treatment of primary PH is still debatable. A previ- ously published anatomic study showed that the pregan- glionic fibers to the arm originate mostly from the third to the sixth spinal segments, and the third and fourth segments were considered as main lesions [11]. Lin and Wu reported that few or none of the fibers from T2 or T3 innervate the hands, whereas fibers from T4 to the skin of the palm definitely pass through T2 and T3 [12]. The current study showed that either T2-T3 or T4 sympathectomy produces resolution of the palmar symp- toms with good satisfaction of the patients. Mild mois- ture hands occurred commonly in T4 TS group; this was well tolerated by the patients. Slight moistness occurred in hot weather mainly, and the satisfaction rate in pa- tients with moist hands was even higher than those with dry hands. The occurrence of slight hand moistness was also reported by Liu et al., who compared T3 versus T4 sympathectomy and it was noted in 59.4% of patients who had T4 sympathectomy vs. 25.8% of those who had The most appropriate segment to be interrupted for the treatment of primary PH is still debatable. A previ- ously published anatomic study showed that the pregan- glionic fibers to the arm originate mostly from the third to the sixth spinal segments, and the third and fourth segments were considered as main lesions [11]. Lin and Wu reported that few or none of the fibers from T2 or T3 innervate the hands, whereas fibers from T4 to the skin of the palm definitely pass through T2 and T3 [12]. Various techniques have been attempted to reduce the occurrence rates of compensatory sweating. Limitation of the study Hand over dryness is a potential side effect of TS sur- gery. In the current study, this was not documented in any of the cases in T4 group but was common (35%) in group T2-T3. A similar high rate (35%) of over dryness of limbs was also observed in of cases following T2 sym- pathectomy for PH by Mahdy et al. [17]. We acknowledge the limitations of the current study; the number is relatively small in the subgroups of pa- tients. The impact of performing bilateral TS at the same time instead of doing it sequentially on the compensa- tory sweating rate needs to be investigated. p y y y The occurrence of Horner’s syndrome is one of the serious potential side effects following the operation. Injury to the sympathetic fibers in T1 (stellate gan- glion) leads to its development. Such injury may occur during the operation resulting from transmitted heat via the diathermy or may be due to traction on the ganglion [18]. This complication was reported by Wait et al. in 5% of 322 patients after sympathectomy and in only 0.9% when sympathicotomy (interruption of the sympathetic trunk by its complete transection) was used. This may point to sympathicotomy being a better option since it avoids extensive manipulation and traction on the sympathetic chain [18]. A study of 85 T2-T3 TH procedures done in 44 children re- ported the occurrence of transient Horner’s syndrome in 18% of cases [19]. Only one case of temporary Horner’s syndrome developed in our study among the T2-T3 group, and it resolved spontaneously within few weeks. The limited development of such side ef- fect in our study could be owed to the limited num- ber of cases in comparison with previous studies; also, much care was given during manipulation at T2 level during surgery. We believe that T4 TS away from stellate ganglion can obviate the occurrence of Horner’s syndrome. Also, the use of bipolar diathermy is always advocated when available rather than mono- polar diathermy which is associated with more heat dispersion. Availability of data and materials Th l t d d t d t i l f th Availability of data and materials The related data and material of the current study are available from the corresponding author on a reasonable request. The related data and material of the current study are available from the corresponding author on a reasonable request. Ethics approval and consent to participate Ethics approval and consent to participate This study was approved by the Research Ethics Committee and quality assurance unit of the faculty of medicine, Tanta University, on February 2019, protocol number 32967/02/19. A written informed consent for participation at this study was obtained from parents/guardians. T4 TS though theoretically means the least denerv- ation of the hands, it produces resolution of the over- sweating of the hands, and the majority of patients would be satisfied by its results [13]. For such reason, and because it is associated with less morbidity, T4 sym- pathectomy is recommended to be the operation of choice since the goal of the treatment of PH is to im- prove the QOL of the patients, rather than the full elim- ination of sweating function of hands. Discussion The level and extent of resection of the sympathetic chain are thought to be the factors behind the frequency and se- verity of compensatory sweating by many authors [14, 15]. The more sympathetic segments excised, especially those including T2, the greater the incidence of severe compensatory symptoms. The current study showed that either T2-T3 or T4 sympathectomy produces resolution of the palmar symp- toms with good satisfaction of the patients. Mild mois- ture hands occurred commonly in T4 TS group; this was well tolerated by the patients. Slight moistness occurred in hot weather mainly, and the satisfaction rate in pa- tients with moist hands was even higher than those with dry hands. The occurrence of slight hand moistness was also reported by Liu et al., who compared T3 versus T4 sympathectomy and it was noted in 59.4% of patients who had T4 sympathectomy vs. 25.8% of those who had In Liu et al.’s study, the overall occurrence rate of compensatory sweating was 66.4% with those in the T3 group being more affected than patients in T4 group. Sweating was found to be mild, and patients did not feel any discomfort in their daily activities [13]. Page 7 of 8 Page 7 of 8 Elhalaby et al. Annals of Pediatric Surgery (2021) 17:26 Elhalaby et al. Annals of Pediatric Surgery (2021) 17:26 Conclusion Based on our study, we may conclude that (1) both T2- T3 and T4 TS are safe and effective treatment options for PH in children and adolescents. (2) Postoperative CH seems to be more frequent with bilateral T2-T3 sympathectomy compared to bilateral T4 sympathec- tomy. (3) Postoperative CH may be less frequent when TS is done sequentially, and it appears to develop only after bilateral sympathectomy is done. (4) Hand over dryness may occur after T2-T3 TS, while mild moisture hands may be expected after T4 TS. (5) Due to the effi- cacy of both approaches and potential less postoperative complications and better patients satisfaction in group II, T4 is more preferred than T2-3 TS. Acknowledgements Not applicable Acknowledgements Not applicable Consent for publication: A written consent for publication of the results of this study was obtained from parents/guardians. Authors’ contributions All authors have read and approved the manuscript and agree with its submission to Annals of Pediatric Surgery. E. E: principle investigator, study design, participated in surgery, collection of data, data analysis, writing the draft and final version of the manuscript. MAM, AI MT, approved study design, participated in surgery and follow-up of the patients, reviewed and approved the final version of the manuscript HFA, senior supervisor, study design, review the draft and final version of the manuscript. Although T4 ganglion is the main source of sympa- thetic innervation of the hand, some sympathetic fiber from T3 and T2 ganglions also add to hand innervation sequentially as impulses ascend along the sympathetic chain. Ablation at T2 level, therefore, blocks the impulse from T3 and T4 ganglions to the hand. Likely, ablation at T3 level blocks the impulse from T4 ganglions. T2 sympathectomy results in the most thorough sympa- thetic denervation of the hands; however, it induces the most common and severe side effects, which makes it a poor option. Abbreviations PH P l h Abbreviations PH: Palmar hyperhidrosis; TS: Thoracoscopic sympathectomy; CH: Compensatory hyperhidrosis; QOL: Quality of life Abbreviations PH: Palmar hyperhidrosis; TS: Thoracoscopic sympathectomy; CH: Compensatory hyperhidrosis; QOL: Quality of life Competing interests Th h d l h The authors declare that they have no competing interests. The authors declare that they have no competing interests. Page 8 of 8 Page 8 of 8 Elhalaby et al. Annals of Pediatric Surgery (2021) 17:26 References Endoscopic t4-sympathetic block by clamping (ESB4) in treatment of hyperhidrosis palmaris et axillaries, experiences of 165 cases. Ann Chir Gynaecol. 2001;90(3):167–9. 13. Liu Y, Yang J, Liu J, et al. Surgical treatment of primary palmar hyperhidrosis: a prospective randomized study comparing T3 and T4 sympathicotomy. Eur J Cardiothorac Surg. 2009;35(3):398–402. 13. Liu Y, Yang J, Liu J, et al. Surgical treatment of primary palmar hyperhidrosis: a prospective randomized study comparing T3 and T4 sympathicotomy. Eur J Cardiothorac Surg. 2009;35(3):398–402. 14. Zacherl J, Imhof M, Huber ER, et al. Video assistance reduces complication rate of thoracoscopic sympathicotomy for hyperhidrosis. Ann Thorac Surg. 1999;68(4):1177–81. 14. Zacherl J, Imhof M, Huber ER, et al. Video assistance reduces complication rate of thoracoscopic sympathicotomy for hyperhidrosis. Ann Thorac Surg. 1999;68(4):1177–81. 15. Fredman B, Zohar E, Shachor D, Bendahan J, Jedeikin R. Video-assisted transthoracic sympathectomy in the treatment of primary hyperhidrosis: friend or foe? Surg Laparosc Endosc Percutan Tech. 2000;10(4):226–9. 16. Youssef T, Soliman M. Unilateral sequential endoscopic thoracic sympathectomy for palmar hyperhidrosis: a proposed technique to overcome compensatory hyperhidrosis and improve plantar hyperhidrosis. J Laparoendosc Adv Surg Tech A. 2015;25(5):370–4. 17. Mahdy T, Youssef T, Elmonem HA, Omar W, Elateef AA. T4 sympathectomy for palmar hyperhidrosis: looking for the right operation. Surgery. 2008; 143(6):784–9. 17. Mahdy T, Youssef T, Elmonem HA, Omar W, Elateef AA. T4 sympathectomy for palmar hyperhidrosis: looking for the right operation. Surgery. 2008; 143(6):784–9. 18. Wait SD, Killory BD, Lekovic GP, et al. Thoracoscopic sympathectomy for hyperhidrosis: analysis of 642 procedures with special attention to Horner’s syndrome and compensatory hyperhidrosis. Neurosurgery. 2010;67(3):652– 65718. 19. Sinha CK, Kiely E. Thoracoscopic sympathectomy for palmar hyperhidrosis in children: 21 years of experience at a tertiary care center. Eur J Pediatr Surg. 2013;23:486–9. References 1. Cohen Z, Shinar D, Levi I, Mares AJ. Thoracoscopic upper thoracic sympathectomy for primary palmar hyperhidrosis in children and adolescents. J Pediatr Surg. 1995;30(3):471–3. 1. Cohen Z, Shinar D, Levi I, Mares AJ. Thoracoscopic upper thoracic sympathectomy for primary palmar hyperhidrosis in children and adolescents. J Pediatr Surg. 1995;30(3):471–3. 2. Krasna MJ, Demmy TL, McKenna RJ, Mack MJ. Thoracoscopic sympathectomy: the U.S. experience. Eur J Surg Suppl. 1998;164(580):19–21. 3. Laje P, Rhodes K, Magee L. Mary Kate Klarich Thoracoscopic bilateral T3 sympathectomy for primary focal hyperhidrosis in children. J Pediatr Surg. 2017;52:313–6. 3. Laje P, Rhodes K, Magee L. Mary Kate Klarich Thoracoscopic bilateral T3 sympathectomy for primary focal hyperhidrosis in children. J Pediatr Surg. 2017;52:313–6. 4. Steiner Z. Cohen, Kleiner O, Matar I, Mogilner J. Do children tolerate thoracoscopic sympathectomy better than adults? Pediatr Surg Int. 2008;24: 343–7. 4. Steiner Z. Cohen, Kleiner O, Matar I, Mogilner J. Do children tolerate thoracoscopic sympathectomy better than adults? Pediatr Surg Int. 2008;24: 343–7. 5. Reisfeld R, Nguyen R, Pnini A. Endoscopic thoracic sympathectomy for hyperhidrosis: experience with both cauterization and clamping methods. Surg Laparo Endo Per Tech. 2002;12(4):255–67. 5. Reisfeld R, Nguyen R, Pnini A. Endoscopic thoracic sympathectomy for hyperhidrosis: experience with both cauterization and clamping methods. Surg Laparo Endo Per Tech. 2002;12(4):255–67. 6. Lin TS, Fang HY. Transthoracic endoscopic sympathectomy in the treatment of palmar hyperhidrosis with emphasis on perioperative management (1360 case analyses). Surg Neurol. 1999;52(5):453–7. 6. Lin TS, Fang HY. Transthoracic endoscopic sympathectomy in the treatment of palmar hyperhidrosis with emphasis on perioperative management (1360 case analyses). Surg Neurol. 1999;52(5):453–7. 7. Chiou TS, Liao KK. Orientation landmarks of endoscopic transaxillary T-2 sympathectomy for palmar hyperhidrosis. J Neurosurg. 1996;85(2):310–5. 8. Drott C, Gothberg G, Claes G. Endoscopic transthoracic sympathectomy: an efficient and safe method for the treatment of hyperhidrosis. J Am Acad Dermatol. 1995;33(1):78–81. 9. Drott C. Results of endoscopic thoracic sympathectomy (ETS) on hyperhidrosis, facial blushing, angina pectoris, vascular disorders and pain syndromes of the hand and arm. Clin Auton Res. 2003;13(1):26–30. 10. Schmidt J, Bechara FG, Altmeyer P, Zirngibl H. Endoscopic thoracic sympathectomy for severe hyperhidrosis: impact of restrictive denervation on compensatory sweating. Ann Thorac Surg. 2006;81(3):1048–55. 11. Gray H. The sympathetic nerves. In: Lewis WH Anatomy of the human body. 20th ed; 2000. p. 1292–9. 12. Lin CC, Wu HH. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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Erythropoiesis-stimulating agent dosing, haemoglobin and ferritin levels in UK haemodialysis patients 2005–13
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Birnie, K., Caskey, F. J., Ben-Shlomo, Y., Sterne, J. A. C., Gilg, J., Nitsch, D., & Tomson , C. (2017). Erythropoiesis-stimulating agent dosing, haemoglobin and ferritin levels in UK haemodialysis patients 2005–13. Nephrology Dialysis Transplantation, 32(4), 692-698. https://doi.org/10.1093/ndt/gfw043 Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1093/ndt/gfw043 Link to publication record on the Bristol Research Portal PDF-document © The Author 2016. Published by Oxford University Press on behalf of ERA-EDTA. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. © The Author 2016. Published by Oxford University Press on behalf of ERA-EDTA. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. © The Author 2016. Published by Oxford University Press on behalf of ERA-EDTA. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Birnie, K., Caskey, F. J., Ben-Shlomo, Y., Sterne, J. A. C., Gilg, J., Nitsch, D., & Tomson , C. (2017). Erythropoiesis-stimulating agent dosing, haemoglobin and ferritin levels in UK haemodialysis patients 2005–13. Nephrology Dialysis Transplantation, 32(4), 692-698. https://doi.org/10.1093/ndt/gfw043 Birnie, K., Caskey, F. J., Ben-Shlomo, Y., Sterne, J. A. C., Gilg, J., Nitsch, D., & Tomson , C. (2017). Erythropoiesis-stimulating agent dosing, haemoglobin and ferritin levels in UK haemodialysis patients 2005–13. Nephrology Dialysis Transplantation, 32(4), 692-698. https://doi.org/10.1093/ndt/gfw043 University of Bristol – Bristol Research Portal General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/brp-terms/ 9. Daugirdas JT, Chertow GM, Larive B et al. Effects of frequent hemodialysis on measures of CKD mineral and bone disorder. J Am Soc Nephrol 2012; 23: 727–738 19. ANSI/AAMI. Quality of dialysis fluid for hemodialysis and related therapies. Standard 11663. ANSI/AAMI, 2009 20. Grassmann A, Gioberge S, Moeller S et al. ESRD patients in 2004: global overview of patient numbers, treatment modalities and associated trends. Nephrol Dial Transplant 2005; 20: 2587–2593 10. Chan CT, Greene T, Chertow GM et al. Determinants of left ventricular mass in patients on hemodialysis: Frequent Hemodialysis Network (FHN) Trials. Circ Cardiovasc Imaging 2012; 5: 251–261 p p 21. European Best Practice Guidelines Expert Group on Hemodialysis ERA. Section II. Haemodialysis adequacy. Nephrol Dial Transplant 2002; 17 (Suppl. 7): 16–31 11. Pauly RP, Gill JS, Rose CL et al. Survival among nocturnal home haemodial- ysis patients compared to kidney transplant recipients. Nephrol Dial Transplant 2009; 24: 2915–2919 22. Benedict FG, Root HF. Insensible perspiration: its relation to human physi- ology and pathology. Arch Intern Med 1926; 38: 1–5 12. Young BA, Chan C, Blagg C et al. How to overcome barriers and establish a successful home HD program. Clin J Am Soc Nephrol 2012; 7: 2023–2032 23. Cox P. Insensible water loss and its assessment in adult patients: a review. Acta Anaesthesiol Scand 1987; 31: 771–776 13. European Best Practice Guidelines Expert Group on Hemodialysis, European Renal Association. Section V. Chronic intermittent haemodialysis and prevention of clotting in the extracorporal system. Nephrol Dial Transplant 2002; 17 (Suppl. 7): 63–71 24. Guyton AC. Extracellular and intracellular fluids; interstitial fluid and edema. In: Guyton AC, ed. Textbook of Medical Physiology. Philadelphia: Saunders; 1991, pp. 274–285 14. Davenport A. What are the anticoagulation options for intermittent hemo- dialysis? Nat Rev Nephrol 2011; 7: 499–508 25. Daugirdas JT, Depner TA, Greene T et al. Standard Kt/Vurea: a method of calculation that includes effects of fluid removal and residual kidney clear- ance. Kidney Int 2010; 77: 637–644 15. Daugirdas JT. Second generation logarithmic estimates of single-pool varia- ble volume Kt/V: an analysis of error. J Am Soc Nephrol 1993; 4: 1205–1213 26. Daugirdas JT, Leypoldt JK, Akonur A et al. Improved equation for estimat- ing single-pool Kt/V at higher dialysis frequencies. Erythropoiesis-stimulating agent dosing, haemoglobin and ferritin levels in UK haemodialysis patients 2005–13 Kate Birnie1, Fergus Caskey1,2,3, Yoav Ben-Shlomo1, Jonathan A.C. Sterne1, Julie Gilg2, Dorothea Nitsch and Charles Tomson5 ey1,2,3, Yoav Ben-Shlomo1, Jonathan A.C. Sterne1, Julie Gilg2, Dorothea Nitsch4 1School of Social and Community Medicine, University of Bristol, Canynge Hall, 39 Whatley Road, Clifton, Bristol BS8 2PS, UK, 2UK Renal Registry, Southmead Hospital, Bristol, UK, 3Renal Unit, Southmead Hospital, North Bristol NHS Trust, Bristol, UK, 4Department of Non- communicable Disease Epidemiology, London School of Hygiene & Tropical Medicine, London, UK and 5Department of Renal Medicine, Freeman Hospital, Newcastle upon Tyne, UK Correspondence and offprint requests to: Kate Birnie; E-mail kate.birnie@bristol.ac.uk University of Bristol – Bristol Research Portal General rights Nephrol Dial Transplant 2013; 28: 2156–2160 16. Leypoldt JK. Urea standard Kt/Vurea for assessing dialysis treatment adequacy. Hemodial Int 2004; 8: 193–197 17. Leypoldt JK, Jaber BL, Zimmerman DL. Predicting treatment dose for novel therapies using urea standard Kt/V. Semin Dial 2004; 17: 142–145 27. Leypoldt JK, Bernardo A, Muller M et al. Evaluation of standard urea Kt/V for frequent haemodialysis prescriptions [abstract]. Nephrol Dial Transplant 2014; 29 (Suppl. 3): iii466–iii467 18. National Kidney Foundation. KDOQI Clinical Practice Guidelines and Clinical Practice Recommendations for 2006 Updates: Hemodialysis Adequacy, Peritoneal Dialysis Adequacy and Vascular Access. Am J Kidney Dis 2006; 48 (Suppl. 1): S1–S322 Received: 3.8.2015; Editorial decision: 10.2.2016 Nephrol Dial Transplant (2017) 32: 692–698 doi: 10.1093/ndt/gfw043 Advance Access publication 25 March 2016 METHODS Nearly 57 000 adult patients were receiving renal replacement therapy (RRT) in the UK at the end of 2013 (prevalence 888/ million), and demand for RRT is rising [1]. Anaemia is a com- mon complication for patients with chronic kidney disease (CKD), resulting in reduced quality of life, fatigue, decreased exercise capacity and shortness of breath. Erythropoiesis- stimulating agents (ESAs) are the main treatment for CKD patients with anaemia [2] and are effective in correcting and maintaining haemoglobin (Hb) levels and decreasing the need for blood transfusions. Intravenous (IV) iron supplements are an important adjunct in the treatment of anaemia in patients on haemodialysis. We identified RCTs and treatment guidelines with the potential to affect anaemia management for patients on haemodialysis during the period 2005–13. These were used to construct a timeline depicting when changes in management were expected. p The UK Renal Registry (UKRR) collects clinical and bio- chemical data from all patients 18 years of age receiving RRT in the UK. Data are extracted electronically, on a quarterly basis, and are validated and cleaned prior to analysis [14]. Some centres submit data extracts that include ESA doses. All patients who received haemodialysis in 2005–13 in centres submitting ESA doses were included in the analyses. Centres reporting fewer than 60% of haemodialysis patients being treated with ESAs were considered to have incomplete data and their data for that quarter were excluded. Patients from 37 centres across England, Wales and Northern Ireland were included in the analyses (from a total of 62 centres in these regions). A patient was considered to be incident during their first year of haemodialysis. Even partial treatment of anaemia with ESAs is expensive, and full correction of Hb requires much higher doses than par- tial correction. When ESAs were first introduced into clinical practice, randomized controlled trials (RCTs) were not per- formed, as it was thought unnecessary and even unethical to leave a control group with untreated anaemia. However, the question of whether full correction of anaemia is preferable to partial correction has now been tested in several RCTs. Besarab et al. [3] randomized haemodialysis patients with cardiovascu- lar comorbidity to full or partial correction of anaemia: this trial was terminated early when it became clear that benefit from full correction was highly improbable. ABSTRACT observational studies suggest better outcomes for patients who achieve higher haemoglobin (Hb) levels, randomized controlled trials comparing higher and lower Hb targets have led to safety concerns over higher targets and to changes in treatment guidelines. Background: Erythropoiesis-stimulating agents (ESAs) with intravenous iron supplementation are the main treatment for anaemia in patients with chronic kidney disease. Although V C The Author 2016. Published by Oxford University Press on behalf of ERA- EDTA. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 69 V C The Author 2016. Published by Oxford University Press on behalf of ERA- EDTA. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 692 completed [6–8] and found no evidence of benefits of a higher (compared with lower) Hb target on cardiovascular events or a composite outcome of death, myocardial infarction, hospitaliza- tion for congestive heart failure and stroke, or they found increased risk of adverse events. These conclusions are difficult to reconcile with data from numerous observational studies that demonstrate the best outcomes are in patients who have high Hb concentrations but require only low doses of ESAs (130–140 g/L) [9–11]. One interpretation is that ESA-sensitive patients derive survival benefit from full correction of anaemia, but that patients with ESA resistance (which is a predictor of poor sur- vival) may be harmed by the high doses of ESAs prescribed under the trial protocols in the (often unsuccessful) attempt to normalize Hb concentration. Methods: Quarterly data from 2005 to 2013 were obtained on 28 936 haemodialysis patients from the UK Renal Registry. We examined trends in ESA use and average dose, Hb and ferritin values over time and Hb according to the UK Renal Association guideline range. Results: The average ESA dose declined over time, with sharper decreases of epoetin seen towards the end of 2006 and from 2009. ABSTRACT Average Hb for patients on ESAs was 114.1 g/L [95% con- fidence interval (CI) 113.7, 114.6] in the first quarter of 2005, which decreased to 109.6 g/L (95% CI 109.3, 109.9) by the end of 2013. Average serum ferritin was 353 mg/L (95% CI 345, 360) at the start of 2005, increasing to 386 mg/L (95% CI 380, 392) in the final quarter of 2013. The percentage of patients with Hb in the range of 100–120 g/L increased from 46.1 at the start of 2005 to 57.6 at the end of 2013. The target Hb for patients with CKD in the UK Renal Association Clinical Practice Guidelines (3rd edition, 2002–07) was >100 g/L with no suggested maximum [12]. These were altered to 105–125 g/L (4th edition, 2007–09) [13] and lowered to 100–120 g/L in the latest version (5th edition, 2010) [2] fol- lowing publication of the RCTs in 2006 [6, 8] and 2009 [7]. The impact of these guideline changes on ESA dosing and hence on achieved Hb levels remains unclear. We, therefore, examined trends in ESA dosing in UK haemodialysis patients between 2005 and 2013 and corresponding trends in achieved Hb levels. We related these trends to changes in the national guidelines for management of anaemia. Conclusions: Anaemia management patterns for haemodialysis patients changed in the UK between 2005 and 2013. These pat- terns most likely reflect clinician response to emerging trial evi- dence and practice guidelines. Registries play an important role in continued observation of anaemia management and will monitor further changes as new evidence on optimal care emerges. Keywords: anaemia management, erythropoiesis-stimulating agents, ESA, haemodialysis, haemoglobin E S A d o s e , H b a n d f e r r i t i n l e v e l s i n U K H D p a t i e n t s 2 0 0 5 – 1 3 METHODS The US Food and Drug Administration later classified the trial results as showing increased risk of all-cause mortality for the group randomized to the higher Hb target [4] and a later reanalysis of the full trial report showed no improvement in quality of life [5]. Three large RCTs in patients with CKD not yet on dialysis have been Patients were defined as being on an ESA if a drug type (dar- bepoetin, epoetin-a, -b or not otherwise specified, or methoxy polyethylene glycol-epoetin b) and/or a dose was recorded. ESA dose is presented as the weekly epoetin dose in our analysis. Doses of 150 IU/week (likely to be darbepoetin) were harmonized with epoetin data by multiplying by 200. No adjustments were made with respect to route or frequency of administration. The measurement of Hb (g/L) that was closest to the end of the quarter was extracted. Serum ferritin was E S A d o s e , H b a n d f e r r i t i n l e v e l s i n U K H D p a t i e n t s 2 0 0 5 – 1 3 693 measured in mg/L. Data were also extracted on age, sex, race (White, South Asian, Black, Chinese, other or missing), year of starting RRT and primary renal disease (diabetes, glomerulo- nephritis, hypertension, polycystic kidneys, pyelonephritis, renal vascular disease, other or uncertain [15]). Hb target of 135 or 113 g/L. There was evidence of increased risk of the primary outcome (a composite of death, myocardial infarction and hospitalization for heart failure or stroke) in the high-Hb compared with the low-Hb group [hazard ratio (HR) 1.34 (95% CI 1.03, 1.74)]. In the CREATE trial, also published in 2006 [6], 603 patients with CKD not on RRT were random- ized to a target Hb of 130–150 or 105–115 g/L. Early complete correction of anaemia did not reduce the risk of cardiovascular events. In the TREAT trial, which was the largest RCT and was published in 2009 [7], 4038 patients with diabetes, CKD and anaemia were randomly assigned to achieve a Hb level of 130 g/L with the use of darbepoetin alfa or placebo (patients in the placebo group received darbepoetin alfa as a rescue agent if Hb <90 g/L). Statistical methods For each calendar year, we derived descriptive statistics sum- marizing the demographic and clinical characteristics of the cohort. ESA dose and ferritin were log transformed, as they had positively skewed distributions. The percentage of patients on the different ESA drug types were calculated for each quarter. To examine trends in anaemia management over time, we cal- culated the geometric mean of the ESA dose [with 95% confi- dence interval (CI)] per quarter, stratified by drug type [(i) darbepoetin and (ii) epoetin-a, -b or not otherwise specified; there were not enough numbers to also include a methoxy poly- ethylene glycol-epoetin b group]. The doses were also examined restricting to incident patients. The mean achieved Hb levels were calculated separately for ESA-treated and non-treated patients. We calculated the proportion of all patients on ESA treatment over time and the proportion of patients with Hb in the categories <100, 100–120 and >120 g/L. Patients were also put into categories according to their ESA status: those always receiving ESAs over the time period, patients changing on/off ESAs over the time period and those never receiving ESAs. Trends in these statistics over time were compared with the timeline of events with the potential to affect anaemia manage- ment. All means and proportions were standardized by year using direct standardization according to the age, sex, race and primary renal disease distribution for all years of data com- bined. The numbers with missing values for these factors were reported and people with missing values were excluded from the analyses. All analyses were carried out using Stata version 13 (StataCorp, College Station, TX, USA). METHODS The risk of a composite outcome of death or cardio- vascular event was similar in the two groups [HR for darbepoe- tin alfa versus placebo 1.05 (95% CI 0.94, 1.17)], but there was a higher incidence of stroke in the darbepoetin alfa arm [HR 1.92 (95% CI 1.38, 2.68)]. Following publication of these RCTs, the target Hb for patients with CKD was lowered to 100–120 g/L in UK Renal Association and National Institute for Health and Care Excellence (NICE) treatment guidelines [2, 16]. The Kidney Disease: Improving Global Outcomes (KDIGO) 2012 guidelines suggest that ESA therapy should be started when Hb is between 90 and 100 g/L for adult dialysis patients and that ESAs should not be used to maintain an Hb concentration >115 g/L in adult patients with CKD, but that individualization of therapy will be necessary as some patients may have improvements in quality of life at an Hb concentration >115 g/ L and will be prepared to accept the risks [17]. Characteristics of the study cohort A total of 28 936 haemodialysis patients were included in the cohort. A total of 1659 patients (5.9%) never received ESAs within the study period (2005–13), 6648 (23.0%) changed on/ off ESAs over the time period and 20 593 (71.2%) always received ESA therapy. Darbepoetin was the drug used most fre- quently (51.3%), followed by epoetin-b (21.8%), epoetin-a (17.3%), epoetin not otherwise specified (7.4%) and methoxy polyethylene glycol-epoetin b (2.3%). Demographic characteris- tics of the yearly cohorts are shown in Table 1. The number of patients included in the cohort almost doubled over time, but the age, sex and race distributions changed very little. In 2013, the median age of patients was 66.9 years (interquartile range 54.1–76.8), with 7726 (61.3%) male and 10 095 (80.7%) of K. Birnie et al. Timeline of events that may have affected anaemia management The number of incident patients included increased steadily over time from N ¼ 1423 in 2005 to N ¼ 2193 in 2013. Timeline of events that may have affected anaemia management The dashed vertical lines indicate the publication of the CHOIR and CREATE RCTs (2006) and TREAT (2009). FIGURE 2: Geometric mean ESA weekly dose and 95% CI. The dashed vertical lines indicate the publication of the CHOIR and CREATE RCTs (2006) and TREAT (2009). ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||| White race. The proportion of patients with diabetes as the pri- mary renal disease increased over time, from 17.4% of patients h b f d epoetin drugs and showed a steady increase for darbepoetin (Figure 2), while the proportion of patients on ESAs remained relatively constant (Figure 3). Near the end of 2006 and in early 2007, there appears to be a decline in the proportion of patients on ESAs, with a modest dose reduction, most likely in response to the joint publication of the NICE guideline [18] and the Table 1. Timeline of events that may have affected anaemia management Characteristics of the cohort by year 2005 2006 2007 2008 2009 2010 2011 2012 2013 Number of patients Incident 1423 1645 1703 1840 1895 1870 1842 1960 2193 Total (including incident) 6930 7896 8637 9419 10 326 10 648 10 824 11 462 12 609 Age, years, median (IQR) 65.5 (51.5–75.0) 66.0 (52.1–75.4) 65.9 (52.4–75.5) 66.1 (52.9–75.7) 66.4 (53.2–75.9) 66.7 (53.7–76.2) 66.8 (53.5–76.5) 67.0 (53.9–76.6) 66.9 (54.1–76.8) Sex, n (%) Male 4216 (60.8) 4880 (61.8) 5346 (61.9) 5784 (61.4) 6262 (60.6) 6471 (60.8) 6579 (60.8) 7037 (61.4) 7726 (61.3) Female 2714 (39.2) 3016 (38.2) 3291 (38.1) 3635 (38.6) 4064 (39.4) 4177 (39.2) 4245 (39.2) 4425 (38.6) 4883 (38.7) Race, n (%) White 5477 (83.6) 6286 (84.1) 6884 (83.4) 7580 (83.4) 8419 (83.2) 8783 (83.2) 8822 (81.9) 9489 (82.9) 10 095 (80.7) Black 295 (4.5) 328 (4.4) 367 (4.5) 400 (4.4) 467 (4.6) 457 (4.3) 514 (4.8) 499 (4.4) 779 (6.2) South Asian 677 (10.3) 751 (10.1) 882 (10.7) 983 (10.8) 1073 (10.6) 1143 (10.8) 1263 (11.7) 1266 (11.1) 1403 (11.2) Chinese 39 (0.6) 41 (0.6) 46 (0.6) 45 (0.5) 50 (0.5) 55 (0.5) 51 (0.5) 49 (0.4) 68 (0.5) Other 64 (1.0) 70 (0.9) 75 (0.9) 81 (0.9) 105 (1.0) 125 (1.2) 128 (1.2) 138 (1.2) 160 (1.3) Missing 378 420 383 330 212 85 46 21 104 Primary renal disease, n (%) Diabetes 1182 (17.4) 1400 (18.0) 1581 (18.6) 1792 (19.4) 2004 (19.7) 2065 (19.6) 2191 (20.4) 2351 (20.7) 2718 (21.8) Glomerulonephritis 1027 (15.1) 1184 (15.2) 1287 (15.2) 1389 (15.0) 1509 (14.8) 1565 (14.9) 1577 (14.7) 1700 (15.0) 1875 (15.1) Hypertension 386 (5.7) 400 (5.2) 458 (5.4) 510 (5.5) 599 (5.9) 637 (6.1) 642 (6.0) 747 (6.6) 864 (6.9) Polycystic kidneys 491 (7.2) 565 (7.3) 605 (7.1) 656 (7.1) 704 (6.9) 732 (7.0) 702 (6.5) 772 (6.8) 827 (6.6) Pyelonephritis 725 (10.6) 814 (10.5) 948 (11.2) 992 (10.7) 1056 (10.4) 1035 (9.8) 1024 (9.6) 1046 (9.2) 1141 (9.2) Renal vascular disease 561 (8.2) 640 (8.2) 678 (8.0) 702 (7.6) 736 (7.2) 780 (7.4) 769 (7.2) 801 (7.1) 767 (6.2) Other 965 (14.2) 1113 (14.3) 1184 (13.9) 1345 (14.5) 1485 (14.6) 1598 (15.2) 1667 (15.5) 1776 (15.6) 1955 (15.7) Uncertain 1477 (21.7) 1652 (21.3) 1751 (20.6) 1865 (20.2) 2079 (20.4) 2115 (20.1) 2156 (20.1) 2167 (19.1) 2314 (18.6) Missing 116 128 145 168 154 121 96 102 148 FIGURE 3: Percentage use of ESAs with 95% CIs, in all haemodialy- sis patients. Timeline of events that may have affected anaemia management The dashed vertical lines indicate the publication of the CHOIR and CREATE RCTs (2006) and TREAT (2009). FIGURE 2: Geometric mean ESA weekly dose and 95% CI. The dashed vertical lines indicate the publication of the CHOIR and CREATE RCTs (2006) and TREAT (2009). ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Table 1. Characteristics of the cohort by year FIGURE 3: Percentage use of ESAs with 95% CIs, in all haemodialy- sis patients. The dashed vertical lines indicate the publication of the CHOIR and CREATE RCTs (2006) and TREAT (2009). FIGURE 2: Geometric mean ESA weekly dose and 95% CI. The dashed vertical lines indicate the publication of the CHOIR and CREATE RCTs (2006) and TREAT (2009). FIGURE 3: Percentage use of ESAs with 95% CIs, in all haemodialy- sis patients. The dashed vertical lines indicate the publication of the CHOIR and CREATE RCTs (2006) and TREAT (2009). FIGURE 2: Geometric mean ESA weekly dose and 95% CI. The dashed vertical lines indicate the publication of the CHOIR and CREATE RCTs (2006) and TREAT (2009). epoetin drugs and showed a steady increase for darbepoetin (Figure 2), while the proportion of patients on ESAs remained relatively constant (Figure 3). Near the end of 2006 and in early 2007, there appears to be a decline in the proportion of patients on ESAs, with a modest dose reduction, most likely in response to the joint publication of the NICE guideline [18] and the CHOIR and CREATE trials [4, 6] (see Figure 1). The decline in patients treated with ESAs continues until 2013 [percentage of patients receiving ESAs decreases from 92.8 (95% CI 92.0, 93.5) in the first quarter of 2005 to 87.1 (95% CI 86.5, 87.8) in the final quarter of 2013]. There is an overall decline in average dar- bepoetin dose from mid-2006. Average doses of epoetin drugs declined from 2006, followed by a rise in 2008–09, followed by a White race. The proportion of patients with diabetes as the pri- mary renal disease increased over time, from 17.4% of patients in 2005 to 21.8% in 2013. The number of incident patients included increased steadily over time from N ¼ 1423 in 2005 to N ¼ 2193 in 2013. White race. The proportion of patients with diabetes as the pri- mary renal disease increased over time, from 17.4% of patients in 2005 to 21.8% in 2013. Timeline of events that may have affected anaemia management Events that may have affected anaemia management in the UK are summarized in Figure 1. The CHOIR trial, published in 2006 [8], randomized 1432 patients with CKD not on RRT to a FIGURE 1: Timeline of events. FIGURE 1: Timeline of events. FIGURE 1: Timeline of events. K. Birnie et al. 694 ||||||||||||||||||||||||||||| White race. The proportion of patients with diabetes as the pri- mary renal disease increased over time, from 17.4% of patients in 2005 to 21.8% in 2013. The number of incident patients included increased steadily over time from N ¼ 1423 in 2005 to N ¼ 2193 in 2013. epoetin drugs and showed a steady increase for darbepoetin (Figure 2), while the proportion of patients on ESAs remained relatively constant (Figure 3). Near the end of 2006 and in early 2007, there appears to be a decline in the proportion of patients on ESAs, with a modest dose reduction, most likely in response to the joint publication of the NICE guideline [18] and the CHOIR and CREATE trials [4, 6] (see Figure 1). The decline in patients treated with ESAs continues until 2013 [percentage of patients receiving ESAs decreases from 92.8 (95% CI 92.0, 93.5) in the first quarter of 2005 to 87.1 (95% CI 86.5, 87.8) in the Table 1. Timeline of events that may have affected anaemia management Characteristics of the cohort by year 2005 2006 2007 2008 2009 2010 2011 2012 2013 Number of patients Incident 1423 1645 1703 1840 1895 1870 1842 1960 2193 Total (including incident) 6930 7896 8637 9419 10 326 10 648 10 824 11 462 12 609 Age, years, median (IQR) 65.5 (51.5–75.0) 66.0 (52.1–75.4) 65.9 (52.4–75.5) 66.1 (52.9–75.7) 66.4 (53.2–75.9) 66.7 (53.7–76.2) 66.8 (53.5–76.5) 67.0 (53.9–76.6) 66.9 (54.1–76.8) Sex, n (%) Male 4216 (60.8) 4880 (61.8) 5346 (61.9) 5784 (61.4) 6262 (60.6) 6471 (60.8) 6579 (60.8) 7037 (61.4) 7726 (61.3) Female 2714 (39.2) 3016 (38.2) 3291 (38.1) 3635 (38.6) 4064 (39.4) 4177 (39.2) 4245 (39.2) 4425 (38.6) 4883 (38.7) Race, n (%) White 5477 (83.6) 6286 (84.1) 6884 (83.4) 7580 (83.4) 8419 (83.2) 8783 (83.2) 8822 (81.9) 9489 (82.9) 10 095 (80.7) Black 295 (4.5) 328 (4.4) 367 (4.5) 400 (4.4) 467 (4.6) 457 (4.3) 514 (4.8) 499 (4.4) 779 (6.2) South Asian 677 (10.3) 751 (10.1) 882 (10.7) 983 (10.8) 1073 (10.6) 1143 (10.8) 1263 (11.7) 1266 (11.1) 1403 (11.2) Chinese 39 (0.6) 41 (0.6) 46 (0.6) 45 (0.5) 50 (0.5) 55 (0.5) 51 (0.5) 49 (0.4) 68 (0.5) Other 64 (1.0) 70 (0.9) 75 (0.9) 81 (0.9) 105 (1.0) 125 (1.2) 128 (1.2) 138 (1.2) 160 (1.3) Missing 378 420 383 330 212 85 46 21 104 Primary renal disease, n (%) Diabetes 1182 (17.4) 1400 (18.0) 1581 (18.6) 1792 (19.4) 2004 (19.7) 2065 (19.6) 2191 (20.4) 2351 (20.7) 2718 (21.8) Glomerulonephritis 1027 (15.1) 1184 (15.2) 1287 (15.2) 1389 (15.0) 1509 (14.8) 1565 (14.9) 1577 (14.7) 1700 (15.0) 1875 (15.1) Hypertension 386 (5.7) 400 (5.2) 458 (5.4) 510 (5.5) 599 (5.9) 637 (6.1) 642 (6.0) 747 (6.6) 864 (6.9) Polycystic kidneys 491 (7.2) 565 (7.3) 605 (7.1) 656 (7.1) 704 (6.9) 732 (7.0) 702 (6.5) 772 (6.8) 827 (6.6) Pyelonephritis 725 (10.6) 814 (10.5) 948 (11.2) 992 (10.7) 1056 (10.4) 1035 (9.8) 1024 (9.6) 1046 (9.2) 1141 (9.2) Renal vascular disease 561 (8.2) 640 (8.2) 678 (8.0) 702 (7.6) 736 (7.2) 780 (7.4) 769 (7.2) 801 (7.1) 767 (6.2) Other 965 (14.2) 1113 (14.3) 1184 (13.9) 1345 (14.5) 1485 (14.6) 1598 (15.2) 1667 (15.5) 1776 (15.6) 1955 (15.7) Uncertain 1477 (21.7) 1652 (21.3) 1751 (20.6) 1865 (20.2) 2079 (20.4) 2115 (20.1) 2156 (20.1) 2167 (19.1) 2314 (18.6) Missing 116 128 145 168 154 121 96 102 148 FIGURE 3: Percentage use of ESAs with 95% CIs, in all haemodialy- sis patients. Use of ESAs, 2005–13 Prior to 2007, when existing guidelines had no upper limit to Hb levels, the geometric mean dose of ESA remained stable for 695 E S A d o s e , H b a n d f e r r i t i n l e v e l s i n U K H D p a t i e n t s 2 0 0 5 – 1 3 (Figure 4). The mean Hb for patients on ESA in the first quarter of 2005 was 114.1 g/L (95% CI 113.7, 114.6) and was 109.6 g/L FIGURE 7: Geometric mean ferritin with 95% CI in all haemodialy- sis patients. The dashed vertical lines indicate the publication of the CHOIR and CREATE RCTs (2006) and TREAT (2009). FIGURE 6: Percentages in the Hb groups (<100, 100–120 and >120 g/L) in all haemodialysis patients. Dark grey represents Hb <100 g/ L, mid-grey represents Hb 100–120 g/L and light grey represents Hb >120 g/L. FIGURE 6: Percentages in the Hb groups (<100, 100–120 and >120 g/L) in all haemodialysis patients. Dark grey represents Hb <100 g/ L, mid-grey represents Hb 100–120 g/L and light grey represents Hb >120 g/L. FIGURE 4: Mean Hb levels over time with 95% CIs in all haemodial- ysis patients. Circles represent patients not receiving ESAs; triangles represent patients receiving ESAs. The dashed vertical lines indicate the publication of the CHOIR and CREATE RCTs (2006) and TREAT (2009). b l l h C ll h d FIGURE 6: Percentages in the Hb groups (<100, 100–120 and >120 g/L) in all haemodialysis patients. Dark grey represents Hb <100 g/ L, mid-grey represents Hb 100–120 g/L and light grey represents Hb >120 g/L. FIGURE 4: Mean Hb levels over time with 95% CIs in all haemodial- ysis patients. Circles represent patients not receiving ESAs; triangles represent patients receiving ESAs. The dashed vertical lines indicate the publication of the CHOIR and CREATE RCTs (2006) and TREAT (2009). FIGURE 7: Geometric mean ferritin with 95% CI in all haemodialy- sis patients. The dashed vertical lines indicate the publication of the CHOIR and CREATE RCTs (2006) and TREAT (2009). FIGURE 5: Mean Hb levels over time in all haemodialysis patients by ESA status. Crosses represent patients always receiving ESAs; tri- angles represent patients changing on/off ESAs over the time period; circles represent patients never receiving ESAs. Use of ESAs, 2005–13 The decrease in average Hb levels is then observed for many years—when further guidelines and the TREAT trial [7] were published—until a settling of Hb levels in 2012 and 2013. second decline from around 2010 after the publication of the TREAT trial [7] and further published guidelines. The average darbepoetin dose declined from 6431 IU/week (95% CI 6221, 6649) in the first quarter of 2005 to 5921 IU/week (95% CI 5780, 6066) in the last quarter of 2013. The average epoetin dose declined from 8107 IU/week (95% CI 7884, 8336) in the first quarter of 2005 to 7310 IU/week (95% CI 7133, 7491) in the last quarter of 2013. The percentage of patients on the differ- ent ESA drug types is shown in the online supplement (Supplementary data, Figure S1). Similar ESA dose patterns were observed in incident patients (Supplementary data, Figure S2). Use of ESAs, 2005–13 The dashed vertical lines indicate the publication of the CHOIR and CREATE RCTs (2006) and TREAT (2009). FIGURE 7: Geometric mean ferritin with 95% CI in all haemodialy- sis patients. The dashed vertical lines indicate the publication of the CHOIR and CREATE RCTs (2006) and TREAT (2009). FIGURE 5: Mean Hb levels over time in all haemodialysis patients by ESA status. Crosses represent patients always receiving ESAs; tri- angles represent patients changing on/off ESAs over the time period; circles represent patients never receiving ESAs. The dashed vertical lines indicate the publication of the CHOIR and CREATE RCTs (2006) and TREAT (2009). (Figure 4). The mean Hb for patients on ESA in the first quarter of 2005 was 114.1 g/L (95% CI 113.7, 114.6) and was 109.6 g/L (95% CI 109.3, 109.9) by the end of 2013. The decline in mean Hb appears to start in 2007, following NICE guidelines [18] and the publication of the CHOIR [8] and CREATE [6] trials in 2006. The decrease in average Hb levels is then observed for many years—when further guidelines and the TREAT trial [7] were published—until a settling of Hb levels in 2012 and 2013. The lowering in Hb was seen for patients who were receiving ESAs, those not on treatment and across treatment groups (always on ESAs, changing on/off ESAs, never on ESAs; Figure 5), although the numbers of patients never receiving ESAs were small and the pattern showed large fluctuations. The percentage of patients with Hb >120 g/L has decreased over time, from 36.3 at the start of 2005 to 22.4 at the end of 2013 (Figure 6). The percentage of patients with Hb in the range of 100–120 g/L has increased from 46.1 at the start of 2005 to 57.6 at the end of 2013. From the second quarter of 2011 onwards, >55% of haemodialysis patients have had Hb levels in the range of 100–120 g/L. In contrast to average Hb levels, there has been a sharp increase in ferritin levels from 2008 to 2011 (Figure 7), (Figure 4). The mean Hb for patients on ESA in the first quarter of 2005 was 114.1 g/L (95% CI 113.7, 114.6) and was 109.6 g/L (95% CI 109.3, 109.9) by the end of 2013. The decline in mean Hb appears to start in 2007, following NICE guidelines [18] and the publication of the CHOIR [8] and CREATE [6] trials in 2006. Achieved Hb and ferritin, 2005–13 The Hb patterns for patients on ESAs mirror what is seen with ESA dosage, so that we observed an initial small increase in mean Hb until the end of 2006 followed by a steady decline K. Birnie et al. 696 December 2012. The mean prescribed dose of patients receiving IV epoetin decreased from 20 500 U/week in August 2010 to 13 300 U/week in December 2012, with the greatest decline occur- ring during June–August 2011. An increase of iron use was observed in 2011 and serum measures of iron stores increased [22]. However, the increase in average IV iron dose did not per- sist beyond 2011. The sustained increase in ferritin levels in US dialysis patients after policy changes in 2011 appeared to be partly due to reductions in ESA dosing and not solely IV iron dosing practices [23]. In Germany, dialysis procedure rates were changed in 2002 from per-session to weekly flat rate pay- ments, and quality assurance was introduced in 2009 with defined treatment targets for different indices including Hb. DOPPS phases 1–4 (1998–2011) found that Hb levels increased starting in 1998 and remained consistent through 2005, with only 8–10% of patients below 100 g/L. Around 90% of patients were prescribed ESAs, with the dose declining since peaking in 2006. IV iron use was highest in 2011 [24]. with a decline near the beginning of 2011, after which levels appear to have stabilized. The average serum ferritin was 353 mg/L (95% CI 345, 360) in the first quarter of 2005 and 386 mg/L (95% CI 380, 392) in the final quarter of 2013. Summary of findings We examined ESA doses and Hb levels in haemodialysis patients from 2005 to 2013 in the UK. This period included the publication of major clinical trials and changes to treatment practice guidelines. The use of ESAs has decreased over time, but it is not possible to determine which events had the most influence on dosing, as results from RCTs and anaemia man- agement guidelines were also published during this time. The average Hb level decreased over time, with levels after 2011 largely compliant with the UK Renal Association 2010 target range of 100–120 g/L. A decrease in Hb levels over time was observed for both patients who were treated with ESAs and those not receiving ESAs, suggesting that after the reduction in the Hb target thresholds, patients were started on ESAs at lower levels of Hb. A small, but sharp increase in ferritin levels was seen from 2008 to 2011, coinciding with decreases in ESA use. This pattern cannot be directly explained by adherence to revised anaemia management guidelines. Serum ferritin is a marker of iron balance, i.e. iron loss from occult bleeding, blood sampling and loss of blood in the dialysis machine, versus diet- ary absorption plus IV supplementation. The observed pattern of ferritin may have been driven by coincident economic pres- sures to improve the cost-effectiveness of ESA use as well as by emerging evidence that IV iron was safe and effective [19]. Alternatively, it could be that clinicians are administrating the same amount of iron to patients, but it is not being used up so quickly because of reduced ESA doses. Interestingly, the last 2 years have seen a stable pattern with no increase or decrease in levels. Strengths and weaknesses The UK Renal Registry is a large and highly representative database allowing trends in clinical practice patterns to be cap- tured [11]. This study gives an indication that clinicians are responding to changes in guidelines important for patient safety. The analysis of ESA usage is, however, limited by incom- plete data returns, as some renal centres do not routinely record computerized data on ESA dose or drug type. Centres reporting <60% of haemodialysis patients being treated with ESAs were considered to have incomplete data and were excluded from the analysis. These exclusion criteria are relatively arbitrary, but they are based in part on the frequency distribution graph of the centres’ ESA use as it appears in the data. The percentage of patients on ESAs is calculated from these data and incomplete data returns risk impacting on any conclusions drawn. The dose conversion used for darbepoetin may not accurately esti- mate weekly epoetin dose. There is no information on how much iron therapy was being used to treat anaemia in this pop- ulation; however, data were available on ferritin levels, giving an indication of achieved iron stores over the period. Data were only obtained from haemodialysis patients and are not general- izable to peritoneal dialysis patients or other CKD populations. Comparison with other studies Results from RCTs and clinical practice patterns in the USA have influenced anaemia management in recent years. A study of US non-dialysis chronic kidney disease patients found that the emergence of safety concerns following RCTs and the subse- quent changes in product labelling, restrictions to reimburse- ment for ESAs and changes to clinical practice guidelines all appeared to influence physician dosing practices, resulting in less frequent use of ESAs, lower ESA doses and lower achieved Hb levels [20]. A study of US haemodialysis patients from the United States Renal Data System found that ESA dosing decreased from 2007 and that Hb levels mirrored ESA dosing trends [21]. The Dialysis Outcomes and Practice Patterns Study (DOPPS) Practice Monitor was developed to detect and report on trends in dialysis care before, during and after implementa- tion of the End-Stage Renal Disease Prospective Payment System, which was initiated by the US Centers for Medicare and Medicaid Services in January 2011. The percentage of patients with Hb >120 g/L declined from 32 in August 2010 to 14 in E S A d o s e , H b a n d f e r r i t i n l e v e l s i n U K H D p a t i e n t s 2 0 0 5 – 1 3 FUNDING 12. Standards and Audit Subcommittee of the Renal Association. Treatment of Adults and Children with Renal Failure. Standards and Audit Measures, 3rd edn. London: Royal College of Physicians, 2002 K.B. was supported by a Medical Research Council (MRC) UK fellowship (RD1826). J.A.C.S. was supported by National Institute for Health Research (NIHR) Senior Investigator Award NF-SI-0611-10168. Y.B.-.S was supported by the National Institute for Health Research (NIHR) Collaboration for Leadership in Applied Health Research and Care West (CLAHRC West) at University Hospitals Bristol NHS Foun- dation Trust. The views expressed are those of the authors and not necessarily those of the NHS, NIHR, MRC or Depart- ment of Health. K.B. was supported by a Medical Research Council (MRC) UK fellowship (RD1826). J.A.C.S. was supported by National Institute for Health Research (NIHR) Senior Investigator Award NF-SI-0611-10168. Y.B.-.S was supported by the National Institute for Health Research (NIHR) Collaboration for Leadership in Applied Health Research and Care West (CLAHRC West) at University Hospitals Bristol NHS Foun- dation Trust. The views expressed are those of the authors and not necessarily those of the NHS, NIHR, MRC or Depart- ment of Health. 13. Cassidy M, Richardson D, Jones C. Clinical Practice Guidelines. Module 2: Complications, 4th edn. http://www.renal.org/guidelines (1 July 2015, date last accessed). UK Renal Association, 2007 14. Ansell D, Tomson CR. UK Renal Registry 11th Annual Report (December 2008): Chapter 15 The UK Renal Registry, UKRR database, validation and methodology. Nephron Clin Pract 2009; 111 (Suppl 1): c277–c285 15. UK Renal Registry 17th Annual Report: Appendices. Nephron 2015; 129 (Suppl 1): 267–322 16. National Institute for Health and Care Excellence. Anaemia Management in People with Chronic Kidney Disease. NICE Clinical Guideline 114. http:// www.nice.org.uk/guidance/cg114. NICE, 2011. 17. Kidney Disease: Improving Global Outcomes (KDIGO) Anemia Work Group. KDIGO Clinical Practice Guideline for Anemia in Chronic Kidney Disease. Kidney Int Suppl 2012; 2: 279–335 CONFLICT OF INTEREST STATEMENT 18. National Institute for Health and Care Excellence. Anaemia Management in People with Chronic Kidney Disease. NICE Clinical Guideline 39. https:// www.nice.org.uk/guidance/cg39. NICE, 2006. None declared. The results presented in this paper have not been published previously in whole or part, except in abstract format. 19. Susantitaphong P, Alqahtani F, Jaber BL. Efficacy and safety of intravenous iron therapy for functional iron deficiency anemia in hemodialysis patients: a meta-analysis. Am J Nephrol 2014; 39: 130–141 20. Regidor D, McClellan WM, Kewalramani R et al. Changes in erythropoiesis-stimulating agent (ESA) dosing and haemoglobin levels in US non-dialysis chronic kidney disease patients between 2005 and 2009. Nephrol Dial Transplant 2011; 26: 1583–1591 CONCLUSIONS Anaemia management patterns for haemodialysis patients have changed in the UK between 2005 and 2013, with these changes likely to have been in response to results from RCTs and changing clinical practice guidelines. There has been a decrease in ESA use, dose and average achieved Hb levels in recent years, with fewer patients having Hb levels >120 g/L and more patients having Hb levels within the 100–120 g/L target range. Average serum ferritin levels have increased in the same time period. Previous research suggests that clini- cians are responsive to clinical guidelines, though the degree to which new practices are adopted can vary considerably depending on the context [25, 26]. It is likely that in this case, economic considerations due to the price of ESAs further E S A d o s e , H b a n d f e r r i t i n l e v e l s i n U K H D p a t i e n t s 2 0 0 5 – 1 3 697 6. Drueke TB, Locatelli F, Clyne N et al. Normalization of hemoglobin level in patients with chronic kidney disease and anemia. N Engl J Med 2006; 355: 2071–2084 facilitated adoption of the guidelines, as clinicians could reduce unit expenditure by reducing ESA dosage and treating fewer patients. The continued monitoring of anaemia management through registry data is important for patient safety and enhanc- ing patient quality of life and enables us to ensure that any new evidence on the optimal use of ESA therapy for haemodialysis patients is translated into everyday clinical care. 7. Pfeffer MA, Burdmann EA, Chen CY et al. A trial of darbepoetin alfa in type 2 diabetes and chronic kidney disease. N Engl J Med 2009; 361: 2019–2032 8. Singh AK, Szczech L, Tang KL et al. Correction of anemia with epoetin alfa in chronic kidney disease. N Engl J Med 2006; 355: 2085–2098 9. Levin A, Djurdjev O, Duncan J et al. Haemoglobin at time of referral prior to dialysis predicts survival: an association of haemoglobin with long-term outcomes. Nephrol Dial Transplant 2006; 21: 370–377 10. Regidor DL, Kopple JD, Kovesdy CP et al. Associations between changes in hemoglobin and administered erythropoiesis-stimulating agent and survival in hemodialysis patients. J Am Soc Nephrol 2006; 17: 1181–1191 ACKNOWLEDGEMENTS We thank all the UK renal centres for providing data to the UK Renal Registry. 11. Macdougall IC, Tomson CR, Steenkamp M et al. Relative risk of death in UK haemodialysis patients in relation to achieved haemoglobin from 1999 to 2005: an observational study using UK Renal Registry data incorporating 30,040 patient-years of follow-up. Nephrol Dial Transplant 2010; 25: 914–919 SUPPLEMENTARY DATA Supplementary data are available online at http://ndt.oxford journals.org. 21. Freburger JK, Ng LJ, Bradbury BD et al. Changing patterns of anemia man- agement in US hemodialysis patients. Am J Med 2012; 125: 906–914.e9 22. Fuller DS, Pisoni RL, Bieber BA et al. The DOPPS practice monitor for U.S. dialysis care: update on trends in anemia management 2 years into the bun- dle. Am J Kidney Dis 2013; 62: 1213–1216 Received: 12.7.2015; Editorial decision: 10.2.2016 REFERENCES 23. Karaboyas A, Zee J, Morgenstern H et al. Understanding the recent increase in ferritin levels in United States dialysis patients: potential impact of changes in intravenous iron and erythropoiesis-stimulating agent dosing. Clin J Am Soc Nephrol 2015; 10: 1814–1821 1. Rao A, Casula A, Castledine C. UK Renal Registry 17th Annual Report: Chapter 2 UK Renal Replacement Therapy Prevalence in 2013: national and centre-specific analyses. Nephron 2015; 129 (Suppl 1): 31–56 1. Rao A, Casula A, Castledine C. UK Renal Registry 17th Annual Report: Chapter 2 UK Renal Replacement Therapy Prevalence in 2013: national and centre-specific analyses. Nephron 2015; 129 (Suppl 1): 31–56 p y p pp 2. Mikhail A, Shrivastava R, Richardson D. Clinical Practice Guidelines. Anaemia of CKD, 5th edn. http://www.renal.org/guidelines (1 July 2015, date last accessed). UK Renal Association, 2010 24. Kleophas W, Karaboyas A, Li Y et al. Changes in dialysis treatment modal- ities during institution of flat rate reimbursement and quality assurance pro- grams. Kidney Int 2013; 84: 578–584 3. Besarab A, Bolton WK, Browne JK et al. The effects of normal as compared with low hematocrit values in patients with cardiac disease who are receiving hemodialysis and epoetin. N Engl J Med 1998; 339: 584–590 25. Grimshaw JM, Russell IT. Effect of clinical guidelines on medical practice: a systematic review of rigorous evaluations. Lancet 1993; 342: 1317–1322 26. Grol R, Dalhuijsen J, Thomas S et al. Attributes of clinical guidelines that influence use of guidelines in general practice: observational study. BMJ 1998; 317: 858–861 4. FDA Drug Safety Communication: modified dosing recommendations to improve the safe use of Erythropoiesis-Stimulating Agents (ESAs) in chronic kidney disease. http://www.fda.gov/Drugs/DrugSafety/ucm259639.htm2011 (11 August 2015, date last accessed) 5. Coyne DW. The health-related quality of life was not improved by targeting higher hemoglobin in the Normal Hematocrit Trial. Kidney Int 2012; 82: 235–241 K. Birnie et al. 698
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Early Genital Surgery in Disorders/Differences of Sex Development: Patients’ Perspectives
Archives of sexual behavior
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Early Genital Surgery in Disorders/Differences of Sex Development: Patients’ Perspectives Received: 1 November 2019 / Revised: 8 February 2021 / Accepted: 10 February 2021 / Published online: 12 March 2021 © The Author(s) 2021 Archives of Sexual Behavior (2021) 50:913–923 https://doi.org/10.1007/s10508-021-01953-6 Archives of Sexual Behavior (2021) 50:913–923 https://doi.org/10.1007/s10508-021-01953-6 ORIGINAL PAPER Abstract Controversy continues over a proposed moratorium on elective genital surgery in childhood for disorders/differences of sex development (DSD). Empirical evidence on patient preference is needed to inform decision-making. We conducted a multi- centre survey by cross-sectional questionnaire in 14 specialized clinics in six European countries. The sample comprised 459 individuals (≥ 16 years) with a DSD diagnosis, including individuals with congenital adrenal hyperplasia (CAH) (n = 192), XY DSD with prenatal androgen effect (A) (n = 150), and without (nA) (n = 117). Main outcome measures were level of agreement with given statements regarding genital surgery, including clitoris reduction, vaginoplasty, and hypospadias repair. A total of 66% of individuals with CAH and 60% of those with XY DSD-A thought that infancy or childhood were the appropriate age for genital surgery. Females with XY DSD were divided on this issue and tended to prefer vaginoplasty at a later age (XY DSD-A 39%, XY DSD-nA 32%). A total of 47% of males preferred early hypospadias surgery. Only 12% (CAH), 11% (XY DSD-A), and 21% (XY DSD-nA) thought they would have been better off without any surgery in childhood or adolescence. Individuals who had early genital surgery were more likely to approve of it. Outcome data failed to support a general moratorium on early elective genital surgery. Participant perspectives varied considerably by diagnostic category, gender, history of surgery, and contact with support groups. Case-by-case decision-making is better suited to grasping the ethical complexity of the issues at stake. Trial registration: German Clinical Trials Register DRKS00006072. Abstract Keywords  Disorders of sex development · Congenital adrenal hyperplasia · Genital surgery · Pediatrics · Ethics Keywords  Disorders of sex development · Congenital adrenal hyperplasia · Genital surgery · Ped Vol.:(0123456789) 1 3 * Claudia Wiesemann cwiesem@gwdg.de 1 Department of Paediatric Endocrinology and Diabetology, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany 2 Sozialpädiatrisches Zentrum, Center for Chronically Sick Children, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany 3 Department of Paediatrics, Göttingen University Medical Center, Göttingen, Germany 4 Department of Pediatrics, Penn State College of Medicine, Hershey, PA, USA 5 Department of Plastic, Reconstructive and Hand Surgery, Amsterdam University Medical Center (VUmc), Amsterdam, The Netherlands 6 Department of Medical Psychology and Sexology, Amsterdam University Medical Center, Amsterdam (VUmc), The Netherlands 7 Pediatric Surgery, Karolinska University Hospital and Department of Women’s and Children’s Health, and Center of Molecular Medicine, Karolinska Institutet, Stockholm, Sweden 8 Klinik für Kinder‑ und Jugendmedizin, Universität zu Lubeck, Lubeck, Germany 9 Androgen Insensitivity Syndrome Support Group, London, UK 10 Institute of Biomedical Ethics and History of Medicine, University of Zurich, Zürich, Switzerland 11 Department of Medical Ethics and History of Medicine, Göttingen University Medical Center, Humboldtallee 36, 37073 Göttingen, Germany 6 Department of Medical Psychology and Sexology, Amsterdam University Medical Center, Amsterdam (VUmc), The Netherlands * Claudia Wiesemann cwiesem@gwdg.de * Claudia Wiesemann cwiesem@gwdg.de Department of Medical Psychology and Sexology, Amsterdam University Medical Center, Amsterdam (VUmc), The Netherlands 7 Pediatric Surgery, Karolinska University Hospital and Department of Women’s and Children’s Health, and Center of Molecular Medicine, Karolinska Institutet, Stockholm, Sweden 8 Klinik für Kinder‑ und Jugendmedizin, Universität zu Lubeck, Lubeck, Germany 9 Androgen Insensitivity Syndrome Support Group, London, UK 10 Institute of Biomedical Ethics and History of Medicine, University of Zurich, Zürich, Switzerland 11 Department of Medical Ethics and History of Medicine, Göttingen University Medical Center, Humboldtallee 36, 37073 Göttingen, Germany 1 Department of Paediatric Endocrinology and Diabetology, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany 2 Sozialpädiatrisches Zentrum, Center for Chronically Sick Children, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany 3 Department of Paediatrics, Göttingen University Medical Center, Göttingen, Germany 4 Department of Pediatrics, Penn State College of Medicine, Hershey, PA, USA 5 Department of Plastic, Reconstructive and Hand Surgery, Amsterdam University Medical Center (VUmc), Amsterdam, The Netherlands 1 Department of Paediatric Endocrinology and Diabetology, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany 7 Pediatric Surgery, Karolinska University Hospital and Department of Women’s and Children’s Health, and Center of Molecular Medicine, Karolinska Institutet, Stockholm, Sweden 2 Sozialpädiatrisches Zentrum, Center for Chronically Sick Children, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany 8 Klinik für Kinder‑ und Jugendmedizin, Universität zu Lubeck, Lubeck, Germany 9 Androgen Insensitivity Syndrome Support Group, London, UK 3 Department of Paediatrics, Göttingen University Medical Center, Göttingen, Germany 10 Institute of Biomedical Ethics and History of Medicine, University of Zurich, Zürich, Switzerland 4 Department of Pediatrics, Penn State College of Medicine, Hershey, PA, USA 11 Department of Medical Ethics and History of Medicine, Göttingen University Medical Center, Humboldtallee 36, 37073 Göttingen, Germany 5 Department of Plastic, Reconstructive and Hand Surgery, Amsterdam University Medical Center (VUmc), Amsterdam, The Netherlands (0123 1 3456789) 3 914 Archives of Sexual Behavior (2021) 50:913–923 Participants Controversy continues over elective surgery in childhood for disorders/differences of sex development (DSD) (Carmack, Notini, & Earp, 2016; Hughes, Houk, Ahmed, Lee, & LWPES/ ESPE Consensus Group, 2006; Lee et al., 2016; Mouriquand et al., 2016). Critics demand a general moratorium on surgery designed to “normalize” ambiguous genitalia, as in congeni- tal adrenal hyperplasia (CAH), partial androgen insensitivity syndrome (pAIS), and gonadal dysgenesis (GD), buttressing their arguments by patients’ experience of pain, humiliations, and poor functional outcome, as well as the perceived viola- tion of human rights (Diamond & Garland, 2014; Organisation Intersex International Australia et al., 2017). Surgery that is “cosmetic rather than vital for health” and, thus, considered “medically unnecessary” should be postponed until the age of informed consent. Major political institutions, such as the European Parliament, the Council of Europe, Commissioner for Human Rights, and the Special Rapporteur of the United Nations on torture and other cruel, inhuman or degrading treatment or punishment, have recently adopted this position (Council of Europe, Commissioner for Human Rights, 2015; European Parliament, 2013; Human Rights Council, 2013). German and Swiss ethical boards, along with other interdisci- plinary ethical committees, have taken a more cautious stand, allowing for case-by-case decision-making in early childhood (German Ethics Council, 2012; Swiss National Advisory Committee, 2012). However, given the political weight of the moratorium advocates, an outright ban on “normalizing” early surgery may be implemented into national laws sooner or later. The question arises whether substantial evidence on patients’ perspectives is available to support such a significant change in medical practice. Dsd-LIFE is a multicentre cross-sectional clinical evalua- tion study mobilizing 16 partners in Germany, France, the Netherlands, Poland, Sweden, and the United Kingdom, of whom 14 were active recruiting sites. The methodological approach, including the process to ensure valid translation, is described in Rohle et al. (2017). Adolescents (≥ 16 years) and adults with DSD were recruited from February 2014 to September 2015 via medical centers, the project website, and support group contact. All had a clinically confirmed DSD diagnosis conforming to the Chicago Consensus Conference Guidelines (Hughes et al., 2006). The first part of the dsd-LIFE study included a medical interview, a retrospective chart review, and medical examination. The second part of the study was a patient-reported outcome questionnaire. Informed consent was obtained from all individual participants included in the study. Procedure In this substudy, we evaluated 6 items related to views on elective surgery of participants with 46,XX karyotype and CAH, 45,X/46,XY and 46,XY DSD as they often undergo genital surgery in childhood and/or adolescence. We clas- sified them into three subgroups according to diagnosis and exposure to prenatal androgen effect: those with CAH and 46,XX karyotype (CAH), those with 46,XY or 45,X/46,XY karyotype and prenatal androgen effect (XY DSD-A), and those with 46,XY karyotype or 45,X/46,XY without pre- natal androgen effect (XY DSD-nA) (Table 1). In the XY DSD group, we chose exposure to prenatal androgen effect as a diagnostic criterion as it induces atypical develop- ment of the external and/or internal genitalia. Thus, the XY DSD-A group and CAH group often undergo feminizing or masculinizing surgery in childhood and/or adolescence. In XY DSD-nA, the external genitalia phenotype is typi- cally female. Medical decision-making involves value judgements on well-being, identity, and normality, not only by healthcare professionals but also by patients (Bennecke et al. 2020; Brink- mann, Schuetzmann, & Richter-Appelt, 2007; Köhler et al., 2012). It is unclear whether DSD patients are as dissatisfied with early genital surgery as critics maintain (Meyer-Bahlburg et al., 2004; Zucker, 2002). Given the potential impact of a moratorium, we felt an urgent need for more substantial evi- dence of patients’ views for and against such surgery (Sandberg, Callens, & Wisniewski, 2015). The objective of this study was to assess the impact of gender, DSD condition, experience of early surgery, and support group contact on these perspectives using data from a quantitative cross-sectional patient-reported outcome (PRO) study by the dsd-LIFE Network in six Euro- pean countries. Data on DSD surgery (vaginoplasty, clitoris reduction, hypospadias repair) were collected by combining clini- cal report form and patient-reported data. Discrepancies between self-reported data and clinical report file were reviewed manually.i We classified gender as male, female, or other than male or female based on how participants identified at the medi- cal interview. Procedure 1 3 915 Archives of Sexual Behavior (2021) 50:913–923 Table 1   Sample description GD gonadal dysgenesis, pAIS partial AIS, cAIS complete AIS a Nonclassical CAH was not included in this study b Androgen synthesis defects included 3β-Hydroxylase deficiency (n = 2) 17β-HSD III deficiency (n = 11), 5α-Reductase II deficiency (n = 4), 17α-Hydroxylase/17,20 lyase deficiency (n = 1), unknown androgen synthesis defects (n = 2) cI t ( 4) ( 2) th ( 1) CAH XY DSD, with androgen effect (A) XY DSD, no androgen effect (nA) n = 192 n = 150 n = 117 Salt-wasting (n = 111), simple virilising (n = 66), other forms of CAH (n = 15)a, M age (SD) 30.5 (10.9) Partial GD (n = 37), ovotestes (n = 5) pAIS (n = 35), hypospadia (n = 25), andro- gen synthesis defects (n = 19)b, mixed GD (n = 21), other unknown XY DSD (n = 7), M age (SD) 26.0 (10.1) Mixed GD (n = 24), complete GD (n = 21), cAIS (n = 71), other XY DSD (n = 1), M age (SD) 32,4 (13.8) n % n % n % Gender  Female 188 97.9 59 39.3 114 97.4  Male 4 2.1 87 58.0 0 –  ­Otherc 0 – 4 2.7 3 2.6 Country  Germany 74 38.5 36 24.0 29 24.8  France 58 30.2 36 24.0 24 20.5  The Netherlands 17 8.9 12 8.0 38 32.5  Poland 14 7.3 50 33.3 14 12.0  Sweden 12 6.3 13 8.7 10 8.6  United Kingdom 17 8.9 3 2.0 2 1.7 Genital surgery (missing n = 5)  No 15 8.0 6 4.0 17 14.5  Yes 173 92.0 143 96.0 100 85.5  Clitoris surgery 129 68.6 36 24.2 1 0.9  Vaginoplasty 128 68.1 35 23.5 15 12.8  Hypospadias surgery 5 2.7 77 51.7 0 – Contact to sup- port group (last 12 months) (missing n = 24)  No 163 88.6 120 87.0 89 78.8  Yes 21 11.4 18 13.0 24 21.2 y b Androgen synthesis defects included 3β-Hydroxylase deficiency (n = 2) 17β-HSD III deficiency (n = 11), 5α-Reductase II deficiency (n = 4), 17α-Hydroxylase/17,20 lyase deficiency (n = 1), unknown androgen synthesis defects (n = 2) c repair) relevant only for a part of the sample. Procedure To simplify the display of data on statements 2–6, we adapted answers from the 5-point rating scale (strongly/partly agree, neither agree nor disagree, partly/strongly disagree) into a 3-point one (agree, neither agree nor disagree, disagree). Sample Medical diagnoses, age, gender, country, history of geni- tal surgery, and contact with support groups are shown in Table 1. Response rate in the dsd-LIFE study was 36.1% (Rohle et al., 2017). Measures For the patient-reported outcome questionnaire, we devel- oped items addressing the ethical evaluation of the need for and timing of genital surgery. We identified ethically con- tested issues via analysis of the relevant ethical and legal literature and via an explorative questionnaire sent to DSD support groups in associated countries as well as personal interviews with members of support groups. From this material, we constructed one general question and related answers and five statements to be rated on a five-item Likert scale. Three statements (3–5) referred to specific surgical procedures (clitoris reduction, vaginoplasty, hypospadia 1. “When do you think is the appropriate time for genital surgery?” 2. “Any decision about these surgical procedures should be postponed until the affected person reaches the age of legal responsibility.” 3. “Reduction of an enlarged clitoris is necessary in girls.” 1 3 916 Archives of Sexual Behavior (2021) 50:913–923 4. “Vaginoplasty (surgical construction of a vagina) in ado- lescence or adulthood with patient’s consent is better than before 6 months of age (infancy).” procedures should be postponed until the affected person reaches the age of legal responsibility” (Fig. 1). In the CAH group, 88 (51.2%) participants disagreed with this statement; in the XY DSD-A group, 76 (56.3%) participants and 31 (28.7%) participants disagreed in the XY DSD-nA group. There was an association between views of participants and diagnostic grouping (p = .013). Participants with CAH or XY DSD-A were more inclined to disagree, whereas those with XY DSD-nA tended to agree with the statement. 5. “Hypospadias repair with patient’s consent is better than before 2 years of age (infancy).”f 6. “I think I would have been better off without any of the surgeries performed in my childhood/adolescence.” Data Analysis In general, participants responses are displayed as percent- ages per diagnostic group, participant’s contact with support group within the last 12 months, gender, and/or history of genital surgery. For the XY DSD-A group, the association between responses and female or male gender was analyzed; results regarding persons with other gender are reported sep- arately due to the small number of individuals concerned (n = 4); no association was analyzed between gender and CAH or XY DSD-nA due to the small number of individu- als with male gender. All group differences were tested using Pearson χ2 tests. Due to the exploratory character of the anal- yses, all p values were interpreted as non-confirmatory and post hoc tests were omitted. SPSS Version 22 was used for all analyses. Appropriate Time for Genital Surgery A total of 415 participants answered the question “When do you think is the appropriate time for genital surgery?” Altogether, 27 (6.5%) participants stated that adulthood was the right age for such a procedure. There was an association between participants’ choice of age and diagnostic grouping (p < .001) (Fig. 2a). Of the CAH group, 80 (46.2%) partici- pants stated that infancy (1 month–3 years) was the appro- priate age for genital surgery and 35 (20%) chose childhood (4–12 years). Of the XY DSD-A group, 47 (35.3%) partici- pants preferred infancy and 12 (25%) preferred childhood. In contrast, participants in the XY DSD-nA group preferred ado- lescence (13–17 years; n = 33, 30%), adulthood (≥ 18 years; n = 12, 11%), or any age if the patient decides him-/herself (n = 20, 18%). Within the XY DSD-A group, there was an association between responses and gender (p = .007; Fig. 2b), since 44% of males preferred infancy and 31% childhood over other stages of life, whereas females slightly preferred adolescence (31%) over infancy (26%) and childhood (19%). There was an association between having had support group contact within the last 12 months and a preference for surgery at a later age (p = .001) (Fig. 2c). General Postponement of Surgical Procedures until the Age of Legal Responsibility 3   Views of participants with female gender on the need for clitoris reduction surgery Fig. 3   Views of participants with female gender on the need for clitoris reduction surgery Fig. 3   Views of participants with female gender on the need for clitoris reduction surgery Fig. 3   Views of participants with female gender on the need for clitoris reduction surgery (14%) disagreed with this statement, 59 (18.8%) were unde- cided, and 91 (29%) answered “don’t know.” There was an association between participants’ views and diagnostic grouping (p < .001) (Fig. 3). Most partici- pants in favor of clitoris reduction were in the CAH group, whereas those undecided mostly belonged to the XY DSD- nA group. Those members of the CAH group who had had (14%) disagreed with this statement, 59 (18.8%) were unde- cided, and 91 (29%) answered “don’t know.” (14%) disagreed with this statement, 59 (18.8%) were unde- cided, and 91 (29%) answered “don’t know.” clitoris surgery more often preferred clitoris reduction in girls (p = .048) (see Table 2). There was an association between participants’ views and diagnostic grouping (p < .001) (Fig. 3). Most partici- pants in favor of clitoris reduction were in the CAH group, whereas those undecided mostly belonged to the XY DSD- nA group. Those members of the CAH group who had had There was an association between participants’ views and diagnostic grouping (p < .001) (Fig. 3). Most partici- pants in favor of clitoris reduction were in the CAH group, whereas those undecided mostly belonged to the XY DSD- nA group. Those members of the CAH group who had had General Postponement of Surgical Procedures until the Age of Legal Responsibility A total of 314 participants rated the statement “Reduction of an enlarged clitoris is necessary in girls” (Fig. 3, only persons identifying as female). Of those, 120 (38.2%) agreed, and 44 In total, 415 participants rated their agreement or disagree- ment with the statement “Any decision about these surgical 1 3 Fig. 1   Participants’ views on postponing genital surgery until the age of legal responsibility . 1   Participants’ views on tponing genital surgery until age of legal responsibility Fig. 1   Participants’ views on postponing genital surgery until the age of legal responsibility 1 3 917 Archives of Sexual Behavior (2021) 50:913–923 (14%) disagreed with this statement, 59 (18.8%) were unde- cided, and 91 (29%) answered “don’t know.” clitoris surgery more often preferred clitoris reduction in girls (p = .048) (see Table 2). Fig. 2   Participants’ views on the appropriate time for genital surgery. a By diagnostic group, b by gender, only XY DSD-A group, c by contact to support group (total cohort) Fig. 3   Views of participants with female gender on the need for clitoris reduction surgery tatement, 59 (18.8%) were unde- red “don’t know.” on between participants’ views p < .001) (Fig. 3). Most partici- duction were in the CAH group, mostly belonged to the XY DSD- of the CAH group who had had clitoris surgery more often preferred clitoris reduction in girls (p = .048) (see Table 2). Vaginoplasty A total of 323 participants rated the statement “Vaginoplasty (surgical construction of a vagina) in adolescence or adult- hood with patient’s consent is better than before six months Fig. 2   Participants’ views on the appropriate time for genital surgery. a By diagnostic group, b by gender, only XY DSD-A group, c by contact to support group (total cohort) (14%) disagreed with this statement, 59 (18.8%) were unde- cided, and 91 (29%) answered “don’t know.” There was an association between participants’ views and diagnostic grouping (p < .001) (Fig. 3). Most partici- pants in favor of clitoris reduction were in the CAH group, whereas those undecided mostly belonged to the XY DSD- nA group Those members of the CAH group who had had clitoris surgery more often preferred clitoris reduction in girls (p = .048) (see Table 2). Vaginoplasty A total of 323 participants rated the statement “Vaginoplasty (surgical construction of a vagina) in adolescence or adult Fig. Fig. 3   Views of participants with female gender on the need for clitoris reduction surgery Fig. 2   Participants’ views on the appropriate time for genital surgery. a By diagnostic group, b by gender, only XY DSD-A group, c by contact to support group (total cohort) Vaginoplasty A total of 323 participants rated the statement “Vaginoplasty (surgical construction of a vagina) in adolescence or adult- hood with patient’s consent is better than before six months 1 918 Archives of Sexual Behavior (2021) 50:913–923 Table 2   Views of participants with female gender with and without a history of clitoris surgery on the need for clitoris reduction Hypospadias Repair of age (infancy)” (Fig. 4, only persons identifying as female). One hundred and three (31.9%) agreed, 40 (12.4%) neither agreed nor disagreed, and 84 (26.0%) disagreed with the statement (“don’t know”: 96/29.7%). There was an asso- ciation between participants’ views and diagnostic group- ing (p < .001), with participants in the CAH group tending to disagree with the statement (61/37%) more often than participants of the XY DSD-A and nA groups (12/21% and 11/11%). There was an association between participants’ views and history of surgery in the XY DSD-nA group (p = .014) (see Table 3). Altogether, 74 participants who identified as male and had hypospadias surgery responded to the statement “Hypo- spadias repair with patient’s consent is better than before two years of age (infancy)” (Fig. 5, data are descriptive). A total of 13 (17.6%) participants agreed with the statement, eight (10.9%) neither agreed nor disagreed, and 35 (47.3%) disagreed. Table 2   Views of participants with female gender with and without a history of clitoris surgery on the need for clitoris reduction Females with CAH Females with XY DSD-A Clitoris surgery No clitoris surgery Clitoris surgery No clitoris surgery n = 113 n = 30 n = 27 n = 21 n (%) n (%) n (%) n (%) “Reduction of an enlarged clitoris is necessary in girls”  Don’t know 12 (10.6) 8 (26.7) 9 (33.3) 5 (23.8)  Agree 76 (67.3) 12 (40.0) 9 (33.3) 2 (9.5)  Neither agree nor disagree 20 (17.7) 6 (20.0) 6 (22.2) 4 (19.1)  Disagree 5 (4.4) 4 (13.3) 3 (11.1) 10 (47.6) Table 2   Views of participants with female gender with and without a history of clitoris surgery on the need for clitoris reduction Females with CAH Females with XY DSD-A Clitoris surgery No clitoris surgery Clitoris surgery No clitoris surgery n = 113 n = 30 n = 27 n = 21 n (%) n (%) n (%) n (%) “Reduction of an enlarged clitoris is necessary in girls”  Don’t know 12 (10.6) 8 (26.7) 9 (33.3) 5 (23.8)  Agree 76 (67.3) 12 (40.0) 9 (33.3) 2 (9.5)  Neither agree nor disagree 20 (17.7) 6 (20.0) 6 (22.2) 4 (19.1)  Disagree 5 (4.4) 4 (13.3) 3 (11.1) 10 (47.6) Fig. Views on Surgery, in General In total, 351 participants rated their level of agreement or disagreement with the statement: “I think I would have been better off without any of the surgeries performed in my child- hood/adolescence.” A total of 48 (13.7%) participants partly or strongly agreed, while 250 (71.2%) partly or strongly disagreed with the statement, most of them strongly (197, 56.1%) (Fig. 6a). There was an association between views and diagnostic grouping (p = .001) since participants of the XY DSD-nA group were less inclined to disagree with this statement. Hypospadias Repair 4   Views of participants with female gender on appropri- ate time of vaginoplasty Table 3   Views of participants with female gender with and without a history of vaginoplasty on the appropriate time Females with CAH Females with XY DSD-A Females with XY DSD-nA Vaginoplasty No vaginoplasty Vaginoplasty No vaginoplasty Vaginoplasty No vaginoplasty Fig. 4   Views of participants with female gender on appropri- ate time of vaginoplasty Fig. 4   Views of participants with female gender on appropri- ate time of vaginoplasty Fig. 4   Views of participants with female gender on appropri- ate time of vaginoplasty pants appropri- Table 3   Views of participants with female gender with and without a history of vaginoplasty on the appropriate time Table 3   Views of participants with female gender with and without a history of vagino Table 3   Views of participants with female gender with and without a history of vaginoplasty on the appropriate time Females with CAH Females with XY DSD-A Females with XY DSD-nA Vaginoplasty No vaginoplasty Vaginoplasty No vaginoplasty Vaginoplasty No vaginoplasty n = 113 n = 21 n = 30 n = 22 n = 14 n = 70 n (%) n (%) n (%) n (%) n (%) n (%) “Vaginoplasty (surgical construction of a vagina) in adolescence or adulthood with patient’s consent is better than before 6 months of age (infancy)”  Don’t know 17 (15.0) 8 (38.1) 8 (26.7) 7 (31.8) 1 (7.1) 38 (54.3)  Agree 30 (26.6) 7 (33.3) 13 (43.3) 9 (40.9) 7 (50.0) 19 (27.1)  Neither agree nor disagree 13 (11.5) 2 (9.5) 3 (10.0) 2 (9.1) 4 (28.6) 5 (7.1)  Disagree 53 (46.9) 4 (19.1) 6 (20.0) 4 (18.2) 2 (14.3) 8 (11.4) 1 919 Archives of Sexual Behavior (2021) 50:913–923 Fig. 5   Views of participants with male gender on the appropriate time of hypospadias repair ospadias Fig. 6   Participants’ views on surgeries performed in their childhood/adolescence. a By diagnostic group; b by gender, only XY DSD-A group Principal Findings Overall, individuals tend to favor the age at which they underwent the surgery they experienced in their own lives.i p g Participants with 46,XX CAH living in the female gender (a population that usually has vaginoplasty and/or clitoris reduction early in life) predominantly (66%) approved of surgery in infancy or childhood. Slightly more individuals thought that surgical construction of a vagina was better done in infancy than in adolescence or adulthood with patient’s consent (37% vs. 29%). Nowadays, some clinicians tend to postpone the time of vaginoplasty to adolescence because it is considered functional only during or after puberty; oth- ers recommend infancy, based on anatomical patterns that seem to promise better outcome (Creighton, 2005; Creighton, Chernausek, Romao, Ransley, & Salle, 2012). Our results indicate that surgeons should continue to seek functional out- come data as well as explore their patients’ rationale which may differ from their own evaluation. Yet, it should be made clear that the justification of elec- tive genital surgery in early childhood is fundamentally an ethical problem; solutions for an ethical problem should not simply be based on the attitude of majorities (Mertz et al., 2014; Salloch, Vollmann, & Schildmann, 2014). Obviously, the large variety of views expressed within each DSD group indicates a fundamental conflict of values that must be addressed. Notwithstanding these cautionary remarks, and precisely for the same reasons, a general moratorium would not be justified either. Patient autonomy is rightly claimed to play a major role in medical decision-making, and, unfor- tunately, very young children cannot exercise autonomous decision-making. However, at the very least, the diversity of options preferred by adult patients should be considered when a moratorium is discussed at the legislative level.l f The majority of individuals with CAH (61%) and 24% of individuals of the XY DSD-A group agreed that a reduction of an enlarged clitoris was necessary in girls, an operation that is highly contested in the DSD debate, since this type of surgery is understood as having mainly cosmetic function and carries the risk for complications like reduced sensitivity. Those who had clitoris surgery themselves were more likely to approve of clitoris reduction in girls. Principal Findings Our data show that persons with DSD have a much more differentiated view on early genital surgery than might be deduced from current publicized views. The evaluation of the appropriate time for the surgery of ambiguous genitalia dif- fered significantly between the groups with CAH, XY DSD with and without androgen effect (A/nA). A total of 66% of individuals with CAH and 60% of individuals with XY 1 3 Fig. 6   Participants’ views on surgeries performed in their childhood/adolescence. a By diagnostic group; b by gender, only XY DSD-A group Fig. 6   Participants’ views on surgeries performed in their childhood/adolescence. a By diagnostic group; b by gender, only XY DSD-A group 1 3 920 Archives of Sexual Behavior (2021) 50:913–923 Persons with male gender and XY DSD who had hypo- spadias surgery also predominantly agree that hypospadias repair is better done in infancy (47.3%). This may be the case because severe instances of hypospadias in boys can it make difficult to urinate upright and cause significant shame and humiliation (Rynja, de Jong, Bosch, & de Kort, 2011). But many (35%) were undecided, while 18% preferred a later age. More qualitative research is needed to explore the underlying motives of the persons concerned. DSD-A said that infancy or childhood was the appropriate age for genital surgery, whereas 23% and 33%, respectively, preferred adolescence or adulthood, or any time if the patient decides herself/himself. Individuals in the XY DSD-A group who identified as males were more likely (75%) than persons living in the female gender (45%) to favor infancy or child- hood. In general, individuals who had early genital surgery were more likely to approve of it than those who had not. Only 12% of participants from the CAH group, 11% of the XY DSD-A group, and 21% of the XY DSD-nA group said that they would have been better off without any surgeries performed in their childhood or adolescence, thus demon- strating the prevalent acceptance of surgical interventions, independent of timing. Many persons in the XY DSD groups were undecided on the issue of interventions in infancy or childhood. It is noteworthy that, although some advocacy groups have tire- lessly campaigned for a general moratorium, the persons in our study still mostly have not made up their minds. Principal Findings More qualitative research is needed to understand personal motives; one rea- son may be that from a subjective point of view, for females with CAH, typical female appearance might signify more than just cosmetic improvement, as it may be more closely associated with bodily integrity and appropriate functioning. Moreover, the views of this particular group were probably not adequately represented in the public discourse because a large number of adult persons with CAH do not identify as intersex or having a DSD later in life, although they are often born with ambiguous genitalia and early genital surgery is performed in this group. For example, in an online survey by the German Ethics Council from 2011, nearly all participants with CAH responded that they did not feel that they were intersex (German Ethics Council, 2012). As a consequence, their preferences are less likely to be taken into account. The framing of the underlying conflict by some political institutions and advocacy groups that favor a ban is prob- lematic since it fails to acknowledge the complexity of the issues at stake. Critics maintain that elective surgery in DSD is “medically unnecessary” and must therefore be postponed until the age of informed consent. Yet, the bioethicist Mor- reim (2001) warns that medical choices, in general, mostly “require a level of clinical complexity that is not reflected in simplistic notions like necessity, and that should not be hidden under blanket categories connoting a façade of preci- sion.” Even life-saving treatments for cancer are evaluative in the sense that different options have to be weighed against each other. This is precisely the realm where patient-reported outcome and patient preferences can make all the difference. Comparison with Other Studies Other studies on patient evaluation of the appropriate timing of elective genital surgery, in general, support our findings. However, samples sizes were too small to allow for general- izable conclusions. Most refer to only one DSD condition, employ a strictly local approach, and/or often included just 1 3 3 921 Archives of Sexual Behavior (2021) 50:913–923 one question regarding the relevant ethical aspects. In a retro- spective evaluation of 55 adult XY DSD patients (age range 18–60), 67% did not agree that corrective surgery should generally be postponed to adulthood (Meyer-Bahlburg et al., 2004). In a questionnaire-based survey from the Johns Hop- kins School of Medicine on women affected by CAH due to 21-hydroxylase deficiency, patients were asked when femin- izing surgery should be performed (age range at participation, 21 to 71). Fifteen out of 31 chose infancy and nine toddler age or elementary age (Wisniewski, Migeon, Malouf, & Gearhart, 2004). A follow-up study on 62 women with CAH from Sweden found that 20 patients preferred early timing; however, 34 patients did not answer this question (Nordensk- jold et al., 2008). In a survey from Finland, of 24 patients 19 had undergone clitoris reduction and 21 reconstruction of the vaginal introitus in childhood. None of them believed that the operation was performed too early (Fagerholm et al., 2011). In a French questionnaire-based survey with 21 patients with CAH (M age, 27) who all had had clitoris reduction and vagi- nal surgery 90% said that genitoplasty should be performed during the first year of life (Binet, Lardy, Geslin, Francois- Fiquet, & Poli-Merol, 2016). involved in DSD medical treatment, as a general moratorium would entail. Case-by-case decision making, as suggested by several ethical boards (German Ethics Council, 2012; Gillam, Hewitt, & Warne, 2010; Karkazis, Tamar-Mattis, & Kon, 2010; Wiesemann, Ude-Koeller , Sinnecker, & Thyen 2010), is better suited to grasping the remarkable variety of (former) patients’ views, the very complexity of the issues at stake, and the ethical values underlying elective genital surgery in early childhood. Instead of promoting a polarizing morato- rium, more efforts should be invested in improving informa- tion on long-term outcomes, informed consent and assent, and contact with support groups on both the individual and the institutional level to support decision making. Conclusions and Policy Implications Our data suggest that a general moratorium on elective genital surgery in early childhood would not appropriately reflect the views of persons with DSD. This is notably true for persons with CAH and for persons with XY DSD and hypospadias who predominantly favor interventions in child- hood. Persons with XY DSD living in the female gender are divided on this issue with many having no clear-cut view. Thus, our data from patient-reported outcome do not justify a straightforward, one-for-all solution of the ethical dilemmas Funding  Open Access funding enabled and organized by Projekt DEAL. dsd-Life—EU FP7/2007-2013, Grant Agreement No. 305373. Strengths and Limitations The study was part of a large European quantitative cross- sectional outcome study and, to date, the largest and most comprehensive study evaluating (former) patients’ views. Major ethical aspects were included in study design, recruit- ment of participants, and interpretation of data. In this study, it was not asked whether participants knew about the ethi- cal debate on early genital surgery, so they might have been biased toward surgery as the only possible solution. Further- more, a selection bias cannot be ruled out, as participants were partly contacted through medical personnel, a fact that might have suggested compliance with medical authority in these debates. Also, the title of the study “dsd-LIFE” may have deterred those who object to the term “disorders of sex development.” In DSD, loss to follow-up is frequent, so we may have predominantly attracted the “care-minded” and possibly more satisfied patients. The authors publish this paper in memoriam and with the greatest thanks to Privatdozentin (PD) Dr. Birgit Koehler (Charité Universitäts- medizin, Klinik für Pädiatrie m. S. Endokrinologie und Diabetologie), the principal investigator of the European consortium dsd-LIFE and ini- tiator and coauthor of this paper, who died in March 2019 from a severe illness. We honor her dedicated leadership, energy, and enthusiasm for the dsd-LIFE project and the collaboration of clinicians, patients, and support groups, which aim to improve clinical care for “differences/ disorders of sex development.” The authors are in deep grief about this loss and state their gratefulness for the outstanding work of Dr. Koehler. The authors are grateful to the participants of dsd-LIFE and to all of the study centers for their enthusiasm and dedication in contacting potential participants and collecting high-quality data. They especially thank the support groups in the different countries for their help. Authors’ contributions  EB drafted and revised the manuscript. SB, AN, MR, and UT designed the data collection instruments, collected data, carried out the initial analyses, reviewed and revised the manu- script. PL, TCG, JCS, and MS reviewed and revised the manuscript. CW conceptualized and designed the sub-study and critically reviewed the manuscript for important intellectual content. All authors approved the final manuscript as submitted and agreed to be accountable for all aspects of the work. Comparison with Other Studies Overall, the considerable uncertainty about the best time for genital surgery, if considered at all, among (former) patients justi- fies, if not a ban, then at least deliberate attempts to motivate parents to postpone elective genital surgery, thus relieving themselves as much as possible of the responsibility for tak- ing the “right” choices. Acknowledgements  For an overview of all contributors, see the study protocol. 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USING 3D LASER SCANNING TO ANALYZE HERITAGE STRUCTURES: THE CASE STUDY OF EGYPTIAN PALACE
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Journal of Civil Engineering and Management ISSN 1392-3730 / eISSN 1822-3605 2020 Volume 26 Issue 1: 53–65 https://doi.org/10.3846/jcem.2020.11520 USING 3D LASER SCANNING TO ANALYZE HERITAGE STRUCTURES: THE CASE STUDY OF EGYPTIAN PALACE Mohamed MARZOUK * Structural Engineering Department, Faculty of Engineering, Cairo University, Giza, Egypt Received 29 August 2019; accepted 4 October 2019 Abstract. Preservation of heritage buildings should be carried out to get a better understanding of the behavior of their structures and keep them in a good condition. As such, corrective diagnosis of heritage buildings health conditions would help to identify potential risks and take remedy actions. This paper presents a framework for heritage Building Information Modeling (HBIM) application in Egyptian Heritage buildings. The framework is capable of utilizing processed point clouds using 3D laser scanning to create different purpose BIM models at the different levels of development to simulate the structural performances under different types of actions. The paper illustrates an extensive structural analysis for Tosson palace in Cairo – Egypt to assess its health state to assure its sustainability for future use. Keywords: heritage buildings, building information modeling, 3D laser scanning, structural analysis, health conditions. 1. Literature review 1.1. Digital documentation of heritage using LiDAR The 3D laser scanning advances have been presented in the field of surveying and can obtain 3D data about physical objects of different shapes and sizes in a cost and time effective way. Laser scanners enable a large number of points to be recorded in a few minutes. As a result of their common sense and flexibility, these sorts of instruments can be broadly utilized in architectural, archeological, and environmental surveying fields (Valanis & Tsakiri, 2004). Nex and Rinaudo (2010) worked on an algorithm that automatically combines information gathered from digital imaging and LiDAR to segment and detect heritage building features, such as building break lines. The automation algorithm, using high density point clouds, is time saving and it decreases human intervention in the process of documenting heritage buildings. Hesse (2010) developed a data processing method for the extraction of Local Relief Models from airborne LiDAR high density point clouds, which results in color-coded maps of local Relief Models. The proposed method was applied on LiDAR data (acquired by airborne 3D laser scanners) of Baden-Württemberg and proved valuable and accurate in mapping large archeological sites scanned with airborne 3D laser scanners. Haala, Peter, Cefalu, and Kremer (2008) utilized a kinematic terrestrial laser scanning approach to capture the dense point cloud for a historical town. The “Street Mapper” approach, on the other hand, depends on four vehicle-mounted 2D laser scanners and a high performance GNSS/inertial navigation system. Accurately providing the required dereferenced information, this method enables the automatic combination of the four 2D dense point clouds. The proposed approach achieved a feasible 3D laser scanning approach for large heritage sites with an accepted level of accuracy for many urban mapping applications of historical towns. Coren, Visintini, Prearo, and Sterzai (2005) integrated LiDAR data with hyper-spectral data to evaluate irregular behavior of major ground indices, which in turn improves the discovery of new archeological sites. LiDAR provided the accurate surface geometrical data while the hyperspectral survey provided the specific humidity, vegetation, and thermal conditions of the target area. The integration of both technologies generated an accurate Digital Terrain Model (DTM). Sánchez-Aparicio, Del Pozo, Ramos, Arce, and Fernandes (2018) diagnosed the historical structures through multilayered point cloud analysis using the technology of laser scanning to observe the built pathologies. Radiometric and geometric data provide a 3D methodology to extract and quantify the different deformations and *Corresponding author. E-mail: mm_marzouk@yahoo.com Copyright © 2020 The Author(s). Published by VGTU Press This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. M. Marzouk. Using 3D laser scanning to analyze heritage structures: the case study of Egyptian Palace 54 biological Conquest on the masonry. The Fortress of Almeida, in Portugal was examined for restoration activity. The complex diagnostic results of the radiometer were validated and compared with realistic photographs. Presence of salts and moisture were deduced from the deformation layers within the analysis results that revealed the direct relation between the visual diagnosis and that of the point cloud. Głowienka and Michałowska (2017) worked on a project named “Cultural Heritage through time” as a basis for web-based published material to provide 4D data that include time interventions of architectural and structural alterations. The UNESCO-listed historical site of Krakow was examined to forecast and analyze the facility modifications over time. Using LiDAR, UAV, TLS, and archived data, accurate 3D models were obtained which enhanced restoration even that of the underground unseen parts of the facility. Accurate data was provided for the excavation work that included deepness, slope, and thickness of buried masonry. Further research efforts utilized 3D laser scanning (LiDAR) to document heritage sites. Cheng, Wang, Chen, and Li (2016) developed a method to automatically detect ancient walls features and extract its geometry from LiDAR point clouds. They applied their method to Nanjing city wall, along with the eight city walls of Min and Qing dynasties in China. Their work was used in understanding the current state of the historical walls, and added to their digital document, which represents the means of the wall’s construction, rehabilitation, exhibition, and promotion. The integration of LiDAR and unmanned airborne vehicles (UAV) proved efficient in mapping large heritage sites in china. However, due to the vibrations, the collected data suffers from some distortions. Li, Yan, Jing, and Zhao (2015) designed, developed, and tested two platforms which are most suitable for mounting LiDAR to collect high density point clouds for heritage mapping projects in terms of stability, capacity, reliability, and vibrations. The limitations of traditional geometric data acquisition are listed in Table 1. 1.2. 3D parametric HBIM models This section displays the previous efforts directed towards the process of generating 3D parametric HBIM models from point clouds, using terrestrial 3D laser scanners, along with some efforts in documentation, analysis, and dissemination of several historical sites using HBIM models generated from high density point clouds. Barazzetti et al. (2015) described the procedures of developing the HBIM model starting from point cloud data. The developed models were then reformatted to be displayed in mobile apps using augmented and virtual reality visualization technologies. Their purpose was to deliver HBIM models through widely available platforms for both expert and non-expert operators, which is valuable in the tourism and heritage dissemination sectors. Dore et al. (2015) deployed the Dublin historical BIM frame work to perform structural and conservation analysis for the Four Dublin courts. The analysis was intended to assess the damage subjected to the structure during the war. They developed a model using historical data, and another model using 3D laser scanning. The two models were simulated to assess the structural damage that still affects the structure till now, as well as to serve conservation and documentation purposes. Table 1. Weaknesses of traditional geometrical data acquisition techniques Acquisition method Weakness Survey points • Get the coordinates of a few selected no. of control points • Impractical to generate dense point cloud • Prone to human error • Difficult to document the data acquisition process Direct measurements • Impractical to measure every possible dimension • Prone to human error • Difficult to document the data acquisition process • Difficult to document the data acquisition process • Missing information about depth, i.e. 2D information • Highly dependent on the quality of the camera, and operator High quality digital imagery • Highly affected by light and weather conditions • Less accurate information in the distorted parts of the photo • Difficult to document the data acquisition process Photogrammetry 2D Projections • Highly dependent on the quality of photos (refer to the previous entities) • Inherits the weakness of high quality digital imagery • Cannot generate accurate high dense point cloud • Needs immense post processing efforts to get the coordinates of selected points (sparse point cloud) • Difficult to understand/interpret by non-practitioners • Difficult to visualize to non-practitioners • More challenging to visualize to practitioners compared with 3D models • Prone to coordination errors • Impractical to draw every possible section and/or elevation Journal of Civil Engineering and Management, 2020, 26(1): 53–65 Aside from buildings and terrestrial sites, some naval artifacts are considered of great heritage value, and it is of great difficulty to display and effectively disseminate a multitude of naval vehicles out of their context (water masses) in museums. Accordingly, Wetherelt, Cooper, and Zazzaro (2014) processed the LiDAR high density point cloud and generated photorealistic and accurate 3D digital models for the dhows. Their work is displayed in 2D, 3D, and virtual visualization in the National Museum of Qatar. Oreni, Brumana, Della Torre, Banfi, and Previtali (2014) created an HBIM model using terrestrial 3D laser scans and 3D images of the Basilica di Collemaggio in L’Aqualia as part of the restoration process of the site after the earthquake of 2009. The resulted model was used for structural analysis purposes to assess the feasibility of restoration methods. Brumana, Georgopoulos, Oreni, Raimondi, and Bregianni (2013) carried out a 3D laser scanning and a photogrammetric survey for the Church of S. Maria di Scaria (Vall’ Intelvi). They processed the raw data from the survey and generated an HBIM model for the church which displays the construction methods and the various rehabilitations and expansions from the Romans era till the baroque period. Also, they developed an object library that can be used for modelling such buildings across Europe. The resulting model was easy to interpret and was used for dissemination purposes in the multimedia section of a local museum. Amans, Beiping, Ziggah, and Daniel (2013) proposed a framework for documenting heritage sites in West Africa. The framework pivots on the generation of 3D HBIM model from LiDAR high density point clouds. Their approach overcomes the limitations of the traditional methods in terms of accuracy and time consumption. The resulting digital documentation provides a guide to the preservation of heritage sites along with 2D, 3D and virtual visualization, and dissemination of heritage sites. Dore and Murphy (2012) outlined the use of HBIM in modelling historic structures in the Dublin institute of technology. This approach depends on acquiring special data using 3d laser scanning and photogrammetry to develop an HBIM model using modelling objects from libraries of historical buildings elements. Moreover, the developed model is integrated with GIS data through the CityGML framework, thus creating accurate 3D GIS models. Such approach bridges the gap between 3D GIS and HBIM in documenting heritage sites. Murphy, McGovern, and Pavia (2009) outlined in detailed workflow of HBIM system which starts with point cloud and digital imagery, and results in a textured 3D parametric model. They extended the ability of the geometric descriptive language (GDL) to generate 3D parametric objects that they used to develop a library for historical 3D parametric objects of historical buildings’ elements. Rubinowicz and Czynska (2015) examined the possibilities of applying LiDAR data, coupled with digital 3D-city models, in evaluating city landscape parameters for heritage rich contempo- 55 rary urban areas. Such parameters will enable the evaluation of strategic city views, mapping landscape absorption limits, and delineation of heritage rich zones that needs protection from modern urbanization. Their efforts were crowned by developing the computational analysis method Visual Protection Surface (VPS) which enables the automatic collection and evaluation of city landscape parameters, and was successfully implemented in the city of Dresden. Fernández-Lozano, Gutiérrez-Alonso, and Fernández-Morán (2015) used 3D laser scanning in archeological works for discovering man-made structures and building ruins. They developed a detailed map of Roman ancient mining works in the areas of Las Medulas and Omanas along with Duerna and Eria river valleys. Their work provided insights into the Romans’ exploitation hydraulic engineering techniques and geometry; which in turn highlighted the scope and impact of the Roman mining works that turned out to be more important than previously estimated. 1.3. Structural analysis of heritage buildings The following section displays the previous efforts directed towards the structural analysis of heritage buildings specially the masonry structures. Many of the efforts were directed towards the accurate modelling of the structural material, others were directed towards the alignment between the accepted level of approximation and the purpose of the analysis while others presented Finite Element Analysis (FEA) on different heritage assets. The conclusion that can be drawn from the following literature review is the consensus on the importance of following the most accurate structural analysis methods to assess and strengthen heritage structures. Lourenço (2002) addressed the possibilities of analysis of historic structures and proposed a set of guidelines. They investigated what type of analysis should be used. It seems that all methods are of interest, depending on the actual constraints of the engineering problem. And they concluded that all the available techniques are of use and importance. Giordano, Mele, and De Luca (2002) investigated the applicability of different numerical techniques for the analysis of masonry structures by comparing the computed results with the experimental test data obtained on a full-scale masonry specimen. Three approaches are taken into account, including: 1) The standard FEM modelling strategy, based on the concepts of homogenized material and smeared cracking constitutive law, is used in the version implemented in the commercial code ABAQUS by HKS. 2) The program Visual CASTEM 2000 is used for the application of the finite element method with discontinuous elements, which are intended to simulate the presence of vertical and horizontal mortar joints. 3) Finally, the UDEC software by the ITASCA Group is adopted for the Discrete Element modelling. 56 M. Marzouk. Using 3D laser scanning to analyze heritage structures: the case study of Egyptian Palace They also provided a “ready-to-use” input parameter values, which are usually hard to decide. However, parameters are suggested, based on both extensive sensitivity analyses, and experimental validation. They also proved that the proposed values give reasonable results in the modelling of masonry structure under monotonously increasing loads. Kappos, Penelis, and Drakopoulos (2002) evaluated the relative accuracy of different models. Mainly intended for use by practicing engineers, the study focused on the analysis of unreinforced masonry buildings, and on determining whether or not a simple equivalent frame model can be used for design and/or assessment purposes, and under what conditions. They performed several parametric analyses including finite element (FE) models of 2D and 3D structures. Furthermore, their results shed some light on the feasibility of using simplified and cost-effective analytical models as a tool for practical design and/or assessment of typical masonry structures. Lourenço, Mendes, Ramos, and Oliveira (2011) approved the notion that the assessment of the seismic performance of structures is still a challenge because historic masonry structures exhibit peculiar properties, low tensile strength and lack of box behavior, which makes the task of the analyst even more difficult. Traditional design and assessment methods, similar to the ones currently used for reinforced concrete structures, are not applicable. Thus, the authors provided a review of the seismic analysis of masonry structures without box behavior. They discussed different methods of structural analysis, and performed a comparison between pushover methods and non-linear dynamic analysis with time integration. They displayed their results through three cases studies, San Torcato church, Qutb Minar, and “Gaioleiro” buildings. The results show that traditional, adaptive or modal pushover analyses are not totally in agreement with nonlinear dynamic analysis or experimental observations. Oyarzo Vera (2012) exhaustively investigated the dynamic characterization of unreinforced masonry (URM) buildings and the use of their distinctive modal properties in identifying damage. Their numerical and physical models were used to analyze the system applicability and identify damage in non-slender URM buildings. Model tests were conducted on two undamaged URM panels and an undamaged model of a one-story URM house. Impacts with a calibrated hammer were used to excite the specimens during the modal test, and also horizontal harmonic excitations in the case of the house model. Two system identification techniques, SSI and FDD, were considered for extracting the modal properties. The experimental results were then compared with numerical predictions obtained from finite element models originally generated based on geometrical and mechanical properties obtained by standardized tests. The numerical predictions were improved by applying sensitivity-based model updating techniques. The evolution of the modal properties of the URM panels and house model due to artificially induced damage were also investigated. Vibration-based and model updating-based damage identification techniques were employed to detect the damage distribution in the specimens. Finally, a seismic assessment was conducted on a typical New Zealand URM building by applying time-history analysis on finite element models. An original seismic hazard zonation and methodology for selecting appropriate ground-motion records in the North Island of New Zealand is described. A typical stand-alone two-story URM building was simulated using different finite element models. Material and geometrical properties estimated from traditional standardized test results, and properties estimated by a process of model updating based on the experimental data recorded in the vibration tests were considered for these models. The finite element models were analyzed using a time-history methodology for a building in Auckland and Wellington. The response of the building was compared to admissible limits established based on a stability criterion, taking into account three levels of intensity for the ground motion. Betti, Galano, and Vignoli (2014) performed a comparison between different numerical structural analysis methods to predict the seismic behavior of unreinforced masonry heritage structures with flexible diaphragms. As a prototype for the analysis, the authors tested a two-story building on a shaking table undergoing increasing natural ground motion to analyze its seismic response starting from the prototype initial conditions until the mildly damaged reaching extensive damage states. The first method was the finite element analysis technique presenting the near exact approach while the second was a simplified macro-element method, presenting the simplified and less computationally demanding method. The results from the two methods along with the empirical prototype were compared resulting in the following observation. The finite element model was able to predict the damaged areas and the incipient collapse mechanism, as well as the collapse load. The macro-element model was able to predict the collapse load but due to some limitations of the approach, a satisfactory reconstruction of the actual collapse mechanism was not obtained. Nevertheless, the simplified model was able to deduce a fairly accurate estimate of the accelerations at the top floor measured in the tests. Elyamani and Roca Fabregat (2018a, 2018b) presents a literature review carried out to cover the current state-of-the-art of a number of investigation activities carried out integrally for the seismic safety assessment of historical structures, with minimal intervention due to the perseverance nature of the heritage buildings, that constraints the strengthening and structural interventions. 2. Case study Tosson palace is a heritage building that was built in 1886 by Prince Omar Tosson in Shubra, Road El-Farag, north of Cairo, Egypt (see Figure 1). The palace is a 3,200-square meter building on three stories, with an arcade façade and an extended terrace on its upper floor. To diagnose Tosson Journal of Civil Engineering and Management, 2020, 26(1): 53–65 57 • Planning acquisition procedure Phase 1 Phase 2 • 3D laser scanning • Processing of the High -Density Point Cloud Phase 3 Figure 2. Proposed heritage digital documentation procedure Figure 1. Ariel view of Tosson palace palace health condition, two main stages are performed; heritage digital documentation and structural model analysis. These stages are described in the following subsections. 2.1. Heritage digital documentation Heritage digital documentation is composed of three tiers: 1) acquiring raw LiDAR data; 2) processing point cloud; and 3) generating the model. The output and benefits of every tier is inherited to the following because every tier is dependent on the product of the previous tier. The implementation process is divided into three phases as shown in Figure 2. Phase 1: planning acquisition procedure; Phase 2: 3D laser scanning of the site; Phase 3: processing of the High-Density Point Cloud (HDPC). Phase 1 is initiated by acquiring the layout of the site with all information collected via a total station survey and manual distance measurements. The team utilized a Z+F 3D laser scanner to acquire the HDPC. The planning depends on the number of scans required to capture the features of the palace from all directions internally and externally, while maintaining an adequate level of accuracy (HDPC density). A total of 160 scans were required to achieve the mission as depicted in Figure 3. The adopted resolution in Tosson palace was the super High resolution with 3.1 mm point spacing at 10 m distance. Phase 3 was performed utilizing a Dell OptiPlex 7020 Dell Alienware Area 51 workstation for the processing purposes. Phase 3 started with the reg- Figure 3. Layout of the 160 required scans istration of the individual HDPC generated from multiple scans. The data was imported in Autodesk Recap for registration and noise removing. Auto-Registration feature in Autodesk Recap was used. The registered raw HDPC was produced, then, the noise data was removed from the scans as shown in Figure 4. It is worth noting that the average percentage of overlapping points within 1/4” (or 6 mm) of the corresponding feature in Tosson project obtained from Autodesk Recap during registration is 99.88%. The second step in processing phase is to create a 3D model of the palace. Two modelling techniques were used: 1) high detailed modelling for virtual reality purpose, and 2) medium detailed modelling to produce HBIM model for accurate structural analysis and Energy analysis. The HBIM modelling step started once the 3D point cloud was cleaned. Autodesk Revit 2018 was used for modelling as it provides quick, easily modifiable modelling that includes a high standard of structure documents. In addition, Leica CloudWorks, Revit plug-in, was used to help in modelling using as-built point cloud data captured by the laser scanner, directly within Revit for better BIM modelling of existing buildings. Leica CloudWorks used for importing point cloud as a database into the Autodesk Revit project is shown in Figure 4. Then, the point cloud is clipped to show the first and second floor separately. Afterwards, the modelling steps start with building the palace’s external and internal walls two floors (see Figure 5). Modelling structural elements was very important to enabling the calculation of loads on the walls and the floors for structural analysis. Figure 4. Point cloud of Tosson palace M. Marzouk. Using 3D laser scanning to analyze heritage structures: the case study of Egyptian Palace 58 Figure 5. Modelling of structural elements in Tosson palace The walls were analysed under full dead and live loads from different levels. The dead loads consist of the weight of all permanent construction and were computed based on the roofing layers used. The live loads were input in accordance with the Egyptian code (ECP, 2012). Hence, live loads were considered equal to 0.2 t/m2. With resepct to seismic loads, the response spectrum and time-history methods were used to evaluate the additional stresses due to earthquake loading. The mechanical properties for both clay bricks and limestone block that are considered in the analysis are listed in Table 2. 2.3. Horizontal elastic response spectrum As for the response spectrum analysis, it was conducted according to the provisions of the Egyptian code (ECP, 2012). Accordingly, Tosson palace is located in the second zone, so the models will be analysed considering a peak ground acceleration level equal to 0.12 g and using type (1) response spectrum in the Egyptian code. According to the previous researches, soil type (B) is considered in the analysis. A time-history analysis was performed to assess the seismic vulnerability of Tosson palace, despite being time-consuming, 100 hrs per run in this model. The time-history analysis is an accurate and reliable analysis to predict all possible failure mechanisms due to seismic actions as a result of its capability to evidence both in-plane and out-of-plane, local and global failure mechanisms of the structures. A dynamic analysis was carried out using an artificial accelerogram compatible with the elastic re- sponse spectrum of the Egyptian codes. It is worth noting that the artificial accelerogram is developed for assessing the dynamic response of structures due to seismic actions. The corresponding acceleration history applied at the basis of the palace, having a total duration of 10.06 sec., is illustrated in Figure 6. The seismic behaviour of the palace, the seismic, horizontal actions along the longitudinal (X) direction only is considered. The transversal (Z) direction was not analysed according to the long time consumed in the longitudinal (X) direction. In addition, the longitudinal (X) direction is considered a more critical direction. 2.4. Structural assessment In order to establish the numerical model, information is gained from the geometrical survey as well as material properties in order to construct a finite element model. 0.15 0.1 Acceleration, g 2.2. Load assessment and materials properties 0.05 0 –0.05 –0.1 –0.15 –0.2 0 2 4 6 Time, s 8 10 Figure 6. Acceleration history artificially generated (accelerogram) Table 2. Mechanical properties for both clay bricks and limestone block Unit weight, kN/m3 Compressive strength, MPa Tensile strength, MPa Modulus of elasticity (E), MPa Poisson ratio (υ) Walls 23 8.67 0.86 1460 0.2 Columns 24 9.47 0.94 1500 0.2 Vaults 16 9.32 0.93 1900 0.2 Woods 7 – – 10000 0.3 Item 12 Journal of Civil Engineering and Management, 2020, 26(1): 53–65 The finite element model is used to simulate the behaviour of the structure and estimate the stresses in different elements under various loading conditions. The structural model is used to examine various loading scenarios and predict the crack patterns based on estimating the maximum tensile stresses in the structural elements. The model shall also be used to examine the dynamic characteristics of the structure, natural frequencies and corresponding mode shapes. Analysis of the model output is used to draw conclusions and recommendations to judge the safety of the structure. A 3D model is used to analyse Tosson palace walls under various loads. The 3D model is used to simulate the response of the structural elements under static loads and used for checking settlement and internal stresses in walls. ANSYS software is used to conduct the finite element model. Solid elements with variable thickness are used to represent the walls and floors. Solid elements are removed from opening locations such as doors and windows. Modelling of the monument considers geometrical and loading conditions aspects. As for the geometrical aspect, these models are built using the given AutoCAD drawings coming from the 3D laser scanning techniques then extracted to the used software analysis program. Besides, horizontal restraints are assigned to the location of the floors as per site observations. The results of the analysis of the Palace focus on estimating the stresses in various structural elements. The assessment is performed considering vertical and lateral loading. Evaluation of tensile stresses in the walls is performed using the finite element method. The stresses are computed from the various load combinations will be compared with the acceptable levels of stress for such construction. Quadratic solid elements with variable thickness are used to represent the walls. Hence, each node has six degrees of freedom, three translational and three rotational. Shell element size was restricted to 0.5 m, and the ratio between element sides is kept in the range 1:1 as possible. Modeling of the monument considers geometrical, loading, and construction materials conditions aspects. As for 59 the geometrical aspect, these models are built using the HBIM model created from 3D laser scanning techniques, then, extracted to the used software analysis program. Besides, horizontal restraints are assigned to the location of the floors as per site observations. All members of the palace, walls and floors, are simulated using 3D 8-noded brick elements. The element is defined by eight nodes, each having three degrees of freedom, translations in the nodal x, y, and z directions. In ANSYS, the solid element is called SOLID65. The element is capable of plastic deformation and cracking in three orthogonal directions, hence, it is used to model all elements. The palace elements are all assigned elastic behaviour where the resulted values of stresses are lower than the allowable. For this reason, nonlinear materials are not used in the analysis to avoid the unnecessarily long analysis time. Figure 7 illustrates the geometry of the whole model. It is worth noting that the mesh consists of 468,076 elements and 1,408,551 nodes. The model uses standard fixities. As such, the displacements at the lower surface of the walls are prevented in the horizontals and vertical directions (Ux= 0, Uz= 0, and Uy= 0). 2.5. Structural analysis results The results of the analysis of the palace focused on estimating the stresses in various structural elements. The assessment is performed considering vertical and lateral loading. Evaluation of tensile stresses in the walls is performed using the finite element method. The geometry of the wall, as well as the mechanical properties, are taken as previously discussed in this report and are used to produce the numerical models representing the various structural elements of Tosson palace. The models are subjected to loads and deformations as seen relevant, and the stresses are computed for various load combination scenarios. The stresses computed from the various load combinations are compared with the acceptable levels of stress for such construction. Table 3 lists the results of the numerical models for different load cases. Figure 7. The complete geometry of the FE model for Tosson palace M. Marzouk. Using 3D laser scanning to analyze heritage structures: the case study of Egyptian Palace 60 Table 3. The stresses due to different cases in walls and circular columns Walls Circular Columns Horizontal Vertical Horizontal Vertical Compression (MPa) Tension (MPa) Compression (MPa) Tension (MPa) Compression (MPa) Tension (MPa) Compression (MPa) Tension (MPa) Vertical loads (D + L) –0.69 0.7 –1.46 0.53 –1.24 0.27 –0.44 0.50 Response Spectrum –0.90 0.90 –0.96 0.96 –0.53 0.53 –0.74 0.74 Figure 8 shows a plot for the deformed shape under vertical loads, whereas, Figures 9 and 10 show the deformed shape under seismic loading in X-direction and Zdirection, respectively. Figure 11 illustrates the equivalent stress (Von-Mises) and the stresses in the three principal directions (X, Y, and Z) under vertical loading. Figure 12 illustrates the equivalent stress (Von-Mises) and the stresses in the three principal directions (X, Y, and Z) under seismic loading in X-direction, whereas Figure 13 depicts the same under seismic loading in Z-direction. Five mode shapes resulting from free vibration analysis are illustrated in Figure 14. Table 4 lists the natural frequencies for the first 5 vibration modes in the three principal directions (the transversal, longitudinal and vertical direction). It is a) Vertical deformation worth noting that the time-history of the horizontal displacements at the top of the building in X-direction under seismic loads effect in X-direction. The resulted maximum horizontal displacement in X-direction is about 30 mm. Table 4. Natural periods and frequencies for the three principal direction MODE Frequency Period Mode type 1 2 3 4 5 3.7989 4.48123 4.51313 5.30252 5.67505 0.26323 0.22315 0.22158 0.18859 0.17621 Translation in transversal Translation in longitudinal Translation in longitudinal Torsional Torsional b) Horizontal deformation (X-direction) c) Horizontal deformation (Z-direction) Figure 8. Deformation shapes under static loads effect Figure 9. Horizontal deformation (X-direction) shape under seismic loads effect in X-direction Figure 10. Horizontal deformation (Z-direction) shape under seismic loads effect in Z-direction Journal of Civil Engineering and Management, 2020, 26(1): 53–65 61 a) Equivalent stresses (Von-Mises) b) Vertical stresses (Sy) c) Horizontal stresses (Sx) d) Horizontal stresses (Sz) Figure 11. Stresses under static loads effect a) Equivalent stresses (Von-Mises) b) Horizontal stresses (Sx) c) Horizontal stresses (Sz) d) Vertical stresses (Sy) Figure 12. Stresses under seismic load effect in X-direction 62 M. Marzouk. Using 3D laser scanning to analyze heritage structures: the case study of Egyptian Palace a) Equivalent stresses (Von-Mises) b) Horizontal stresses (Sx) c) Horizontal stresses (Sz) d) Vertical stresses (Sy) Figure 13. Stresses under seismic load effect in Z-direction a) Mode shape 1 b) Mode shape 2 Figure 14. To be continued Journal of Civil Engineering and Management, 2020, 26(1): 53–65 63 c) Mode shape 3 d) Mode shape 4 e) Mode shape 5 Figure 14. Five mode shapes resulting from free vibration analysis Conclusions This paper presented a framework for heritage Building Information Modeling (HBIM) application in Egyptian heritage buildings. It reviewed the digital documentation of heritage using LiDAR, 3D parametric HBIM models, and structural analysis of heritage buildings. The proposed framework has been implemented in Tosson palace, a 19th century heritage building located in Cairo, Egypt. The procedure for heritage digital documentation is composed of three tiers: 1) Aquire Raw LiDAR data, 2) Process point cloud, and 3) Generate the model. CloudWorks, an Autodesk Revit plug-in, was used to help in tracing the as-built point cloud data captured by the laser scanner. The generated HBIM model was used to perform structural analysis using the finite element model. The analysis showed that, in general, the range of stresses in the walls and columns are relatively low, even less than the acceptable values for such construction. This confirms the initial assessment that from a structural point of view, the monument is in good condition. However, there are some locations where there is visible deterioration of materials and mortar, probably due to environmental conditions that affected the structure throughout its lifetime. The current situation of cracks is generally stable but it is necessary to take concrete steps to replace the damaged materials and 64 M. Marzouk. Using 3D laser scanning to analyze heritage structures: the case study of Egyptian Palace repair the cracks. Many deterioration signs in the palace’s walls and finishes are related to moisture. The current measured moisture content in the lower part of the walls is rather low; consequently, the deterioration is probably due to an earlier time when the moisture content was high, due to the rise of groundwater, water leakage from the water supply, or collection networks. Acknowledgements This research was financially supported by Egypt-UK Newton-Musharafa Fund: Institutional Links; STDF (the Science and Technology Development Fund), Egypt, Grant No. 26150. Funding This research was funded by Egypt-UK Newton-Musharafa Fund: Institutional Links; STDF (the Science and Technology Development Fund), Egypt, Grant No. 26150. Disclosure statement The author declares that he does not they have any competing financial, professional, or personal interests from other parties. References Amans, O. C., Beiping, W., Ziggah, Y. Y., & Daniel, A. O. (2013). The need for 3D laser scanning documentation for select Nigeria cultural heritage sites. European Scientific Journal, 9(24), 75-91. 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Using LiDAR for digital documentation of ancient city walls. Journal of Cultural Heritage, 17, 188-193. https://doi.org/10.1016/j.culher.2015.04.005 Coren, F., Visintini, D., Prearo, G., & Sterzai, P. (2005, May). Integrating LiDAR intensity measures and hyperspectral data for extracting of cultural heritage. In Proceedings of Italy–Canada 2005 Workshop on 3D Digital Imaging and Modeling: Applications of Heritage, Industry, Medicine and Land, Padova, Italy. Dore, C., & Murphy, M. (2012, September). Integration of Historic Building Information Modeling (HBIM) and 3D GIS for recording and managing cultural heritage sites. In 18th International Conference on Virtual Systems and Multimedia (VSMM) (pp. 369-376), Milan, Italy. https://doi.org/10.1109/VSMM.2012.6365947 Dore, C., Murphy, M., McCarthy, S., Brechin, F., Casidy, C., & Dirix, E. (2015). Structural simulations and conservation analysis-historic building information model (HBIM). The International Archives of Photogrammetry, Remote Sensing and Spatial Information Sciences, 40(5/W4), 351-357. https://doi.org/10.5194/isprsarchives-XL-5-W4-351-2015 ECP. (2012). Egyptian code for calculating loads and forces in structural work and masonry. Housing and Building National Research Center, Ministry of Housing, Utilities and Urban Planning, Cairo, Egypt. Elyamani, A., & Roca Fabregat, P. (2018a). A review on the study of historical structures using integrated investigation activities for seismic safety assessment. Part I: dynamic investigation. Scientific Culture, 4, 1-27. Elyamani, A., & Roca Fabregat, P. (2018b). A review on the study of historical structures using integrated investigation activities for seismic safety assessment. Part II: Model updating and seismic analysis. Scientific Culture, 4, 29-51. Fernández-Lozano, J., Gutiérrez-Alonso, G., & FernándezMorán, M. Á. (2015). Using airborne LiDAR sensing technology and aerial orthoimages to unravel roman water supply systems and gold works in NW Spain (Eria valley, León). Journal of Archaeological Science, 53, 356-373. https://doi.org/10.1016/j.jas.2014.11.003 Giordano, A., Mele, E., & De Luca, A. (2002). Modelling of historical masonry structures: comparison of different approaches through a case study. Engineering Structures, 24(8), 1057-1069. https://doi.org/10.1016/S0141-0296(02)00033-0 Głowienka, E., & Michałowska, K. (2017). 4D reconstruction and visualisation of Krakow Fortress. In 2017 Baltic Geodetic Congress, Gdansk, Poland. Haala, N., Peter, M., Cefalu, A., & Kremer, J. (2008, October). Mobile LiDAR mapping for urban data capture. In Proceedings of the 14th International Conference on Virtual Systems and Multimedia, Limassol, Cyprus. Hesse, R. (2010). Extraction of archaeological features from high-resolution LiDAR data. In 14th International Congress “Cultural Heritage and New Technologies” (pp. 636-642), Vienna, Austria. Kappos, A. J., Penelis, G. G., & Drakopoulos, C. G. (2002). Evaluation of simplified models for lateral load analysis of unreinforced masonry buildings. Journal of Structural Engineering, 128, 890-897. https://doi.org/10.1061/(ASCE)0733-9445(2002)128:7(890) Li, Z., Yan, Y., Jing, Y., & Zhao, S. G. (2015). The design and testing of a LiDAR Platform for a UAV for heritage mapping. The International Archives of Photogrammetry, Remote Sensing and Spatial Information Sciences, 40(1/W4), 17-24. https://doi.org/10.5194/isprsarchives-XL-1-W4-17-2015 Lourenço, P. B. (2002). Computations on historic masonry structures. Progress in Structural Engineering and Materials, 4, 301319. https://doi.org/10.1002/pse.120 Lourenço, P. B., Mendes, N., Ramos, L. F., Oliveira, D. V. (2011). Analysis of masonry structures without box behavior. International Journal of Architectural Heritage, 5, 369-382. https://doi.org/10.1080/15583058.2010.528824 Murphy, M., McGovern, E., & Pavia, S. (2009). Historic building information modelling (HBIM). Structural Survey, 27(4), 311-327. https://doi.org/10.1108/02630800910985108 Journal of Civil Engineering and Management, 2020, 26(1): 53–65 Nex, F., & Rinaudo, F. (2010). Photogrammetric and LiDAR integration for the cultural heritage metric surveys. International Archives of Photogrammetry, Remote Sensing and Spatial Information Sciences, 38(Part 5), 490-495. Oreni, D., Brumana, R., Della Torre, S., Banfi, F., & Previtali, M. (2014). Survey turned into HBIM: the restoration and the work involved concerning the Basilica di Collemaggio after the earthquake (L’Aquila). ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences, 2(5), 267-273. https://doi.org/10.5194/isprsannals-II-5-267-2014 Oyarzo Vera, C. (2012). Damage identification of unreinforced masonry structures based on vibration response (Doctoral dissertation). The University of Auckland. Rubinowicz, P., & Czynska, K. (2015). Study of city landscape heritage using LiDAR data and 3D-city models. International Archives of Photogrammetry, Remote Sensing and Spatial Information Sciences, 40(7/W3), 1395-1402. https://doi.org/10.5194/isprsarchives-XL-7-W3-1395-2015 Sánchez-Aparicio, L. J., Del Pozo, S., Ramos, L. F., Arce, A., & Fernandes, F. M. (2018). Heritage site preservation with combined radiometric and geometric analysis of TLS data. Automation in Construction, 85, 24-39. https://doi.org/10.1016/j.autcon.2017.09.023 Valanis, A., & Tsakiri, M. (2004, July). Automatic target identification for laser scanners. In Proceedings of XXth ISPRS Congress, Istanbul, Turkey. Wetherelt, A., Cooper, J. P., & Zazzaro, C. (2014, August). 3D laser scanning and modelling of the Dhow heritage for the Qatar National Museum. 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Attitude and Peer Norm Predict How Students Use Lecture Recordings
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Attitude and Peer Norm Predict How Students Use Lecture Recordings Citation for published version: Banas, K, Thejll-Madsen, T, Tobar Henriquez, A & Murzyn, E 2023, 'Attitude and Peer Norm Predict How Students Use Lecture Recordings', Computers & Education, vol. 207, 104933. https://doi.org/10.1016/j.compedu.2023.104933 Citation for published version: Banas, K, Thejll-Madsen, T, Tobar Henriquez, A & Murzyn, E 2023, 'Attitude and Peer Norm Predict How Students Use Lecture Recordings', Computers & Education, vol. 207, 104933. https://doi.org/10.1016/j.compedu.2023.104933 A B S T R A C T The impact of lecture recordings on university students’ learning often depends on whether the recordings are used as a substitute for attending live lectures (watching instead of attending; usually undesirable) or as a supplement to attending (watching in addition to attending; usually desirable). However, little is known about the predictors of how students use the recordings. This study examined the demographic and psychological variables associated with attending live lectures, and with the use of lecture recordings as a substitute or supplement. In particular, we characterised the students’ own opinions about attending live lectures and using lecture recordings (attitude), students’ beliefs about what their classmates thought about these behaviours (peer norm), and students’ beliefs about what their lecturers thought about these behaviours (lecturer norm). Using data gathered in a large introductory psychology course (n = 212), we found that attending live lectures, using recordings as a supplement and using recordings as a substitute were all viewed favourably and perceived to be accepted by peers. The perception of lecturer norm was more nuanced, with substitution perceived as the least acceptable to lecturers. Generally, the more positive the students’ own attitude and the perceived peer norm towards engaging with lectures in a particular way (attending live, using recordings as a supplement, or using recordings as a substitute), the more likely students were to engage with lectures in that way. These findings suggest that attitudes and peer norms may be valuable targets for educational interventions in this area. Attitude and peer norm predict how students use lecture recordings Kasia Banas a,b,*, Tobias Thejll-Madsen b, Anita Tobar-Henríqu Kasia Banas a,b,*, Tobias Thejll-Madsen b, Anita Tobar-Henríquez b, Eva Murzyn b a Usher Institute, College of Medicine and Veterinary Medicine, University of Edinburgh, 9 Little France Road, Edinburgh BioQuarter, Edinburgh, EH16 4UX, United Kingdom b Psychology, University of Edinburgh, 7 George Square, Edinburgh, EH8 9AG, United Kingdom Kasia Banas a,b,*, Tobias Thejll-Madsen b, Anita Tobar-Henríquez b, Eva Murzyn b a Usher Institute, College of Medicine and Veterinary Medicine, University of Edinburgh, 9 Little France Road, Edinburgh BioQuarter, Edinburgh, EH16 4UX, United Kingdom b Psychology, University of Edinburgh, 7 George Square, Edinburgh, EH8 9AG, United Kingdom Kasia Banas a Usher Institute, College of Medicine and Veterinary Medicine, University of Edinburgh, 9 Little France Ro EH16 4UX, United Kingdom b Psychology, University of Edinburgh, 7 George Square, Edinburgh, EH8 9AG, United Kingdom Available online 20 September 2023 0360-1315/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.compedu.2023.104933 Received 12 October 2022; Received in revised form 7 September 2023; Accepted 20 September 2023 * Corresponding author. E-mail addresses: Kasia.Banas@ed.ac.uk (K. Banas), 2590705t@student.gla.ac.uk (T. Thejll-Madsen), Anita.Tobar@ed.ac.uk (A. Tobar- Henríquez), eva.murzyn@gmail.com (E. Murzyn). * Corresponding author. Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 Computers & Education 207 (2023) 104933 1.2. Substitution and supplementation Williams et al. (2012) introduced an important distinction between using lecture recordings as a substitute for live lectures (watching recordings instead of attending live lectures) versus using them as a supplement to live lectures (watching recordings as well as attending live lectures). The authors went further to argue that the use of lecture recordings has a particularly positive association with achievement where recordings are used as a supplement rather than a substitute. However, as their study only included data on total attendance and recordings use, and not lecture-level data, the analysis assumes that students with high lecture attendance use recordings as a supplement and students with low lecture attendance use them as a substitute. In a more recent study, Bos et al. (2016) used individual lecture-level data to examine differences in academic achievement between four groups of students: those who used lecture recordings as a supple­ ment, those who used them as a substitute, students who did not use the recordings at all but attended live lectures, and students who neither attended the lectures nor watched the recordings. The results showed that students who used lecture recordings as a supplement performed better on a course assessment than those who attended the lectures in person but did not watch any recordings afterwards. In contrast, students who just watched the recording did no worse than students who attended the lecture in person. One way of gaining insight into how students use lecture recordings is to use learning analytics in conjunction with data mining techniques to find distinct patterns in how students engage with various learning materials. This approach has been implemented by O’Brien and Verma (2019), who found four distinct clusters of student behaviours regarding lecture resources: traditional (students who attended lectures and consulted lecture notes but did not utilise the lecture recordings), digital (students who did not attend lectures but relied on lecture notes and recordings), minimal (students who relied on lecture notes, with low attendance at live lectures and minimal use of recordings), and phantom (low attendance, low use of lecture recordings and notes). Surprisingly, this study did not identify a cluster of students whose behaviour would fit the supplementation pattern - it seemed that students either attended live lectures or relied on recordings as a substitute. Similar to Williams et al. 1. Introduction Over the past two decades, it has become commonplace in many countries, including the United Kingdom, for live university lectures to be recorded, and for the recordings to be provided to students as a learning aid (Ibrahim et al., 2021; Morris et al., 2019). This is received enthusiastically by the students, who see lecture recordings as a helpful tool for aiding their revision and allowing them to catch up on lectures missed due to illness or schedule clashes (Lokuge Dona et al., 2016). University staff remain divided in their attitudes, with some seeing lecture recording as a welcome technological advancement and others concerned about its impact on attendance and retention of information (Dommett et al., 2020). The existing evidence shows that any links between the use of lecture recordings and academic performance remain tenuous at best (Baillie et al., 2022), and one reason for this may be that students interact with lecture recordings in different ways, but empirical studies do not always capture these differences (Banerjee, 2021; Witchel et al., 2022). One key distinction (Williams et al., 2012) lies in whether the recordings are being used as a substitute for live lectures (i.e., students watch recordings instead of attending live lectures) or as a supplement to live lectures (i.e., students attend live lectures, and subse­ quently use the recordings to complement their learning). Observational studies show that students use lecture recordings in both ways Computers & Education 207 (2023) 104933 K. Banas et al. (Chinnery et al., 2021; MacKay et al., 2022). However, we have limited insight into why exactly students engage with lecture re­ cordings in one way or the other. Thus, the first aim of this paper is descriptive: we set out to describe how students use lecture re­ cordings, with a focus on supplementation and substitution. We also explore what attitudes students hold towards lecture recordings and attending live lectures, and what norms pertaining to lecture attendance and lecture recording use they perceive among their peers and lecturers. The second aim is to put lecture recordings in a social context: learning is often conceptualised as a social process (Salomon & Perkins, 1998), and therefore it is key to include social factors in our model of lecture recording use. 1.1. Lecture recordings and lecture attendance Much of the scholarship on lecture recordings has focused on their effect on attendance, largely because attendance is consistently associated with academic achievement (Cred´e et al., 2010), and so lowering attendance may also lead to a decrease in achievement. However, there seems to be no evidence of a consistent detrimental effect of the introduction or use of lecture recording on students’ attendance (Banerjee, 2021). On one hand, Edwards and Clinton (2019) used a longitudinal approach to compare attendance on a similar course before and after lecture recording was introduced, and found that the introduction of recording was followed by a significant drop in attendance. In contrast, studies examining the correlation between attendance and the use of lecture recordings using student-level data available within a particular course (e.g. Nordmann et al., 2019) have found only a very weak association between the amount of recorded material students viewed and attendance at live lectures. l Reflecting on these inconsistent results, two recent chapters by Robson and Kauffman (2022) and Witchel et al. (2022) highlight that in the era of high-quality lecture recordings, it may be possible for students to perform excellently even without high levels of attendance. Some types of lectures, as Witchel et al. (2022) point out, may be replaced with recordings quite effectively, and some students may be particularly skilled at using the recordings to their advantage. Thus, to investigate how lecture recordings may be facilitating or hindering student achievement, we first need a more nuanced understanding of how students use the recordings in specific contexts. 1. Introduction The third aim is predictive: we examine the demographic and psychological variables that can help us predict whether students will use lecture re­ cordings as a supplement or a substitute, and whether students will attend in-person lectures. 1.2. Substitution and supplementation (2012), a limitation of this study lay in the fact that it did not look at attendance and recording use on a lecture-by-lecture basis. Instead, what was analysed was the average number of lectures attended and recordings accessed. 1.3.3. Social identification i i Social identification refers to “defining part of the self in terms of the social group” (Smyth et al., 2019, p. 409), and it is a key tenet of the Social Identity Approach (Hornsey, 2008). The key relevance of this construct to our theorising lies in the fact that when people highly identify with a social group, they derive significant meaning from that group membership, resulting in a motivation to follow the group norms and engage in behaviours prototypical for that group. Thus, a higher level of identification as a student would be expected to correlate with behaviours that are expected of students in that discipline. Indeed, students who report higher identification tend to engage in more thoughtful learning practices (Smyth et al., 2015), have stronger intentions to continue with academic study (Platow et al., 2013) and achieve higher marks (Bliuc et al., 2011a; Bliuc et al., 2011b). i Apart from the direct association with behaviour, identification has also been shown to moderate the effect of social norms (Turner et al., 1987), with highly identified students more likely to follow the peer and educator norm in favour of deep learning (Smyth et al., 2015, 2018). Our study will investigate both the direct and the moderation effect of student identification on attending live lectures and using lecture recordings as a supplement or substitute. 1.3.1. Attitude Attitude is typically defined as the person’s overall evaluation of the target behaviour. It is often found to be a strong predictor of the intention to engage in the behaviour, and intention tends to be predictive of behaviour (Sheeran et al., 2016). In the domain of education, attitude was found to be strongly associated with intentions to attend peer-assisted study sessions, and was an indirect predictor of attendance, mediated by intention (Allen et al., 2019; White et al., 2008). The literature consistently reports that students’ attitudes towards lecture recordings are overwhelmingly positive (MacKay et al., 2022; Mcgowan & Hanna, 2015; Morris et al., 2019; Nkomo & Daniel, 2021), and this holds for most levels and subjects of study, and also in most demographic groups. The positive attitude is related to the fact that providing lecture recordings helps students remove some of the anxiety around missed lectures or difficulties with understanding the lecturer (Nkomo & Daniel, 2021). Students also report that when they know a recording will be available, they worry less about being able to keep up with the lecture content in their notes (MacKay, 2020). 1.3.2. Social norms Social norms refer to whether other people approve of the target behaviour and whether they themselves engage in that behaviour (Cialdini et al., 1991). Smyth and colleagues have studied the influence of norms in university education (Smyth, Chandra, & Mavor, 2018; Smyth, Mavor, & Platow, 2017; Smyth, Mavor, Platow, Grace, & Reynolds, 2015), showing that perceived learning norms of peers and educators affect student learning behaviours: if students believe that their peers and educators favour learning for under­ standing rather than just memorising, they are more likely to engage in learning for understanding themselves. Similarly, sending students a normative message where staff recommended spending more time on lesson activities (versus a control message) led to an increase in the amount of time spent on lesson activities and to a higher quiz score (Eyink et al., 2020). To our knowledge, there is no literature about the social norms surrounding the use of lecture recordings, and bridging this gap is one of the key aims of this study. i 1.3. Predictors of lecture recording use Consequently, we apply the socio-cognitive framework of Theory of Planned Behaviour (TPB; Ajzen, 1991) to investigate the predictors of how lecture recordings are used. The key constructs in TPB are attitudes, social norm and perceived behavioural control – these three are predictors of behavioural intention, and intention, in turn, predicts behaviour. While TPB has been subject to critique, especially in the health literature (see Sniehotta et al., 2014), its relative simplicity and high pre­ dictive power mean that it remains a key framework for studying predictors of behaviour, including in education (e.g. Allen et al., 2019; Pownall, 2012; White et al., 2008). Some voices have called for improving TPB by adding other known predictors of intention and behaviour (Conner, 2015; Conner & Armitage, 1998). Our work adopted exactly this approach, focusing on the TPB predictors of attitude and social norm, and adding social identification – a construct originating from the Social Identity Approach (Hornsey, 2008). 1.3. Predictors of lecture recording use A few key studies (e.g., Nordmann et al., 2019; O’Brien & Verma, 2019; Sarsfield & Conway, 2018) have sought to understand what student-level characteristics are associated with specific study behaviours, including the use of lecture recordings. The variables used in these studies include demographic characteristics, such as gender, being a native speaker of English, or having learning difficulties that may affect engagement with lectures. While demographic variables are a common starting point for predictive models of behaviour, psychological models of learning behaviour often go beyond this by incorporating socio-cognitive predictors, such as at­ titudes or norms (Cheon et al., 2012). The theoretical framing of this paper is based on the premise that there are both individual and social aspects to learning (Salomon & Perkins, 1998): while university instruction has an element of individual knowledge acquisition, classroom interactions with lec­ turers, tutors and peers are also a key part of learning. Crucially, other people are not only the transmitters of knowledge, but they also 2 2 Computers & Education 207 (2023) 104933 K. Banas et al. act as models and advisors for how learning should be done – lecturers often give advice on how to learn, and other students provide a reference point on study practices. Consequently, we apply the socio-cognitive framework of Theory of Planned Behaviour (TPB; Ajzen, 1991) to investigate the predictors of how lecture recordings are used. The key constructs in TPB are attitudes, social norm and perceived behavioural control – these three are predictors of behavioural intention, and intention, in turn, predicts behaviour. While TPB has been subject to critique, especially in the health literature (see Sniehotta et al., 2014), its relative simplicity and high pre­ dictive power mean that it remains a key framework for studying predictors of behaviour, including in education (e.g. Allen et al., 2019; Pownall, 2012; White et al., 2008). Some voices have called for improving TPB by adding other known predictors of intention and behaviour (Conner, 2015; Conner & Armitage, 1998). Our work adopted exactly this approach, focusing on the TPB predictors of attitude and social norm, and adding social identification – a construct originating from the Social Identity Approach (Hornsey, 2008). act as models and advisors for how learning should be done – lecturers often give advice on how to learn, and other students provide a reference point on study practices. 2.2. Course in study The course where this study was embedded was delivered over 11 weeks–10 teaching weeks with a break in the middle. The teaching included three 50-min-long didactic lectures each week, as well as small-group interactive tutorials with discussion of published articles (four in total) and larger practical labs involving data collection and simple analysis (four in total). There were 327 students enrolled. The course was embedded in the wider university context, where introductory courses are typically large (several hundred students) and follow a similar structure, with a combination of lectures and practical or discussion-based sessions and assessment consisting of both coursework and exams. During the data collection period, most courses (including this one) followed a traditional model, where attendance at live lectures was expected, although not mandatory. Additional materials provided to the students included (1) a course handbook outlining the learning outcomes and course structure, (2) a virtual learning environment where lecture slides, recordings and other course information was posted, and (3) readings (assigned journal articles and a recommended textbook). The students typically had access to lecture slides at least 24 h before the lecture but did not have access to any detailed lecture notes. The textbook was suggested as an optional resource, but lectures often went beyond the content covered by the textbook. i The assessment consisted of two substantive elements: an exam comprised of 100 multiple choice questions (45% of the final mark), testing knowledge of the course topics and based primarily on the lecture content, and a 1200-word essay (23% of the final mark), designed to test the students’ ability to think critically about the published literature, and related loosely to the tutorial content. The remaining 32% of the course mark consisted of attendance at tutorials and labs, research participation and completion of study skills activities – each of these components accounted for 8% of the mark. i Students with additional support needs had access to the University’s Disability Office and might have received learning adjust­ ments, such as additional time at exams, extensions for submitting coursework, or support from a scribe. All students also had access to generic study skills support delivered at the university level. Students who spoke English as a second language had access to university- level support (such as guidance on academic English), but were not given any additional support within the course. 2.1. Participants and procedure We recruited students enrolled in a semester-long introductory psychology course at a large research-intensive Scottish university. The data were collected in Autumn 2019, before the covid-19 pandemic led to the cancellation of in-person lectures. There were 327 students enrolled in this course, approximately 90% female, and 60% were enrolled in Psychology degrees (the Scottish system allows students from other degree programmes to take outside courses in the first two years). i All 327 students were invited to participate in the study, 263 consented to participation and completed the study questionnaire, and a subset of 212 students consented to having their questionnaire data linked with administrative records. The data presented below pertain to the 212 participants who consented to having their questionnaire data linked with the administrative data, and whose data we were able to retrieve. Demographic characteristics of these participants are presented in Table 1. Ethical approval was granted by the Psychology Research Ethics Committee (ref. 1–1920/1). 1 Attendance data were not available for lectures delivered on the following dates: 2019-09-18, 2019-09-20, 2019-10-21, 2019-10-23, 2019-10- 25, 2019-10-28, 2019-11-25, 2019-11-27, 2019-11-29. 1.4. The current study Our study focused on two key research questions. First, what is the uptake of lecture recordings, and are they used primarily as a substitute or a supplement? Second, how do attitudes, norms and identification shape how students engage with lecture recordings? The answers will help educators and researchers understand what drives students’ enthusiasm about lecture recordings, and also to what extent being positive about a particular way of using the recordings (e.g. substituting or supplementing) is then translated into behaviour. The results will also provide preliminary insights into whether social norm messaging would be useful for promoting effective lecture recording use, alongside information and practical guidance such as that provided by Nordmann et al. (2022). To answer these questions, we gathered lecture- and student-level data on attendance and use of lecture recordings. We focused on the role of attitudes and perceived peer and educator norms in shaping three key outcomes: attendance at live lectures, using lecture recordings as a supplement to live lectures, and using lecture recordings as a substitute for live lectures. In line with Theory of Planned Behaviour (Ajzen, 1991), we predicted that attitudes and both types of norms would be positively associated with the corresponding outcomes: for example, that students whose attitudes towards attending live lectures are positive, and who perceive the norm among their peers and lecturers to be favourable towards attending in-person lectures, would attend live lectures more often. Further, in line with social psychological theorising (Turner et al., 1987), we predicted that the influence of both types of norms would be stronger among those students who highly identify with their course cohort. 3 2. Method K. Banas et al. K. Banas et al. Computers & Education 207 (2023) 104933 2. Method 2.1. Participants and procedure 2 The nature of the data we obtained does not show how much of the video each student watched during each w access to is the total proportion of the video duration that was watched. 1 Attendance data were not available for lectures delivered on the following dates: 2019-09-18, 2019-09-20, 2019-10-21, 2019-10-23, 2019-10- 25, 2019-10-28, 2019-11-25, 2019-11-27, 2019-11-29. 2 The nature of the data we obtained does not show how much of the video each student watched during each watching episode. What we have access to is the total proportion of the video duration that was watched. 25, 2019-10-28, 2019-11-25, 2019-11-27, 2019-11-29. 2 The nature of the data we obtained does not show how much of the video each student watched during each watching episode. What we have access to is the total proportion of the video duration that was watched. 1 Attendance data were not available for lectures delivered on the following dates: 2019-09-18, 2019-09-20, 20 25, 2019-10-28, 2019-11-25, 2019-11-27, 2019-11-29. 3 Histograms of each individual attitude score can be found on the project’s OSF page. Table 1 Table 1 2.4. Questionnaire data Table 1 Demographic characteristics of participants in the sample. Variable Mean (SD) or Frequency (%) Age 19.2 (SD) Gender Female 180 (84.9%) Male 29 (13.7%) Prefer not to say 3 (1.4%) Degree enrolled in Psychology (single or joint honours) 138 (65.0%) Other degree at the university 66 (31.1%) Exchange students 8 (3.8%) Native speaker of English Yes 132 (62.2%) No 80 (37.7%) Carer status Yes 6 (3.0%) No 206 (97.0%) Employment status Yes 37 (17.5%) No 175 (82.5%) Disability status Yes 18 (8.5%) No 194 (91.5%) Demographic characteristics of participants in the sample. Variable Mean (SD) or Frequency (%) Age 19.2 (SD) Gender Female 180 (84.9%) Male 29 (13.7%) Prefer not to say 3 (1.4%) Degree enrolled in Psychology (single or joint honours) 138 (65.0%) Other degree at the university 66 (31.1%) Exchange students 8 (3.8%) Native speaker of English Yes 132 (62.2%) No 80 (37.7%) Carer status Yes 6 (3.0%) No 206 (97.0%) Employment status Yes 37 (17.5%) No 175 (82.5%) Disability status Yes 18 (8.5%) No 194 (91.5%) 2.4. Questionnaire data 2.4.1. Attitude We asked students about their attitudes towards three behaviours: (1) attending live lectures in person, (2) attending lectures first and then watching the recordings (i.e. supplementation), and (3) watching recorded lectures online instead of attending them in person (i.e. substitution). For each of these behaviours, we asked five questions on 7-point semantic differential scales anchored at bad- good, harmful-beneficial, unpleasant-pleasant, foolish-wise, and unnecessary-necessary (adapted from Armitage & Conner, 1999). The responses were coded so that a higher score indicated a more positive attitude, and were averaged to form a single attitude score for each behaviour. The Cronbach’s alpha for this scale was satisfactory, with alpha = 0.86 for attitude towards lecture attendance, alpha = 0.85 for attitude towards supplementation, and alpha = 0.86 for attitude towards substitution.3 2.4.3. Lecturer norm We also asked participants to estimate the relevant norm among course lecturers. The questions used were adapted from Bassili (2008): “How useful do you think your lecturers in [this course] consider the *live lectures* to be as a teaching tool?” (attendance), “How useful do you think your lecturers in [this course] consider the *recorded lectures* to be as a teaching tool, when they are watched after attending the lecture?” (supplementation), and “How useful do you think your lecturers in [this course] consider the *recorded lectures* to be as a teaching tool, when watched instead of attending lectures in person?” (substitution). 2.4. Questionnaire data At the start of the semester, we distributed a questionnaire measuring students’ attitudes towards attending live lectures and using lecture recordings, relevant social norms, and identification with the psychology student cohort. Participants responded on 7-point Likert-type scales. 2.4.2. Peer norm For each of the target behaviours, we asked participants a single question to estimate the perceived peer norm (adapted from Bassili, 2008): “How useful do you believe [other students] feel the *live lectures* are in this course?” (attendance), “How useful do you believe [other students] feel the *recorded lectures* are in this course, when they are watched after attending the lecture?” (supplementation), and “How useful do you believe [other students] feel the *recorded lectures* are in this course, when watched instead of attending lectures in person?” (substitution). 2.3. Administrative data Attendance was recorded during most lectures, using the attendance-checking functionality within the active learning system Top Hat (2019). During the lecture, attending students were shown a code on the projector screen and were asked to input that code on their devices to confirm attendance. We gathered attendance data from 24 out of 30 lectures delivered during the semester; for four lectures this was not organised in time, and in two cases the lecturer used questions on Top Hat rather than simple attendance checking.1 These had a lower response rate and therefore could not be compared with the other data, which is why they were sub­ sequently excluded from the analysis. We had access to recording use data from all 30 lectures - these data were collected routinely by the system used for lecture re­ cordings, Echo360 (2019). We were able to inspect data on the number of times that each student accessed each video and the per­ centage of the video that had been watched. When we considered each case of a student watching a video at least once (n = 3077), we found that in 71% of cases (n = 2189), if a student accessed a video, they watched half or more of the recording’s duration (over one or more watching sessions).2 Therefore, the data we use here is the number of videos accessed. Given that we had access to both attendance and recording watching data for 24 lectures, for each participant we calculated two key indices: the number of lectures that the participant attended and then watched (broadly corresponding to supplementing), and the number of lectures that the participant did not attend but watched (broadly corresponding to substituting). Each of these indices ranged from 0 to 24, and they were the key outcome variables in our study. Attendance data were not available for lectures delivered on the following dates: 2019 09 18, 2019 09 20, 2019 10 21, 2019 10 23, 2019 10 25, 2019-10-28, 2019-11-25, 2019-11-27, 2019-11-29. 2 The nature of the data we obtained does not show how much of the video each student watched during each watching episode. What we have access to is the total proportion of the video duration that was watched. 4 K. Banas et al. Computers & Education 207 (2023) 104933 2.4.4. Student identification i i Identification with other students on the course was measured using a 4-item scale adapted from Cameron (2004): “I have a lot in common with other [course] students,” “I feel strong ties to other [course] students,” “I find it difficult to form a bond with other [course] students (reverse-scored),” “I don’t feel a sense of being “connected” with other [course] students” (reverse-coded). The scale was internally consistent (Cronbach’s alpha = .84), and so the responses were averaged, with a higher score indicating a higher level of identification. 5 5 Computers & Education 20 K. Banas et al. Computers & Education 207 (2023) 104933 (caption o (caption on next page) Computers & Education 207 (2023) 104933 Computers & Education 207 (2023) 104933 K. Banas et al. Fig. 1. a) Proportion of students attending each lecture i Note: There are two visible dips in attendance. The first one, on 1st November, is likely related to the fact that many students attended Halloween parties the night before (this dip is consistent with historical data from previous years). The second dip, on 15th November, is likely related to upcoming assessment deadlines (the tutorial essay was due on Monday 18th November). b) Proportion of students who accessed each lecture recording, by attendance Note: The solid black line corresponds to the proportion of students who did not attend the lecture, but watched the recording afterwards (i.e., proportion of students who substituted the recording for a live lecture on this occasion). The dashed grey line corresponds to the proportion of students who did attend the lecture and also watched the recording afterwards (i.e. proportion of students who used the recording as a supplement on this occasion). 3.1.1. Lecture attendance We had access to attendance data from 24 out of 30 lectures delivered in September–December 2019. Lecture attendance decreased over time (see Fig. 1a), with each lecture having on average five fewer students attending than the previous one (linear regression analysis indicated a significantly negative slope, with B = −4.88, p < .001). This pattern aligns with anecdotal evidence from previous years. On average, each student attended 55% of lectures. 3.1.2. Lecture recording use On average, each lecture recording was accessed by 31% of students enrolled in the course. The use of lecture recordings also decreased over time, albeit at a slower rate than attendance: each lecture had on average one fewer student watching the recording than the previous one (linear regression analysis indicated a significantly negative slope, with B = −1.10, p = .002). Students who did not attend a given lecture were more likely to watch its recording than those who did (see Fig. 1b). Most students (80%) accessed at least one recording via the system, while 20% of students accessed no recordings at all. In most cases, students accessed a recording only once and watched it in its entirety. 2.5. Analyses plan To test our hypotheses, we performed binomial regression, a suitable method for analysing count data with a large number of 0s (Theobald et al., 2019). The coefficient estimates are reported on a natural logarithmic scale and must be exponentiated before they can be interpreted. The exponentiated coefficient (Odds Ratio, later abbreviated as OR) expresses the likelihood of the outcome, with 1 being a neutral value (i.e. a predictor with a coefficient of 1 does not affect the outcome at all). Predictors with coefficients larger than 1 increase the likelihood of the outcome, and predictors with coefficients below 1 decrease the likelihood. i We estimated three predictive models for each outcome of interest (lecture attendance, substitution, and supplementation). The first model only included demographic predictors: gender, age, being a native speaker of English, being enrolled in a Psychology degree, being a carer, being employed, and identifying as someone with a disability. The second model also included psychological predictors: attitude towards the behaviour, peer norm about the behaviour, educator norm about the behaviour, and group identi­ fication. In the third model, we added the predicted two-way interactions between norms and identification. All analyses were con­ ducted using R (v4.3.0, R Core Team, 2023) and R Studio (v2022.07.1, RStudio Team, 2022). The data and analysis scripts, including a list of all R packages used, can be found online at https://osf.io/tgp8j/. 3.1. Descriptive statistics 3.1. Descriptive statistics 3.1.3. Attitudes and norms self-reported attitude and perceived norms regarding lecture attendance, using lecture recordings as a supplement shed line indicates the median score. Fig. 2. Distribution of scores for self-reported attitude and perceived norms regarding lecture attendance, using lecture recordings as a supplement and as a substitute Note. The dashed line indicates the median score. 3.1.3. Attitudes and norms The distributions of student attitude, peer norm and lecturer norm towards attending lectures in person were left-skewed, indi­ cating that most students had a positive attitude towards this behaviour and reported that both their peers and their lecturers believed that attending live lectures was useful (see Fig. 2a and Table 2). The number of lectures attended was significantly and positively correlated with the student’s attitude towards attending live lectures (r = 0.29, p < .01) and their level of student identification (r = 0.16, p < .05), but not the peer or lecturer norm (ps > .05). The distributions of attitudes and norms towards supplementation (i.e., attending the lecture in person and then watching the recording) were also left-skewed, though responses were distributed more evenly across the positive response options (see Fig. 2b and Table 3). The number of times a recording was used to supplement a live lecture was significantly and positively correlated with the student’s attitude towards supplementation (r = 0.20, p < .01) and the peer norm (r = 0.20, p < .01). The distribution of attitude towards substitution (i.e., not attending a lecture and then watching its recording) was close to normal, with the median response indicating a neutral attitude (see Fig. 2c and Table 4). The distribution of perceived lecturer norm was also approximately normal, with most responses falling in the middle of the scale. Perceived student norm showed a strong left skew, with most students indicating that their peers felt that substitution was helpful. The number of times a recording was used to substitute a live lecture was significantly and positively correlated only with the student’s attitude towards substitution (r = 0.27, p < .01). 7 Computers & Education 207 (2023) 104933 K. Banas et al. 2. Distribution of scores for self-reported attitude and perceived norms regarding lecture attendance, using lecture recordings as a supplem as a substitute Note. The dashed line indicates the median score. g. 2. Distribution of scores for self-reported attitude and perceived norms regarding lecture attendance, using lecture recordings as a supple d as a substitute Note. The dashed line indicates the median score. Fig. 2. Distribution of scores for self-reported attitude and perceived norms regarding lecture attendance, using lecture recordings as a supplement and as a substitute Note. The dashed line indicates the median score. 3.2. Predicting lecture attendance The demographic model (Model 1a; see Table 5 for full model coefficients) indicated that male students were significantly less likely to attend lectures than female students (OR = 0.73, p = .001) and native speakers were significantly less likely to attend lectures than students for whom English was a second language (OR = 0.67, p < .001). Students enrolled in a psychology degree were significantly more likely to attend lectures than students enrolled in other degrees (OR = 2.22, p < .001), and students with a disability were significantly less likely to attend lectures than students without a disability (OR = 0.61, p < .001). 8 Computers & Education 207 (2023) 104933 K. Banas et al. K. Banas et al. We then added psychological predictors to the model (Model 1b). The demographic predictors that were significant in Model 1a remained significant, except for gender. Looking at psychological variables, we found that students attended more lectures if they had a positive attitude towards lecture attendance (OR = 1.32, p < .001) and if they more strongly identified with other psychology students (OR = 1.07, p = .046). Somewhat surprisingly, we also found that a positive peer norm about lecture attendance was associated with lower odds of attending lectures (OR = 0.86, p < .001). In Model 1c, we also included two-way interactions between norms and identification. We found a significant two-way interaction between the student norm and group identification (OR = 1.09, p = .019), in a direction opposite to what we expected: while there was a negative association between student norm and lecture attendance, the slope of this relationship was the steepest among participants with a low level of student identification. In other words, high levels of student identification buffered the effect of the student norm Table 3 Using lecture recordings as a supplement: Means, standard deviations, and Spearman’s correlations. Variable M SD 1 2 3 4 1. Attitude 5.45 1.16 2. Student norm 5.58 1.37 .49*** 3. Lecturer norm 5.76 1.24 .41*** .41*** 4. Student identification 3.95 1.24 .11 −.09 .00 5. Number of lecture recordings watched as a supplement 3.26 4.49 .24*** .27*** −.01 .05 Note. * indicates p < .05. ** indicates p < .01, *** indicates p < .001. Table 4 Using lecture recordings as a substitute: Means, standard deviations, and Spearman’s correlations. Variable M SD 1 2 3 4 1. Attitude 3.99 1.30 2. 3.2. Predicting lecture attendance Student norm 5.82 1.16 .18** 3. Lecturer norm 4.20 1.59 .28*** .35*** 4. Student identification 3.95 1.24 −.16* .05 .05 5. Number of lecture recordings watched as a substitute 4.17 4.96 .21** .11 .02 −.07 Note. * indicates p < .05. ** indicates p < .01, *** indicates p < .001. Table 2 Attending live lectures: Means, standard deviations, and Spearman’s correlations. Variable M SD 1 2 3 4 1. Attitude 6.06 0.91 2. Student norm 5.90 0.96 .44*** 3. Lecturer norm 6.50 0.86 .29*** .29*** 4. Student identification 3.95 1.24 .16* .15* −.02 5. Number of attended lectures 13.80 6.59 .28*** .08 .04 .15* Note. * indicates p < .05. ** indicates p < .01, *** indicates p < .001. Table 2 Attending live lectures: Means, standard deviations, and Spearman’s correlations. Table 2 Attending live lectures: Means, standard deviations, and Spearman’s correlations. Variable M SD 1 2 3 4 1. Attitude 6.06 0.91 2. Student norm 5.90 0.96 .44*** 3. Lecturer norm 6.50 0.86 .29*** .29*** 4. Student identification 3.95 1.24 .16* .15* −.02 5. Number of attended lectures 13.80 6.59 .28*** .08 .04 .15* Note. * indicates p < .05. ** indicates p < .01, *** indicates p < .001. Attending live lectures: Means, standard deviations, and Spearman’s correlations. Table 3 Using lecture recordings as a supplement: Means, standard deviations, and Spearman’s correlations. Variable M SD 1 2 3 4 1. Attitude 5.45 1.16 2. Student norm 5.58 1.37 .49*** 3. Lecturer norm 5.76 1.24 .41*** .41*** 4. Student identification 3.95 1.24 .11 −.09 .00 5. Number of lecture recordings watched as a supplement 3.26 4.49 .24*** .27*** −.01 .05 Note. * indicates p < .05. ** indicates p < .01, *** indicates p < .001. Table 4 Using lecture recordings as a substitute: Means, standard deviations, and Spearman’s correlations. Variable M SD 1 2 3 4 1. Attitude 3.99 1.30 2. Student norm 5.82 1.16 .18** 3. Lecturer norm 4.20 1.59 .28*** .35*** 4. Student identification 3.95 1.24 −.16* .05 .05 5. Number of lecture recordings watched as a substitute 4.17 4.96 .21** .11 .02 −.07 Note. * indicates p < .05. ** indicates p < .01, *** indicates p < .001. Table 3 Using lecture recordings as a supplement: Means, standard deviations, and Spearman’s correlations. Variable M SD 1 2 3 4 1. Attitude 5.45 1.16 2. Student norm 5.58 1.37 .49*** 3. 3.2. Predicting lecture attendance Lecturer norm 5.76 1.24 .41*** .41*** 4. Student identification 3.95 1.24 .11 −.09 .00 5. Number of lecture recordings watched as a supplement 3.26 4.49 .24*** .27*** −.01 .05 Note. * indicates p < .05. ** indicates p < .01, *** indicates p < .001. Table 3 We found a significant two-way interaction between the student norm and group identification (OR = 1.09, p = .019), in a direction opposite to what we expected: while there was a negative association between student norm and lecture attendance, the slope of this relationship was the steepest among participants with a low level of student identification. In other words, high levels of student identification buffered the effect of the student norm (see Fig. 3). Table 5 Demographic and psychological predictors of live lecture attendance (binomial regression coefficients). Table 3 3 lecture recordings as a supplement: Means, standard deviations, and Spearman’s correlations. Table 4 Using lecture recordings as a substitute: Means, standard deviations, and Spearman’s correlations. Variable M SD 1 2 3 4 1. Attitude 3.99 1.30 2. Student norm 5.82 1.16 .18** 3. Lecturer norm 4.20 1.59 .28*** .35*** 4. Student identification 3.95 1.24 −.16* .05 .05 5. Number of lecture recordings watched as a substitute 4.17 4.96 .21** .11 .02 −.07 Note. * indicates p < .05. ** indicates p < .01, *** indicates p < .001. We then added psychological predictors to the model (Model 1b). The demographic predictors that were significant in Model 1a remained significant, except for gender. Looking at psychological variables, we found that students attended more lectures if they had a positive attitude towards lecture attendance (OR = 1.32, p < .001) and if they more strongly identified with other psychology students (OR = 1.07, p = .046). Somewhat surprisingly, we also found that a positive peer norm about lecture attendance was associated with lower odds of attending lectures (OR = 0.86, p < .001). ii We then added psychological predictors to the model (Model 1b). The demographic predictors that were significant in Model 1a remained significant, except for gender. Looking at psychological variables, we found that students attended more lectures if they had a positive attitude towards lecture attendance (OR = 1.32, p < .001) and if they more strongly identified with other psychology students (OR = 1.07, p = .046). Somewhat surprisingly, we also found that a positive peer norm about lecture attendance was associated with lower odds of attending lectures (OR = 0.86, p < .001). ii In Model 1c, we also included two-way interactions between norms and identification. We found a significant two-way interaction between the student norm and group identification (OR = 1.09, p = .019), in a direction opposite to what we expected: while there was a negative association between student norm and lecture attendance, the slope of this relationship was the steepest among participants with a low level of student identification. In other words, high levels of student identification buffered the effect of the student norm (see Fig. 3). In Model 1c, we also included two-way interactions between norms and identification. Table 5 Table 5 Demographic and psychological predictors of live lecture attendance (binomial regression coefficients). Predictors Model 1a Model 1b Model 1c Odds Ratios CI p Odds Ratios CI p Odds Ratios CI p (Intercept) 1.24 0.66–2.33 0.509 1.34 0.71–2.55 0.364 1.30 0.67–2.52 0.435 Gender [Male] 0.73 0.61–0.89 0.001 0.84 0.69–1.02 0.074 0.76 0.62–0.93 0.008 Gender [Prefer Not To Say] 0.33 0.16–0.62 0.001 0.52 0.25–1.02 0.066 0.17 0.07–0.37 <0.001 Age 0.99 0.96–1.03 0.719 0.99 0.96–1.02 0.553 0.99 0.96–1.03 0.699 Native speaker [Yes] 0.67 0.59–0.77 <0.001 0.69 0.60–0.78 <0.001 0.69 0.60–0.79 <0.001 Psychology degree [Yes] 2.22 1.95–2.52 <0.001 2.11 1.85–2.42 <0.001 2.10 1.83–2.40 <0.001 Carer status [Yes] 1.18 0.81–1.76 0.396 1.30 0.88–1.94 0.188 1.31 0.88–1.96 0.182 Employment status [Yes] 0.96 0.82–1.13 0.652 0.85 0.72–1.01 0.066 0.82 0.69–0.97 0.022 Disability status [Yes] 0.61 0.48–0.77 <0.001 0.74 0.58–0.94 0.014 0.79 0.62–1.01 0.061 Attitude 1.32 1.22–1.43 <0.001 1.36 1.26–1.48 <0.001 Student norm 0.86 0.80–0.93 <0.001 0.83 0.76–0.89 <0.001 Lecturers’ norm 1.03 0.96–1.11 0.410 0.96 0.89–1.04 0.313 Group identification 1.07 1.00–1.14 0.046 1.08 1.00–1.15 0.038 Student norm x Group identification 1.09 1.01–1.17 0.019 Lecturers’ norm x Group identification 1.16 1.08–1.24 <0.001 phic and psychological predictors of live lecture attendance (binomial regression coefficients). 9 9 Computers & Education 207 (2023) 104933 K. Banas et al. Computers & Education 207 (2023) We also found a significant interaction between the lecturer norm and student identification (OR = 1.16, p < .001) in the pred ection: among students reporting high levels of identification, the odds of attending lectures increased with a more po ceived lecturer norm. Among students who reported low levels of identification, the odds of attending lectures decreased w re positive lecturer norm. In this model, we also found a significant effect of student employment, with students in paid employ ing lower odds of attending live lectures (OR = 0.82, p = .022). Fig. 3. Interactive effect of norms and identification on lecture attendance. anas et al. Fig. 3. Interactive effect of norms and identification on lecture attendance. Fig. 3. Interactive effect of norms and identification on lecture attendance. We also found a significant interaction between the lecturer norm and student identification (OR = 1.16, p < .001) in the predicted direction: among students reporting high levels of identification, the odds of attending lectures increased with a more positive perceived lecturer norm. 3.4. Predicting substitution behaviour The demographic model (Model 3a) indicated that native speakers were significantly more likely to use the recordings as sub­ stitutes than students for whom English was a second language (OR = 1.45, p < .001). Students enrolled in a psychology degree were significantly less likely to use recordings as substitutes than students taking psychology as an outside course (OR = 0.44, p < .001), and students with a disability were significantly less likely to use recordings as substitutes (OR = 0.65, p = .01). Other demographic predictors did not have a significant effect (see Table 7 for full model coefficients). i ii We then added psychological predictors to the model (Model 3b). The demographic predictors from Model 3a remained significant. Both attitude and peer norm were significant predictors of substitution behaviour: having a positive attitude towards substitution was associated with higher odds of substituting (OR = 1.47, p < .001), and perceiving a positive peer norm towards substitution was also associated with higher odds of substituting (OR = 1.16, p < .003). i In the third step, we added 2-way interactions to the model (Model 3c, Fig. 5). The main effects from Model 3b remained significant, and we also found two significant two-way interactions: an interaction between peer norms and identification (OR = 0.79, p < .001) and an interaction between lecturer norms and identification (OR = 1.09, p = .042). The interaction between student norm and identification ran contrary to what we predicted: among students who did not strongly identify with the course cohort, a more positive norm towards substitution was associated with higher odds of substitution behaviour. Among students who strongly identified with the course cohort, a more positive norm towards substitution was associated with lower odds of substituting. In other words, identification seemed to act as a buffer against the permissive student norm. i g Looking at student identification and lecturer norm, we found an interaction in the predicted direction: students who highly identified with the course cohort were more likely to follow the norm, engaging in more substitution if they perceived the educator Looking at student identification and lecturer norm, we found an interaction in the predicted dir identified with the course cohort were more likely to follow the norm, engaging in more substitution if Table 6 Demographic and psychological predictors of using lecture recordings as a supplement (binomial regression coefficients). 3.3. Predicting supplementation behaviour The demographic model (Model 2a) indicated that male students were significantly less likely to use the recordings as a supplement to live lectures than female students (OR = 0.69, p = .02). Students enrolled in a psychology degree were significantly more likely to use recordings as a supplement (OR = 1.45, p < .001) and so were students with caring responsibilities (OR = 3.37, p < .001). In contrast, students with a disability were significantly less likely to use recordings as a supplement (OR = 0.36, p < .001). Other de­ mographic predictors did not have a significant effect (see Table 6 for full model coefficients). i ii We then added psychological predictors to the model (Model 2b). The demographic predictors that were significant in Model 2a remained significant, except for gender. Native speaker status in this model became significant, with native speakers more likely to engage in supplementation (OR = 1.33, p = .004). Among the psychological variables, having a positive attitude towards supple­ mentation was associated with higher odds of supplementing (OR = 1.27, p < .001), perceiving a positive peer norm towards sup­ plementation was also associated with higher odds of supplementing (OR = 1.46, p < .001), but perceiving a positive lecturer norm towards supplementation was associated with lower odds of supplementing (OR = 0.81, p < .001). i In the third step, we added 2-way interactions to the model (Model 2c, Fig. 4). The main effects from Model 2b remained significant. However, we also found a significant effect of student identification (OR = 1.12, p = .036) and two significant interactions: an interaction between peer norms and identification (OR = 0.64, p < .001), and an interaction between lecturer norms and identification (OR = 1.18, p < .001). Overall, a more favourable peer norm towards supplementation was associated with higher odds of using lecture recordings as a supplement; however, contrary to what we predicted, this relationship was strongest among students who did not strongly identify with their student cohort. Looking at the interaction between identification and lecturer norm, overall, a more favourable lecturer norm towards supplementation was associated with lower odds of using lecture recordings as a supplement. This relationship was strongest among students who did not strongly identify with their student cohort. Table 5 Among students who reported low levels of identification, the odds of attending lectures decreased with a more positive lecturer norm. In this model, we also found a significant effect of student employment, with students in paid employment having lower odds of attending live lectures (OR = 0.82, p = .022). 10 Computers & Education 207 (2023) 104933 K. Banas et al. 3.4. Predicting substitution behaviour Predictors Model 2a Model 2b Model 2c Odds Ratios CI p Odds Ratios CI p Odds Ratios CI p (Intercept) 0.08 0.03–0.19 <0.001 0.10 0.04–0.26 <0.001 0.10 0.04–0.28 <0.001 Gender [Male] 0.69 0.50–0.94 0.020 0.80 0.57–1.09 0.168 0.77 0.54–1.06 0.119 Gender [PreferNotToSay] 0.41 0.10–1.14 0.140 0.34 0.08–0.97 0.079 0.21 0.05–0.60 0.011 Age 1.02 0.98–1.07 0.326 1.00 0.95–1.05 0.871 1.00 0.95–1.05 0.920 Native speaker [Yes] 1.14 0.95–1.37 0.173 1.33 1.10–1.61 0.004 1.39 1.15–1.70 0.001 Psychology degree [Yes] 1.45 1.19–1.77 <0.001 1.25 1.02–1.55 0.032 1.12 0.91–1.38 0.303 Carer status [Yes] 3.37 2.25–4.98 <0.001 4.66 3.00–7.17 <0.001 6.02 3.81–9.47 <0.001 Employment status [Yes] 0.97 0.76–1.22 0.788 0.94 0.73–1.21 0.648 0.87 0.67–1.12 0.289 Disability status [Yes] 0.36 0.21–0.56 <0.001 0.36 0.21–0.57 <0.001 0.34 0.20–0.54 <0.001 Attitude 1.27 1.13–1.43 <0.001 1.35 1.19–1.53 <0.001 Student norm 1.46 1.29–1.66 <0.001 1.73 1.50–2.02 <0.001 Lecturers’ norm 0.81 0.73–0.90 <0.001 0.79 0.71–0.89 <0.001 Group identification 1.06 0.96–1.17 0.250 1.12 1.01–1.25 0.036 Student norm x Group identification 0.64 0.57–0.72 <0.001 Lecturers’ norm x Group identification 1.18 1.07–1.30 0.001 4. Discussion We found that overall, students had a positive attitude towards all three target behaviours: attending live lectures, using lecture recordings as a substitute and using recordings as a supplement. The peer norm was also largely positive - fellow students were perceived to feel positive all three behaviours. The perceived lecturer norm was more differentiated - very positive towards attending live lectures and using lecture recordings as a supplement, but more mixed towards using lecture recordings as a substitute. Notably, we found that most students accessed lecture recordings at least once in the semester, and that accessing a recording often meant watching that recording in full. This pattern could be interpreted as consistent with substitution. However, an alternative interpre­ tation might be that students found it difficult to search for specific sections due to how the user interface of the recordings was set up or the structure of the lectures themselves, and therefore resorted to watching the entire video. i Including demographic variables in the models allowed us to corroborate and extend previous findings from the literature. We found that students in psychology degrees were more likely than external students to attend live lectures and to use lecture recordings as a supplement, but they were less likely to use recordings as a substitute. This adds nuance to Nordmann et al.’s (2019) finding that there were no significant differences in how much psychology students and external students used lecture recordings - while we also did not find a difference in how much these two groups used lecture recordings, we did find a difference in how they used them, with external students being more likely to skip live lectures and watch the recordings instead. Another notable result pertains to native speakers - we found them to be more likely than non-native speakers to skip live lectures and use the recordings as both supple­ mentation and substitution. This differs from the findings of Nordmann et al. (2019), who found no differences in attendance, but also found that non-native speakers used lecture recordings more. Including psychological theorising and variables novel to the lecture recording literature allowed us to draw new conclusions about the factors that influence how students engage with lectures and lecture recordings. Across all three behaviours, we found a significant effect of student attitude and peer norm. Table 6 11 11 Computers & Education 207 (2023) 104933 12 (caption on next page) K. Banas et al. Computers & Education 207 (2023) 104933 K. Banas et al. p ( ) (caption on next page) 12 12 Computers & Education 207 (2023) 104933 K. Banas et al. Fig. 4. Interactive effect of norms and identification on supplementation behaviour. norm to be permissive. Students who did not highly identify with the course cohort went against the lecturer norm and engaged in more substitution behaviour if they perceived the norm to be less positive. Table 7 Demographic and psychological predictors of using lecture recordings as a substitute (binomial regression coefficients). Predictors Model 3a Model 3b Model 3c Odds Ratios CI p Odds Ratios CI p Odds Ratios CI p (Intercept) 0.14 0.06–0.33 <0.001 0.15 0.07–0.38 <0.001 0.17 0.07–0.42 <0.001 Gender [Male] 1.00 0.79–1.26 0.999 0.93 0.73–1.18 0.559 0.95 0.74–1.20 0.660 Gender [Prefer Not To Say] 1.59 0.81–2.96 0.159 1.49 0.74–2.87 0.242 1.09 0.53–2.13 0.817 Age 1.04 0.99–1.08 0.079 1.03 0.98–1.07 0.212 1.02 0.98–1.07 0.289 Native speaker [Yes] 1.45 1.22–1.72 <0.001 1.55 1.30–1.85 <0.001 1.60 1.34–1.91 <0.001 Psychology degree [Yes] 0.44 0.37–0.51 <0.001 0.45 0.38–0.53 <0.001 0.43 0.36–0.51 <0.001 Carer status [Yes] 1.39 0.88–2.14 0.148 1.60 0.98–2.54 0.055 1.40 0.84–2.27 0.177 Employment status [Yes] 0.84 0.67–1.03 0.099 0.81 0.65–1.01 0.062 0.82 0.66–1.01 0.070 Disability status [Yes] 0.65 0.46–0.90 0.011 0.48 0.34–0.68 <0.001 0.44 0.31–0.63 <0.001 Attitude 1.47 1.35–1.61 <0.001 1.50 1.38–1.64 <0.001 Student norm 1.16 1.05–1.27 0.003 1.16 1.05–1.27 0.002 Lecturers’ norm 0.97 0.89–1.06 0.489 0.96 0.88–1.06 0.434 Group identification 0.94 0.86–1.02 0.132 0.96 0.88–1.04 0.331 Student norm x Group identification 0.79 0.71–0.87 <0.001 Lecturers’ norm x Group identification 1.09 1.00–1.19 0.042 norm to be permissive. Students who did not highly identify with the course cohort went against the lecturer norm and engaged in more substitution behaviour if they perceived the norm to be less positive. norm to be permissive. Students who did not highly identify with the course cohort went against the lecturer norm and engaged in more substitution behaviour if they perceived the norm to be less positive. 4. Discussion Generally, the more positive students’ attitudes were towards the focal behaviour, and the more positive the perceived peer norm, the more likely they were to engage with lectures in a particular way. The alignment between attitude and behaviour fits in with the TPB (Ajzen, 1991) and previous research on the predictors of student attendance at university (Ajzen & Madden, 1986; White et al., 2008). However, we found a somewhat surprising pattern for lecture attendance, as a less positive peer norm was associated with higher odds of attending lectures. One potential explanation for this is that the students who do attend lectures might notice the empty seats and develop a perception that the overall peer norm regarding attendance is quite low. An alternative explanation is that social norm is the weakest of TPB predictors, with a significant proportion of studies showing either no relationship between norms and behaviour, or a relationship in a direction opposite to predicted (Armitage & Conner, 2001; Niemiec et al., 2020). As measurement issues may cause these inconsistencies, and our study included only a single-item measure of norm, future research may look to use a longer measure of social norms to capture any effect more robustly. Finally, as our study was conducted relatively early in the students’ university journey (for most students, this was the beginning of their first semester at the university), the attitudes and norms may not have fully developed yet; future research may look to investigate these relationships among students in later years, where the effects may be more robust. 13 Computers & Education 207 (2023) 104933 K. Banas et al. Computers & Education 207 (2023) 1049 14 (caption on next pag K. Banas et al. Computers & Education 207 (2023) 104 K. Banas et al. (caption on next page) 14 14 Computers & Education 207 (2023) 104933 K. Banas et al. Fig. 5. Interactive effect of norms and identification on substitution behaviour. In each of the predictive models, we found significant interactions between both types of norms and student identification. While the pattern of interactions was complex and not always aligned with our predictions, the overall findings suggest that (1) the level of identification early in the semester matters for how students engage with lectures later, and (2) the level of identification moderates how students respond to the norms present in their environment. 4.2. Limitations Our study focused on a cohort of students enrolled in a single course at a university, and therefore, the generalisability of our findings to other contexts could be questioned. Our response to this issue is twofold. First, we see our descriptive findings as another useful data point in the growing literature on lecture recordings. Second, and perhaps more importantly, our findings on the role of attitudes and social norms, since they are rooted in theory, are likely to be more widely generalisable, not only to other contexts but also to other behaviours. Thus, we would expect the use of lecture recordings at any university to be associated with student attitudes towards supplementation and substitution, and also that the student uptake of any new technology would depend on their attitudes towards that technology and social norms pertaining to it. Related to the above point, the course in question did have an assessment structure that rewarded attendance at small group teaching sessions (16% of the mark) and memorising factual information (45% of the mark). It is possible that this incentive structure encouraged students to prioritise attendance at small group sessions over attendance at lectures, and thus affected how students engaged with lectures and lecture recordings. Any research findings need to be interpreted within their context, and ours may not replicate in environments where the incentive structure is significantly different. Future studies investigating the predictors of lecture recording use in other courses will be able to paint a more complete picture. i While the focus on first-year psychology students in our study was driven by both theoretical (Nordmann et al., 2019) and practical factors, including students from other years and other subjects would have allowed us to compare whether the norms and attitudes towards different uses of lecture recordings are the same both within and across degrees. It is possible that as students progress in their degree programmes, they become more socialised in the university culture and develop a clearer view of their lecturers’ expectations regarding different study behaviours. It is also possible that some programmes are better than others at communicating what they expect of their students and what learning behaviours they recommend. Future studies could test this hypothesis by measuring atti­ tudes and norms towards lecture recording use in different cohorts, comparing both the year of study and the degree subject. 4.1. Strengths Our study contributes to the literature in two key ways. First, it addresses a common methodological gap in both lecture recording (Banerjee, 2021) and educational technology research (Hew et al., 2019) by applying a well-validated theoretical framework to its conception and undertaking. Having a solid grounding in theory gives us insight into the mechanisms behind empirical findings (Mueller & Urbach, 2017) and increases generalisability of the findings to other contexts (Jones & Czerniewicz, 2011). We found some support for the Theory of Planned Behaviour, with attitude and peer norm identified as key predictors of how students used lecture recordings. Second, including socio-cognitive variables sheds light on the students’ attitudes and perceived norms about attending live lectures and using lecture recordings. Student attitudes towards different learning behaviours are often assumed rather than measured explicitly. In our study, asking about attitudes highlighted that the assumptions educators hold are not necessarily correct. For example, it was a novel finding that the attitude towards supplementation was less positive than the attitude towards attending live lectures. As lecture recording researchers, we perceive supplementation to be the best strategy, as it combines the benefits of attending live lectures and the additional revision of difficult content from the recording. However, it is unclear whether the students have a similar view. Finally, responses to the norm questions were somewhat surprising, particularly in the case of lecturer norms about substitution - we expected the students to hold a strong perception that lecturers do not consider substitution to be a useful learning strategy; instead, we found that the responses to this question were distributed relatively evenly, with no positive skew, indicating that there was no clarity among the students about their lecturers’ views on substitution. 4. Discussion Higher identification has been shown to correlate with deep learning practices, such as learning for understanding, looking up suggested readings, and self-testing (Bliuc et al., 2011b). Thus, it is no surprise that higher identification would generally be associated with using lectures in the way recommended by lecturers (e.g., attending live lectures). The premise that the level of identification would moderate the effect of social norms is at the heart of social psychological theorising (Turner et al., 1987), and we did indeed find those moderation effects. The fact that the exact direction of the interaction was not always as expected will require further investigation from future studies in this area. 4.2. Limitations In line with the arguments made by Robson and Kauffman (2022), the effect of lecture attendance on student achievement may have changed since the pre-pandemic times, and may also differ significantly between academic subjects. These relationships would thus best be studied across multiple university courses, or even multiple universities. supplement or substitute, are related to academic outcomes. In line with the arguments made by Robson and Kauffman (2022), the effect of lecture attendance on student achievement may have changed since the pre-pandemic times, and may also differ significantly between academic subjects. These relationships would thus best be studied across multiple university courses, or even multiple universities. 4.3. Conclusion In summary, in line with previous work, we found that the uptake of lecture recordings was high, and that recordings were more likely to be watched by those students who did not attend the live lecture. Our key novel finding was that the “how” of lecture recording use (i.e. whether they were used as a supplement or a substitute to live lectures) depended not only on the students’ attitude towards the way of using the recordings, but also on their peers’ norm around it. Thus, there was robust evidence of lecture recording use being socially determined – students were likely to follow the norm set by their peers. Although quite complex, the pattern of interactions suggested that the level of identification moderates the effect of norms, and that highly identified students tend to engage in the more desirable behaviours (attending live lectures or supplementing with recordings), even if the perceived norms towards those behaviours are negative. These findings suggest potential avenues for further study and educational intervention - both student identification and social norms have previously been studied as targets of intervention and could be targeted in this context to improve effective engagement with lectures. If students feel that they are part of a larger community of learners, and if the peer norms are more positive towards using lecture recordings as a supplement than using them as a substitute, we can expect students to follow those norms and supplement attending live lectures with the recordings, which may in turn lead to more effective learning. Funding We gratefully acknowledge that funding for this PTAS project was provided by the University of Edinburgh Development Trust. Without this ongoing support PTAS would not be able to fund these projects which do so much for the quality of the student learning experience at the University of Edinburgh. We would like to thank all alumni, staff and other donors who support the Development Trust, including through its unrestricted Edinburgh Fund. Author contributions Kasia Banas: Conceptualisation, Methodology, Investigation, Formal analysis, Writing – original draft, Writing – review & editing, Validation. Tobias Thejll – Madsen: Methodology, Investigation, Writing – original draft, Writing – review & editing. Anita Tobar- Henríquez: Methodology, Investigation, Writing – review & editing. Eva Murzyn: Conceptualisation, Methodology, Writing – review & editing. Data availability Data and code are available on the OSF (https://osf.io/tgp8j/). 4.2. Limitations We opted for a single questionnaire at the start of the semester and a one-item measure of student and lecturer norms, which may have been noisy and contributed to the unusual pattern of interactions between norms and identification. In setting up the study, we prioritised participant experience, ensuring that we minimised survey fatigue with a single short questionnaire and clear, succinct questions. This was particularly important given that we asked the same questions about the three target behaviours. We chose to distinguish between the student and lecturer norms, as this distinction was of interest theoretically, but the trade-off was including 15 K. Banas et al. Computers & Education 207 (2023) 104933 only one question about each type of norm. In future studies, we would like to see a more nuanced measurement of normative con­ structs and multiple measurements of attitude and norm over time. This is particularly important because introducing a clearer educator norm may be one promising avenue for intervention in this area. only one question about each type of norm. In future studies, we would like to see a more nuanced measurement of normative con­ structs and multiple measurements of attitude and norm over time. This is particularly important because introducing a clearer educator norm may be one promising avenue for intervention in this area. Finally, it is a limitation that our study did not include academic performance as an outcome. Future research may seek to extend the findings of Williams et al. (2012) and Bos et al. (2016), and investigate how lecture attendance, and using the recordings as a supplement or substitute, are related to academic outcomes. In line with the arguments made by Robson and Kauffman (2022), the i Finally, it is a limitation that our study did not include academic performance as an outcome. 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British Journal of Educational Technology, 47(5), 906–917. https://doi.org/10.1111/bjet.12300 Cameron J E (2004) A three factor model of social identity Self and Identity 3(3) 239 262 https://doi org/10 1080/13576500444000047 , , G , C , gg , J , G , S ( 0 6) p performance: Recorded lectures. British Journal of Educational Technology, 47(5), 906–917. https://doi.org/10.1111/bjet.12300 erformance: Recorded lectures. British Journal of Educational Technology, 47(5), 906–917. https://doi.org/10.1111/bjet.12300 ron, J. E. (2004). A three-factor model of social identity. Self and Identity, 3(3), 239–262. References A meta-analysis. Society & Natural Resources, 33(8), 1024–1040. https://doi.org/10.1080/08941920.2020.1729912 Nkomo, L. M., & Daniel, B. K. (2021). Sentiment analysis of student engagement with lecture recording. TechTrends, 65(2), 213–224 s11528-020-00563-8 Nordmann, E., Calder, C., Bishop, P., Irwin, A., & Comber, D. (2019). 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Lecture capture, attendance, and exam performance in the biosciences: Exploring Rare exceptions to the link between attendance and performance in the era of online teaching. In H. J. Witchel, & M. W. Lee (Eds.), Technologies in biomedical and life sciences education (pp. 343–382). Springer International Publishing. https://doi.org/10.1007/978-3-030-95633-2_12. ., Aikens, M., Eddy, S., & Jordt, H. (2019). Beyond linear regression: A reference for analyzing common data types in discipline b Physical Review Physics Education Research, 15(2), Article 020110. https://doi.org/10.1103/PhysRevPhysEducRes.15.020110 Williams, A., Birch, E., & Hancock, P. (2012). The impact of online lecture recordings on student performance. Australasian Journal of Educational Technology, 28(2). https://doi.org/10.14742/ajet.869 urner, J. C., Hogg, M. A., Oakes, P. J., Reicher, S. D., & Wetherell, M. S. (1987). Rediscovering the social group: A self-categorizat White, K. M., Thomas, I., Johnston, K. L., & Hyde, M. K. (2008). Predicting attendance at peer-assisted study sessions for Statistics: planned behavior. The Journal of Social Psychology, 148(4), 473–492. https://doi.org/10.3200/SOCP.148.4.473-492 Sniehotta, F. F., Presseau, J., & Araújo-Soares, V. (2014). Time to retire the theory of planned behaviour. Health Psychology Review, 8(1), 1–7. https://doi.org/ 10.1080/17437199.2013.869710 Theobald, E. J., Aikens, M., Eddy, S., & Jordt, H. (2019). Beyond linear regression: A reference for analyzing common data types in discipline based education research. Physical Review Physics Education Research, 15(2), Article 020110. https://doi.org/10.1103/PhysRevPhysEducRes.15.020110 Turner, J. C., Hogg, M. A., Oakes, P. J., Reicher, S. D., & Wetherell, M. S. (1987). Rediscovering the social group: A self-categorization theory. Blackwell. White, K. M., Thomas, I., Johnston, K. L., & Hyde, M. K. (2008). Predicting attendance at peer-assisted study sessions for Statistics: Role identity and the theory of planned behavior. The Journal of Social Psychology, 148(4), 473–492. https://doi.org/10.3200/SOCP.148.4.473-492 Williams, A., Birch, E., & Hancock, P. (2012). The impact of online lecture recordings on student performance. Australasian Journal of Educational Technology, 28(2). https://doi.org/10.14742/ajet.869 Witchel, H. J., Guy, R., Torrens, C., Langlands, K., & Doggrell, S. A. (2022). Attendance debate Part 2. Lecture capture, attendance, and exam performance in the biosciences: Exploring Rare exceptions to the link between attendance and performance in the era of online teaching. In H. J. Witchel, & M. W. Lee (Eds.), Technologies in biomedical and life sciences education (pp. 343–382). Springer International Publishing. https://doi.org/10.1007/978-3-030-95633-2_12. Sniehotta, F. F., Presseau, J., & Araújo-Soares, V. (2014). Time to retire the theory of planned behaviour. Health Psychology Review, 8(1), 1–7. https://doi.org/ 10.1080/17437199.2013.869710 Theobald, E. J., Aikens, M., Eddy, S., & Jordt, H. (2019). Beyond linear regression: A reference for analyzing common data types in discipline based education research. Physical Review Physics Education Research, 15(2), Article 020110. https://doi.org/10.1103/PhysRevPhysEducRes.15.020110 Turner, J. C., Hogg, M. A., Oakes, P. J., Reicher, S. D., & Wetherell, M. S. (1987). Rediscovering the social group: A self-categorization theory. Blackwell. White, K. M., Thomas, I., Johnston, K. L., & Hyde, M. K. (2008). Predicting attendance at peer-assisted study sessions for Statistics: Role identity and the theory of planned behavior. The Journal of Social Psychology, 148(4), 473–492. https://doi.org/10.3200/SOCP.148.4.473-492 Williams, A., Birch, E., & Hancock, P. (2012). The impact of online lecture recordings on student performance. Australasian Journal of Educational Technology, 28(2). https://doi.org/10.14742/ajet.869 References Discipline social identification, study norms and students. Educational Psychology, 35(1), 53–72. https://doi.org/10.1080/01443410.2013.822962 17 K. Banas et al. Computers & Education 207 (2023) 104933 Computers & Education 207 (2023) 104933 Williams, A., Birch, E., & Hancock, P. (2012). The impact of online lecture recordings on student performance. Australasian Journal of Educational Technology, 28(2). https://doi.org/10.14742/ajet.869 Witchel, H. J., Guy, R., Torrens, C., Langlands, K., & Doggrell, S. A. (2022). Attendance debate Part 2. Lecture capture, attendance, and exam performance in the biosciences: Exploring Rare exceptions to the link between attendance and performance in the era of online teaching. In H. J. Witchel, & M. W. Lee (Eds.), Technologies in biomedical and life sciences education (pp. 343–382). Springer International Publishing. https://doi.org/10.1007/978-3-030-95633-2_12. 18 18
https://openalex.org/W3014275290
http://www.scielo.br/pdf/ean/v24n3/en_1414-8145-ean-24-3-e20190341.pdf
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Indicadores clínicos para o diagnóstico de enfermagem Síndrome do equilíbrio fisiológico prejudicado para doadores de órgãos
Escola Anna Nery
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ABSTRACT Luciana Nabinger Menna Barreto1  Éder Marques Cabral2,3  Natália Chies3  Miriam de Abreu Almeida3  1 Hospital de Clínicas de Porto Alegre. Porto Alegre, RS, Brasil 2 Hospital São Camilo. Esteio, RS, Brasil 3 Universidade Federal do Rio Grande do Sul. Porto Alegre, RS, Brasil Luciana Nabinger Menna Barreto1  Éder Marques Cabral2,3  Natália Chies3  Miriam de Abreu Almeida3  1 Hospital de Clínicas de Porto Alegre. Porto Alegre, RS, Brasil 2 Hospital São Camilo. Esteio, RS, Brasil 3 Universidade Federal do Rio Grande do Sul. Porto Alegre, RS, Brasil Luciana Nabinger Menna Barreto1  Éder Marques Cabral2,3  Natália Chies3  Miriam de Abreu Almeida3  Objective: To select clinical indicators for the developing nursing diagnosis of Impaired Physiological Balance Syndrome for potential brain-dead organ donors. Method: Expert consensus study, conducted using the Delphi technique, on clinical indicators of a developing nursing diagnosis. The study sample was intentional by invitation and consisted of 37 nurses who met the inclusion criteria. Clinical indicators that reached a consensus equal to or greater than 70% were considered validated. Results: The experts recommended 25 out of 44 evaluated indicators, of which eight belonged to the Endocrine-metabolic changes group, seven to the Hemodynamic and/or cardiovascular changes group, five to the Ventilatory changes group, two to the Nutritional changes group and three to the Coagulation, Inflammatory and/or immune changes group. Conclusion and implications for practice: This new nursing diagnosis can contribute to the development of nursing knowledge in the field of organ donation, thereby contributing to teaching and research. It also has implications for practice, providing diagnostic accuracy and supporting the implementation and evaluation of interventions that affect improving potential donor maintenance. Keywords: Nursing Diagnosis; Tissue and Organ Procurement; Organ Transplantation; Classification. RESUMO Objetivo: Selecionar indicadores clínicos para o diagnóstico de enfermagem em desenvolvimento Síndrome do equilíbrio fisiológico prejudicado para potenciais doadores de órgãos em morte encefálica. Método: Estudo de consenso de especialistas, realizado através da técnica Delphi, dos indicadores clínicos de um diagnóstico de enfermagem em desenvolvimento. A amostra do estudo foi intencional mediante convite e constituída por 37 enfermeiros que atenderam a critérios de inclusão. Os indicadores que obtiveram um consenso igual ou maior que 70% foram considerados validados. Resultados: Especialistas recomendaram 25 dos 44 indicadores avaliados, dos quais oito pertenciam ao grupo Alterações endócrino-metabólicas, sete ao grupo Alterações hemodinâmicas e/ou cardiovasculares, cinco ao grupo Alterações ventilatórias, dois ao grupo Alterações nutricionais e três ao grupo Alterações de coagulação, inflamatórias e/ou imunológicas. Conclusão e implicações para a prática: Esse novo diagnóstico de enfermagem pode colaborar com o desenvolvimento de conhecimento da enfermagem na área de doação de órgãos, contribuindo para o ensino e pesquisa, além de acarretar implicações para a prática, proporcionando acurácia diagnóstica e embasando a implementação e a avaliação de intervenções que impactam na melhora da manutenção do potencial doador. i EAN r/ EAN r/ www.scielo.br/ Esc Anna Nery 2020;24(3):e20190341 Esc Anna Nery 2020;24(3):e20190341 RESEARCH | PESQUISA Esc Anna Nery 2020;24(3):e2019034 DOI: 10.1590/2177-9465-EAN-2019-0341 Corresponding author: Luciana Nabinger Menna Barreto E-mail: luciana.nabinger@gmail.com Submitted on 11/23/2019 Accepted on 01/28/2020. INTRODUCTION 44 possible defining characteristics. The related factor of this ND is brain death.7 The ND definition is: The disproportion between the high demand for transplants and the low supply of organs is a worldwide reality.1,2 It is estimated that the annual number of transplants represents less than 10% of the global need. Thus, the scarcity of organ supply is considered a serious public health problem.1f Impaired state of physiological balance that triggers a set of endocrine-metabolic symptoms, hemodynamic, cardiovascular, ventilatory, nutritional, coagulation, inflammatory and/or immunological symptoms that compromise the health and/or the functions of the organs7:664 Due to various reasons, the percentage of effective organ donors is not higher, as failure to identify and report brain death, family authorization to donate, exclusion criteria, for example, contagious diseases, malignant tumors, among others. Among these reasons there are also failures in maintaining the potential donor. Thus, the proper maintenance of the potential donor through specific care is essential, since any dysfunction is capable to compromise the viability of the organs.2i In order to refine new NDs, it is suggested that experts select their clinical indicators. It is noteworthy that defining characteristics are observable indicators that are grouped as manifestations of an ND (signs or symptoms). In the NANDA-I 2018-2020 edition, two new categories of indicators were introduced: populations at‑risk and associated conditions. “Populations at risk are groups of people who share some characteristic that makes each member susceptible to a specific human response...”. While “the associated conditions correspond to medical diagnoses, injuries, procedures, medical devices or pharmaceutical agents”.6:65 Taking into account the importance of specific care for the proper maintenance of the potential organ donor, the Nursing Process (NP) is a methodology that can help in this context. Nursing, through the use of the NP, guides the health care practices in a systematic way seeking to qualify assistance in the production of care. INTRODUCTION In this context, the use of classifications with standardized language is important for the application of the NP from its stages as they offer a structure to organize nursing diagnoses, interventions and results.3 It is believed that the development and future use of the ND Impaired physiological balance syndrome for potential brain-death organ donators, it will provide diagnostic accuracy favoring implantation of interventions adapted to this specific clinical group with the possibility of achieving the best results; as the proper maintenance of the potential donor contributing to the better viability of the organs offered for transplants. It is noteworthy that to use this ND, the nurse will need to use critical thinking and diagnostic reasoning in order to make an appropriate judgment, since not all brain-dead patients have the proposed ND syndrome. The nursing diagnoses (NDs) that are most used in the maintenance scenario of potential organ donors are: Hyperthermia, Deficient liquid volume risk , Decreased cardiac output risk, Decreased cardiac output, Harmed gas-related exchange, Unstable glycemic risk , Bleeding risk, Infection risk and Diminished intracranial adaptive capacity.4,5 It is observed that most of the NDs that are prevalent for potential organ donors are related to physiological changes. Due to the complexity of brain death, these patients often have all the mentioned NDs. However, it is believed that a very large number of NDs listed on the patient makes it difficult to prioritize care. However, NANDA International (NANDA-I) classification does not have unique ND that approach such condition. Thus, a ND syndrome with the main physiological changes in a single ND could treat them together and with more effective results.i Given the above, the research question of the study is: Which clinical indicators are suitable for developing ND Impaired physiological balance syndrome for potential organ donors in brain death? Thus, this study aimed to select clinical indicators for the developing ND Impaired physiological balance syndrome for potential organ donors in brain death. From this investigation it is expected to qualify the application of the NP in the area of maintenance of the potential organ donor and to promote the production of knowledge in this field. In addition, the synthesis of this knowledge can increase the level of evidence of this ND in the taxonomy. Nursing diagnosis for organ donors Barreto LNM, Cabral EM, Chies N, Almeida MA Barreto LNM, Cabral EM, Chies N, Almeida MA INTRODUCTION ND is defined by NANDA-I as: “a clinical judgment about a human response to health conditions/life processes, or a vulnerability to that response, from an individual, a family, a group or a community”.6:88 ND can be directed to a problem, a health‑promoting state or a potential risk. The use of syndromes can be found in these categories. NANDA-I defines a ND syndrome as: “A clinical judgment related to a certain group of nursing diagnoses that occur together, being better treated through similar interventions”.6:82 RESUMEN Objetivo: Seleccionar indicadores clínicos para el diagnóstico de enfermería en desarrollo Síndrome del equilibrio fisiológico deteriorado en potenciales donantes de órganos con muerte encefálica. Método: Estudio de consenso de expertos, realizado por medio de la técnica Delphi, sobre los indicadores clínicos de un diagnóstico de enfermería en desarrollo. La muestra del estudio fue intencional por invitación y consistió en 37 enfermeros que cumplieron con los criterios de inclusión. Los indicadores clínicos que alcanzaron un consenso igual o superior al 70% se consideraron validados. Resultados: Los expertos recomendaron 25 de los 44 indicadores evaluados, de los cuales, ocho pertenecían al grupo Alteraciones endocrino-metabólicas, siete al grupo Alteraciones hemodinámicas y/o cardiovasculares, cinco al grupo Alteraciones respiratorias, dos al grupo Alteraciones nutricionales y tres al grupo Alteraciones de la coagulación, inflamatorias y/o inmunológicas. Conclusión y implicaciones para la práctica: Este nuevo diagnóstico de enfermería puede colaborar con el desarrollo del conocimiento de la enfermería en el campo de donación de órganos, y así contribuir a la enseñanza y a la investigación, además de tener implicaciones para la práctica, al proporcionar exactitud diagnóstica y apoyar la implementación y evaluación de intervenciones que impactan en la mejora del de mantenimiento del posible donante de órganos. Palabras clave: Diagnóstico de Enfermería; Obtención de Tejidos y Órganos; Trasplante de Órganos; Clasificación. Escola Anna Nery 24(3)2020 1 1 Nursing diagnosis for organ donors RESULTS The online instrument contains two sections. The first is in a form for recording the sample characterization data. The second contains the clinical indicators, their conceptual definitions and a space to indicate “recommend” or “do not recommend” for each of them, in addition to a space for observations below each variable. Before starting up the first round of opinions, a pre-test of the instrument was carried out with five judges who were not part of the sample. The pre-test had the purpose of verifying weaknesses and gaps in the instrument to correct them before definitive data collection. Thirty-seven nurses (n=37) considered specialists for the study evaluated the ND diagnostic content Impaired physiological balance syndrome for potential organ donors. They recommended 25 of the 44 clinical evaluated indicators. A Table 1 displays sample characterization. Table 2 shows the clinical indicators recommended by the specialists for the ND at issue, after two rounds of sending the instrument. A total of 25 indicators were recommended by more than 70% of experts, therefore, considered validated in terms of content.i The variables used in the data collection instrument were 44  clinical indicators identified in an integrative literature review for the developing ND Impaired Physiological Balance Syndrome for potential organ donors in brain death. In this integrative review, the indicators were divided into five groups: Endocrine-metabolic changes, Hemodynamic and/or cardiovascular changes, Ventilatory changes, Nutritional changes and Coagulation changes, inflammatory and/or immunological.7 t is noteworthy that conceptual definitions were constructed for each indicator based on scientific literature in order to ensure the understanding of each variable and enable an adequate judgment of each indicator by the panel of experts. Some experts modified their recommendations in the second round of shipment of the instrument. It is observed that metabolic acidosis and respiratory alkalosis obtained 25 recommendations (67.6%) in the first round. However, in the second round, no specialist recommended them, with the justification that they are included in the acid-base imbalance. Likewise, altered MAP in the first round obtained 31 (83.8%) recommendations and in the second none, justifying that the altered blood pressure already includes the altered MAP. Thus, altered blood pressure in the first round was recommended by 34 (91.9%) of the specialists and in the second round it obtained 100% of recommendation. Decreased ejection fraction obtained 21 recommendations (56.8%) in the first round and none in the second. METHOD This study consisted on selecting by consensus of specialists for the clinical indicators of the ND Impaired physiological balance syndrome through the Delphi technique. The consensus study is proposed as a research method used to develop standards of professional practice improving the professionalism of the area. This method allows nurses to review knowledge content In view of the above, the ND referred to as Impaired physiological balance syndrome is being developed. In the first part of its development, an integrative literature review identified Escola Anna Nery 24(3)2020 2 Nursing diagnosis for organ donors Barreto LNM, Cabral EM, Chies N, Almeida MA as needed to reach consensus. In the first round of opinions, the instrument was sent via e-mail. From its return, the answers were counted and analyzed. In the second round of opinions, the instrument was revised and sent again to the participants with information on the results achieved in the first round. In this way, the experts were invited to carry out a new evaluation of their judgments in view of the statistical forecast of each response of the group, being possible to maintain the opinion or modify it. Thus, the participants filled out the data collection instrument for the selection of clinical indicators applicable to the developing ND Impaired physiological balance syndrome for potential organ donors in brain death. At the end of the collection, a final report with the results from the rounds was sent to the specialists. in their area of expertise and to identify or link relevant terms of nursing taxonomies for specific populations and clinical contexts. Thus, nurses seek to reach consensus or collective opinion on a phenomenon of practice through a research method in which the power to produce knowledge is in the contributions of the participants.8,9 Likewise, the Delphi technique can be used as a method of data collection to reach consensus on a subject through expert opinions. The objective of this technique is to obtain maximum consensus from a group of experts on a given topic.10 The study sample was intentional by invitation and consisted of 37 nurses who met the inclusion criteria: Minimum clinical experience of two years working with potential organ donors in brain death and getting to know the NP and NANDA-I. Exclusion criteria have not en established. METHOD The sample calculation used a 95% confidence level, 70% expert proportion for the consensus and an acceptable sample error of 15%.11 This calculation stipulated a minimum of 36 specialists. Data analysis was descriptive. The clinical indicators of ND that obtained a consensus equal to or greater than 70% among nurses were considered validated. This study was registered on the Brazil platform (CAEE 72793817.6.0000.5327) and approved by the Research Committee of the School of Nursing at the Federal University of Rio Grande do Sul under number 33688 and by the Health Ethics Committee of Hospital de Clínicas de Porto Alegre under the number 170500. Data collection was carried out between April and May 2018 using the Delphi technique. The experts were invited to take part in the study via email. Nurses interested in participating were directed to a virtual environment in Google Forms, where they had access to the Free and Informed Consent Form (FICF). The FICF’s agreement directed to the data collection instrument.i RESULTS The experts pointed out that decreased cardiac output is broader and includes the change in ejection fraction. Data collection took place through two rounds of sending the instrument to the panel of experts, which were carried out Escola Anna Nery 24(3)2020 Escola Anna Nery 24(3)2020 3 Nursing diagnosis for organ donors Barreto LNM, Cabral EM, Chies N, Almeida MA Table 1. Characterizing the sample of specialist nurses (n=37). Variables n (%) Degree* Specialization 27 (73) Master’s degree 22 (59) Doctor’s degree 7 (19) Degree area* Intensive therapy 24 (65) Organ donation and transplants 8 (22) Nursing process 6 (16) Others 9 (24) Time of training 2 - 5 years 2 (5) 6 - 10 years 14 (38) 11 - 15 years 14 (38) 16 - 20 years 6 (16) Over 21 years old 1 (3) Professional Experience Critical care - maintenance of the potential donor 28 (76) CIHDOTT 14 (38) Team for removing multiple organs 11 (30) Clinical OPO 4 (11) Transplant Center 2 (5) Time of experience in donor maintenance, organ donation and/or transplantation 2 - 5 years 10 (27) 6 - 10 years 15 (41) 11 - 15 years 10 (27) 16 - 20 years 2 (5) Use of NANDA-I Clinical Experience 29 (78) Teaching 14 (38) Conducting researches 12 (32) State RS 24 (65) PR 4 (11) RJ 3 (8) SC 2 (5) SP 2 (5) MA 1 (3) ES 1 (3) Source: Author (2018). * Some specialists have more than one degree. Table 1. Characterizing the sample of specialist nurses (n=37). n (%) Impaired gas exchange (00030) The nine clinical indicators that have a numerical code next to them are NDs already existing in NANDA-I. The definition of syndrome stands out for this classification: “clinical judgment regarding a particular cluster of NDs that occur together, being better treated through similar interventions”.6:82 Professional Experience p ( ) Time of experience in donor maintenance, organ donation and/or transplantation 2 - 5 years 10 (27) 6 - 10 years 15 (41) 11 - 15 years 10 (27) 16 - 20 years 2 (5) Use of NANDA-I Clinical Experience 29 (78) Teaching 14 (38) Conducting researches 12 (32) State RS 24 (65) PR 4 (11) RJ 3 (8) SC 2 (5) SP 2 (5) MA 1 (3) ES 1 (3) Source: Author (2018) Nursing diagnosis for organ donors Barreto LNM, Cabral EM, Chies N, Almeida MA Nursing diagnosis for organ donors Table 2. Indicators of the ND Impaired physiological balance syndrome (n=37). Clinical indicators 1st round 2nd round Endocrine-metabolic changes f (%) f (%) Validated Diabetes insipidus 32 (86.5) 32 (86.5) Altered diuresis 31 (83.8) 31 (83.8) Electrolyte imbalance 31 (83.8) 31(83.8) Hypothermia (00006) 30 (81.1) 30 (81.1) Hyperthermia (00007) 29 (78.4) 29 (78.4) Acid Base Imbalance 28 (75.7) 29 (78.4) Hyperglycemia 27 (73) 28 (75.7) Dysfunction of endocrine regulation 27 (73) 26 (70.3) Invalidated Keratinize serum increased 22 (59.5) 22 (59.5) Alteration in liver function 22 (59.5) 22 (59.5) Hyperosmolarity 21 (56.8) 21 (56.8) High blood lactate 19 (51.4) 19 (51.4) Metabolic Acidosis 25 (67.6) 0 (0) Respiratory alkalosis 25 (67.6) 0 (0) Hemodynamic and/or cardiovascular changes Validated Altered blood pressure 34 (91.9) 37 (100) Decreased cardiac output (00029) 28 (86.5) 32 (86.5) Ineffective peripheral tissue perfusion (00204) 28 (75.7) 28 (75.7) Altered cardiac rate 27 (73) 27 (73) Deficient liquid volume (00027) 27 (73) 27 (73) Excessive liquid volume (00026) 26 (70.3) 26 (70.3) Abnormal cardiac rhythm 26 (70.3) 26 (70.3) Invalidated Altered pulse pressure variation 21 (56.8) 21 (56.8) Altered cardiac index 19 (51.4) 19 (51.4) Increased systemic and/or peripheral vascular resistance 17 (45.9) 17 (45.9) Systemic or pulmonary vascular resistance 50% of baseline 16 (43.2) 16 (43.2) Cardiac dysfunction 14 (37.8) 11 (29.7) Altered central venous pressure (CVP) 11 (29.7) 11 (29.7) Left ventricular stroke index <15 × g · m / m2 10 (27) 10 (27) Altered mean arterial pressure (MAP) 31 (83.8) 0 (0) Ejection fraction decreased 21 (56.8) 0 (0) Ventilatory changes Validated Impaired spontaneous ventilation (00033) 29 (78.4) 32 (86.5) Source: Author (2018). Table 2. Indicators of the ND Impaired physiological balance syndrome (n=37). Professional Experience Clinical indicators Endocrine-metabolic changes Escola Anna Nery 24(3)2020 5 Nursing diagnosis for organ donors Barreto LNM, Cabral EM, Chies N, Almeida MA Nursing diagnosis for organ donors Barreto LNM, Cabral EM, Chies N, Almeida MA Nursing diagnosis for organ donors Table 2. Continued... Table 2. Continued... Clinical indicators 1st round 2nd round Impaired gas exchange (00030) 27 (73) 27 (73) Arterial oxygen saturation (AoS2) diminished 26 (70.3) 26 (70.3) Arterial oxygen pressure (AoP2) low 26 (70.3) 26 (70.3) Impaired oxygenation (AoP2/FiO2 < 300) 26 (70.3) 26 (70.3) Invalidated Change in breathing pattern 28 (75.7) 25 (67.6) Pulmonary congestion 18 (48.6) 18 (48.6) Oxygen extraction rate (TeO2) reduced 13 (35.1) 13 (35.1) Pulmonary capillary wedge pressure >12 mmHg 7 (18.9) 7 (18.9) Nutritional changes Validated unbalanced nutrition; less than bodily needs (00002) 27 (73) 27 (73) Anemia 26 (70.3) 26 (70.3) Coagulation, inflammatory and/or immunological changes Validated Coagulation change 23 (70.3) 23 (70.3) Inflammatory changes 23 (70.3) 23 (70.3) Immunological changes 23 (70.3) 23 (70.3) Source: Author (2018).i DISCUSSION CVP monitoring helps to control excessive fluid infusion that can cause pulmonary edema.13,15,17 Thus, the maintenance of the potential organ donor includes full knowledge and immediate handling of the main complications resulting from brain death, in addition to the assistance of the multidisciplinary team. Nursing plays an important role in this scenario, since adequate assistance is essential to make the donation effective. Finally, it is considered that the use of ND is important, since through it the nurse prescribes interventions that will make a difference in patient maintenance and, consequently, in organ viability.4i In the group Ventilatory changes, clinical indicators were recommended Impaired oxygenation, Arterial oxygen pressure (AoP2) altered, Arterial oxygen saturation (AoS2) diminished, Impaired gas exchange and Impaired spontaneous ventilation. The brain death triggers a series of pathophysiological changes (arterial hypotension, hypothermia, diabetes insipidus, hyperglycemia, myocardial injury, coagulopathy), of which the literature highlights with a 62% incidence of moderate to severe respiratory dysfunction in potential organ donors.16 Thus, brain death can cause a series of ventilatory changes, such as: Neurogenic pulmonary edema, increased risk for aspiration, ventilator-associated lung injury, among others. Although all solid organs are affected, the lungs are particularly sensitive to these events. Thus, for organ viability, especially the lung, it is important to control AoP 2, AoS2 and oxygenation (AoP2/FiO2), in addition of gas-state exchange.12,13,19 Taxonomies Nursing Outcomes Classification (NOC) and Nursing Interventions Classification (NIC) have results and interventions that may be applied to this population with the ND proposed in this study. It is noteworthy that making connections between the ND and NOC results can suggest aspects of the problem or the patient’s condition that are expected to be improved or resolved through an intervention, while NIC interventions suggest the relationship between the problem and nursing actions that can solve or alleviate this problem. In this way, in practice, nurses can, with the help of the NOC, control the effectiveness of NIC interventions.24,25 The 25 clinical indicators recommended in this study require greater direction of nursing interventions in maintaining the potential organ donor in patients with the proposed ND syndrome. However, this does not mean that non-validated indicators will receive less attention. The importance of multidisciplinary and interdisciplinary work is emphasized to focus on these indicators that are not recommended and are less sensitive to nursing interventions, but also important for the better viability of the organs offered for transplants. DISCUSSION Escola Anna Nery 24(3)2020 Escola Anna Nery 24(3)2020 6 Nursing diagnosis for organ donors Barreto LNM, Cabral EM, Chies N, Almeida MA and also contribute to the viability of the organs offered for transplantation.12,13 changes, inflammatory changes and immunological changes. Brain injury releases the plasminogen activator which can have consequences on coagulation (thrombocytopenia, thromboplastin time and activated prothrombin) and can lead to hemorrhagic complications.15 In addition, a systemic and local inflammatory response also occurs as a result of brain death. This response consists in the activation of complement and endothelium, in the release of cytokines and chemokines and in the flow of leukocytes to the organs, which can cause tissue damage and ischemia lesions in the organs, collaborating in graft rejection.23 Brain damage can also cause immune changes. Leukocytes may be elevated as a result of systemic inflammatory state or nosocomial infection.15 Gluconeogenesis increases with the “sympathetic storm” caused by brain death due to greater insulin resistance in peripheral tissues and reduced pancreas insulin secretion, causing hyperglycemia. The brain death also causes damage to the pituitary gland which can lead to hormonal deficiencies and also affects the central thermoregulation, and may result both in hypothermia as well as hyperthermia.13-15,17,18 In the group hemodynamic and/or cardiovascular changes, clinical indicators were recommended: Decreased cardiac output, altered cardiac rate, ineffective peripheral tissue perfusion, altered blood pressure, altered cardiac rhythm, deficient fluid volume and excessive fluid volume. The literature highlights that brain death (a related factor of this ED) causes deleterious effects on potential donors, causing several physiological changes. These changes, when not properly treated, are the main causes of the potential donor not being effective, in addition to impacting the quality of the transplanted organs.13,16 The “nice storm” in the donors with brain death releases catecholamines that produces vasoconstriction and initially leads to hypertension and tachycardia and can lead to cardiac arrhythmia. After the same, the result is loss of sympathetic tone, with profound vasodilation which induces hypotension, bradycardia and hypovolemia, which are the main problems during the maintenance of the potential donor. Hypotension should be avoided as it results in hypoperfusion, which contributes to the rapid deterioration of organ function. As a result of high intracranial pressure, a cardiovascular instability occurs, which can lead to myocardial injury. CVP and MAP should be monitored. DISCUSSION This study enabled the selection by consensus by 37 specialists of 25 clinical indicators for developing ND Impaired physiological balance syndrome for potential organ donors in brain death. These indicators may in practice assist nurses in the diagnostic inference process, allowing greater security in the choice of diagnosis. In addition, the results and interventions listed will present greater chances of targeting and effectiveness. The originality of this pioneering study in ND development in the theme of maintaining the potential organ donor stands out. In the endocrine-metabolic disorders group, clinical indicators were recommended: Acid-base imbalance, electrolyte imbalance, diabetes insipidus, altered diuresis, endocrine regulatory dysfunction, hyperglycemia, hypertension and hypothermia. Acid-base disorders impair organ function and can be serious in potential organ donors. Metabolic acidosis leads to hypotension and vasodilation. It is caused by tissue hypoperfusion and causes worsening cardiac contraction and reduced endothelial response to catecholamines. While respiratory alkalosis is commonly due to hyperventilation and diuretic treatment administered in an attempt to decrease intracranial pressure.12-14 This study was relevant since, even if the patient is brain dead, there is a need for targeted care to maintain the potential organ donor in order to achieve the best viability of the organs offered for transplantation. In this sense, studies of this nature contribute to the improvement of nursing classifications taking into account the studied population. The literature shows that among the physiological changes resulting from brain death in the potential organ donor, diabetes insipidus occurs in 46 to 78% of the cases.15,16 Diabetes insipidus without intervention causes polyuria and leads to hypernatremia and hypovolemic shock.15 Studies indicate that, among metabolic disorders, hypernatremia is frequent, being a failure to maintain potential organ donors and subsidizing graft failure . Electrolyte imbalance is usually the result of large urinary losses. It is noteworthy that ions have important roles in cell physiology In relation to the results of the study, the sample of specialists is considered qualified since all nurses are postgraduates. Allied to this, the time of formation was considered expressive (range 6-10 years and 11-16 years obtained 38% predominance, each). It is noteworthy that all specialists reported professional experience in the area of donor maintenance, donation and/or organ transplants. In addition, 78% of the sample reported clinical experience with the use of NANDA-I, in addition to teaching and research experience. REFERENCES 1. Citerio G, Cypel M, Dobb GJ, Dominguez-Gil B, Frontera JA, Greer DM et al. Organ donation in adults: a critical care perspective. Intensive Care Med. 2016;42(3):305-15. http://dx.doi.org/10.1007/s00134-015- 4191-5. PMid:26754754. 2. Siqueira MM, Araújo CC, Roza BA, Schirmer J. Efficiency indicators to assess the organ donation and transplantation process: systematic review of the literature. Rev Panam Salud Publica. 2016;40(2):90-7. PMid:27982363. It is suggested to replicate this research with other related factors such as circulatory death (provided for in the legislation of some countries), for example, as well as with other possible patient populations that this new ND proposal can be applied to. However, using just brain death as a related factor and only in the context of maintaining the potential organ donor was considered a limitation of this study. 3. Adamy EK, Metelski FK, Argenta C, Silva OM, Zocche DAA. Reflection on the interface between patient safety and the nursing process. Rev Enferm Health Care. 2018;7(1):272-8. 4. Bianchia M, Accinelli LG, Silva MA, Menegócio AM. Nursing identification diagnosis to the potential organ donor patient. Uniciências. 2015;19(2):174- 80. 5. Marcelino CAG, Silva ABV, Kobayashi RM, Ayoud AC, Conceição AP. Diagnósticos e intervenções de enfermagem aplicáveis ao potencial doador de órgãos e tecidos. Anais do 10° Simpósio Nacional dos Diagnósticos de Enfermagem; 2010 ago. 4-7; Brasília, Brasil. Brasília: Associação Brasileira de Enfermagem; 2010. p. 343-9.ii It is noteworthy that the results of this investigation portray an advance in nursing care, teaching and research practice. In healthcare practice, the use of this new ND will provide diagnostic accuracy supporting the implementation and evaluation of interventions that impact the improvement of the maintenance of the potential organ donor. In teaching, the use of elements from this ND can contribute to the construction of knowledge about the theme, especially with regard to the physiological changes caused by brain death and the care provided to maintain the potential donor. In addition, this ND can be explored in future research by applying the ND in a real environment. Also, the development of this ND can collaborate with the qualification of the NANDA-I taxonomy, and contribute to the construction of nursing knowledge in the area of maintenance of the potential organ donor. 6. Herdman TH, Kamitsuru S. Nursing diagnoses: definitions & classification 2018-2020. New York: Thieme; 2018. 7. Menna Barreto LN, Chies N, Cabral EM, Nomura ATG, Almeida MA. AUTHORS’ CONTRIBUTIONS quality of care due to its complexity.7 In this way, the ND Impaired physiological balance syndrome may assist the nurse in choosing interventions that enable the achievement of the best results. Study conception and design. Data collection, data analysis and interpretation. Findings discussion. Content writing and/or critical review. Approval of the final version of the article. Responsibility for all aspects of the content and integrity of the published article: Luciana Nabinger Menna Barreto, Miriam de Abreu Almeida. Data analysis and interpretation. Findings discussion. Content writing and/or critical review. Approval of the final version of the article. Responsibility for all aspects of the content and integrity of the published article: Éder Marques Cabral, Natália Chies. REFERENCES Impaired physiological equilibrium syndrome in potential organ donors: identification of defining characteristics. Enfermería Global. 2019;18(3):643-93. http://dx.doi.org/10.6018/eglobal.18.3.358841. 8. Carlson J. Consensus validation process: a standardized research method to identify and link the relevant NANDA, NIC, and NOC terms for local populations. Int J Nurs Terminol Classif. 2006;17(1):23-4. 9. Silva MB, Barreto LNM, Panato BP, Engelman B, Figueiredo MS, Rodríguez-Acelas AL et al. Clinical indicators for evaluation of outcomes of Impaired tissue integrity in orthopedic patients: consensus study. Int J Nurs Knowl. 2019;30(2):81-6. http://dx.doi.org/10.1111/2047- 3095.12204. PMid:29489065. Thus, nursing has investigated best clinical practices and innovative tools to support the care plan. Using this ND may come to provide better care systematization, assisting in the proper maintenance of the potential donor, and consequently contribute to the better viability of the organs offered for transplants, reflecting on the number of used organs, in addition to improving post-transplant survival. 10. Reguant-Álvarez M, Torrado-Fonseca M. El método Delphi. Revista d’Innovació i Recerca en Educació. 2016;9(1):87-102. 11. Santana RF, Passarelles DMA, Rembold SM, Souza PA, Lopes MVO, Melo UG. Diagnóstico de enfermagem risco de recuperação cirúrgica retardada: validação de conteúdo. Rev. Eletr. Nur. 2018;20:v20a34. 12. Maciel CB, Greer DM. ICU management of the potential organ donor: state of the art. Curr Neurol Neurosci Rep. 2016;16(9):86. http://dx.doi. org/10.1007/s11910-016-0682-1. PMid:27498101. 13. Kumar L. Brain death and care of the organ donor. J Anaesthesiol Clin Pharmacol. 2016;32(2):146-52. http://dx.doi.org/10.4103/0970- 9185.168266. PMid:27275040. DISCUSSION Therefore, nurses can intervene with requests for consultations, referrals, as well as request evaluations and support from other professionals when deemed necessary. In the group Nutritional changes, the clinical indicators Anemia and Imbalanced Nutrition were recommended: Less than the bodily needs. The literature points out that coagulopathy, fluid administration and bleeding can cause anemia in the potential organ donor.20 In addition, guidelines for maintaining the potential donor recommend the use of nutritional support21 to provide exogenous fuel in order to maintain lean mass and immune function, as well as avoid metabolic complications. Enteric nutrition should be continued, as it increases the supply of glycogen and has the potential to optimize graft function.12,22l Thus, it is believed that not using NP and standardized languages in care practice, as well as the lack of tools and instruments to direct the maintenance of the potential organ donor, makes clinical reasoning difficult, being able to affect the In the group Coagulation changes, inflammatory and/or immunological, clinical indicators were recommended Coagulation Escola Anna Nery 24(3)2020 7 7 Nursing diagnosis for organ donors Barreto LNM, Cabral EM, Chies N, Almeida MA Nursing diagnosis for organ donors Barreto LNM, Cabral EM, Chies N, Almeida MA ASSOCIATED EDITOR Cândida Caniçali Primo CONCLUSION AND IMPLICATIONS FOR PRACTICE This study made it possible to select, by consensus of experts, the main clinical indicators for developing the ND Impaired physiological balance syndrome. In order to increase the ND evidence level, it is suggested to carry out concept analysis and clinical validation in order to define which indicators give more support and precision to the ND. In addition, given the changes published in NANDA-I 2018-2020, it will be necessary to review which indicators will be conceptualized as defining characteristics or associated conditions or populations at risk. The ND must also be structured within the seven axes of NANDA-I, including domain and class. It is also relevant to develop studies with links between the proposed ND and the NOC and NIC taxonomies. It is noteworthy that the ND Impaired physiological balance syndrome will be forwarded to NANDA-I for publication in order to collaborate with the taxonomy qualification. a Extracted from the doctoral thesis “Development and Validation of Impaired Physiological Balance Syndrome for Patients with Brain Death and Potential Organ Donors Nursing Diagnosis”, sustained by Luciana Nabinger Menna Barreto, in the Postgraduate Program in Nursing, Nursing School of Federal University of Rio Grande do Sul, under the guidance of Prof Dr. Miriam de Abreu Almeida, in 2018. Nursing diagnosis for organ donors Barreto LNM, Cabral EM, Chies N, Almeida MA Nursing diagnosis for organ donors Barreto LNM, Cabral EM, Chies N, Almeida MA FINANCIAL SUPPORT Grant of a scientific initiation scholarship from the National Council for Scientific and Technological Development (CNPQ) to Natália Chies. 14. Westphal GA, Caldeira Fo M, Vieira KD, Zaclikevis VR, Bartz MC, Wanzuita R et al. Diretrizes para manutenção de múltiplos órgãos no potencial doador adulto falecido: parte II. Ventilação mecânica, Escola Anna Nery 24(3)2020 8 Nursing diagnosis for organ donors Nursing diagnosis for organ donors Barreto LNM, Cabral EM, Chies N, Almeida MA Barreto LNM, Cabral EM, Chies N, Almeida MA position statement. Indian J Crit Care Med. 2017;21(5):303-16. http:// dx.doi.org/10.4103/ijccm.IJCCM_160_17. PMid:28584434. position statement. Indian J Crit Care Med. 2017;21(5):303-16. http:// dx.doi.org/10.4103/ijccm.IJCCM_160_17. PMid:28584434. controle endócrino metabólico e aspectos hematológicos e infecciosos. Rev Bras Ter Intensiva. 2011;23(3):269-82. http://dx.doi.org/10.1590/ S0103-507X2011000300004. PMid:23949398. 21 21. Meyfroidt G, Gunst J, Martin-Loeches I, Smith M, Robba C, Taccone FS et al. Management of the brain-dead donor in the ICU: general and specific therapy to improve transplantable organ quality. Intensive Care Med. 2019;45(3):343-53. http://dx.doi.org/10.1007/s00134-019-05551-y. PMid:30741327. 15. Wong J, Tan HL, Goh JPS. Management of the brain dead organ donor. Trends in Anaesthesia and Critical Care. 2017;13(1):6-12. http://dx.doi. org/10.1016/j.tacc.2016.11.004. 16. Essien EO, Fioretti K, Scalea TM, Stein DM. Physiologic features of brain death. Am Surg. 2017;83(8):850-4. PMid:28822390. 22. Carrott P, Cherry-Bukowiec JR, Jones CM, Miller KR, Kiraly L. Nutrition therapy in the organ donor: theoretical benefits and barriers to implementation. Curr Nutr Rep. 2016;5(3):199-203. http://dx.doi. org/10.1007/s13668-016-0170-x. 17. Zambergs K, Vyas H. Management of potential organ donor. Paediatr Child Health. 2015;25(5):234-8. http://dx.doi.org/10.1016/j.paed.2015.01.004. 18. Patel MS, Abt PL. Current practices in deceased organ donor management. Curr Opin Organ Transplant. 2019;24(3):343-50. http:// dx.doi.org/10.1097/MOT.0000000000000638. PMid:31090647. 23. Poppelaars F, Seelen MA. Complement-mediated inflammation and injury in brain dead organ donors. Mol Immunol. 2017;84:77-83. http:// dx.doi.org/10.1016/j.molimm.2016.11.004. PMid:27989433.i 19. Courtwright A, Cantu E. Evaluation and management of the potential lung donor. Clin Chest Med. 2017;38(4):751-9. http://dx.doi.org/10.1016/j. ccm.2017.07.007. PMid:29128023. 24. Moorhead S, Johnson M, Maas ML, Swanson E. Classificação dos resultados de enfermagem (NOC). Rio de Janeiro: Elsevier; 2016.i 25. Bulechek GM, Butcher HK, Dochterman JM, Wagner CM. Classificação das intervenções em enfermagem (NIC). Rio de Janeiro: Elsevier; 2016. 20. Govil D, Patil V, Todi S, Gurav S, Pandit RA, Zirpe KG et al. Management of potential organ donor: Indian Society of Critical Care Medicine: Escola Anna Nery 24(3)2020 9
https://openalex.org/W2005164911
https://www.research.unipd.it/bitstream/11577/2572788/1/Int%20J%20Mol%20Sci%202013%20Mar%201.pdf
English
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Active Silver Nanoparticles for Wound Healing
International journal of molecular sciences
2,013
cc-by
12,544
Int. J. Mol. Sci. 2013, 14, 4817-4840; doi:10.3390/ijms14034817 Int. J. Mol. Sci. 2013, 14, 4817-4840; doi:10.3390/ijms14034817 International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms OPEN ACCESS 1. Introduction For centuries, silver compounds and ions have been extensively used for both hygienic and healing purposes, due to their strong bactericidal effects, as well as a broad spectrum antimicrobial activity [1,2]. Taking advantage of its bactericidal properties, various silver-containing preparations have been used for the treatment of chronic wounds. In the 17th and 18th centuries, silver nitrate was already used for ulcer treatment, and in 1960, it was introduced for the management of burns. After a decrease in the use of silver salts consequent to the introduction of antibiotics in 1940, in more recent years, there has been a renewed interest in silver, due to increased resistance of bacteria to antibiotics and improvements in polymer technology. This has resulted in a large number of silver-containing dressings being available on the market. Silver is applied to burns, either in the form of impregnated bandages or as a cream containing silver sulfadiazine as the active agent, a product that is still considered the benchmark silver product [3]. At the end of the 1990s, several Ag-containing dressings from different manufacturers appeared in commerce. Silver-based dressings are now available as a variety of fibers or polymeric scaffolds impregnated or coated with a Ag salt or metallic Ag in nanoparticulate form. They all exhibit fast and broad spectrum antibacterial activity against both Gram-positive and -negative bacteria [4,5]. In recent years, the mechanism of action of silver has been investigated: it seems that silver shows a multilevel antibacterial effect, due to blockage of respiratory enzyme pathways, as well as alteration of microbial DNA and the cell wall [6]. Silver has been demonstrated to be effective also against multidrug-resistant organisms [7,8], whilst maintaining a low systemic toxicity [9]. Clinically, several studies have confirmed their safety for patients [10,11] and concerns about their cytotoxicity on fibroblasts and keratinocytes have not been confirmed [12–14]. Nanoparticles (NPs) are defined as particles having one or more dimensions in the order of 100 nm or less. Silver NPs (Ag NPs) have been shown to possess unusual physical, chemical and biological properties [15–17]. The effectiveness of Ag NP-containing dressings has been widely tested in vitro, and much research work has been published recently demonstrating that these dressings have a fast and broad spectrum antibacterial activity against both Gram-positive and -negative bacteria [18,19]. However, up to now, the number of in vivo studies has been limited [20,21]. Active Silver Nanoparticles for Wound Healing 2013, 14 4818 observations and experimental data collected here are consistent with previously published articles and support the safety of Ag NP-based dressing in wound treatment. observations and experimental data collected here are consistent with previously published articles and support the safety of Ag NP-based dressing in wound treatment. Keywords: silver; nanoparticles; ICP-MS; SEM; TEM; Acticoat™ Flex 3; in vivo; in vitro; cytotoxicity; mitochondrial toxicity Keywords: silver; nanoparticles; ICP-MS; SEM; TEM; Acticoat™ Flex 3; in vivo; in vitro; cytotoxicity; mitochondrial toxicity Active Silver Nanoparticles for Wound Healing Chiara Rigo 1, Letizia Ferroni 2, Ilaria Tocco 3, Marco Roman 4, Ivan Munivrana 3, Chiara Gardin 2, Warren R. L. Cairns 4, Vincenzo Vindigni 3, Bruno Azzena 3, Carlo Barbante 4 and Barbara Zavan 2,* Chiara Rigo 1, Letizia Ferroni 2, Ilaria Tocco 3, Marco Roman 4, Ivan Munivrana 3, Chiara Gardin 2, Warren R. L. Cairns 4, Vincenzo Vindigni 3, Bruno Azzena 3, Carlo Barbante 4 and Barbara Zavan 2,* 1 Department of Molecular Sciences and Nanosystems, University Ca’ Foscari, Santa Marta, Dorsoduro 2137, 30123 Venice, Italy; E-Mail: chiara.rigo@unive.it 1 Department of Molecular Sciences and Nanosystems, University Ca’ Foscari, Santa Marta Dorsoduro 2137 30123 Venice Italy; E-Mail: chiara rigo@unive i 2 Department of Biomedical Sciences, University of Padova, Viale G. Colombo 3, 35100 Padov Italy; E-Mails: letizia.ferroni@unipd.it (L.F.); chiara.gardin@unipd.it (C.G.) 3 Burns Centre, Division of Plastic Surgery, Hospital of Padova, via Giustiniani 2, 35128 Padova, Italy; E-Mails: ilaria.toccotussardi@gmail.com (I.T.); ivan.munivrana@unipd.it (I.M.); vincenzo.vindigni@unipd.it (V.V.); bruno.azzena@sanita.padova.it (B.A.) 4 CNR-IDPA c/o Department Environmental Sciences Informatics and Statistics, University Ca’ Foscari, Dorsoduro 2137, 30123 Venezia, Italy; E-Mails: marco.roman@unive.it (M.R.); cairns@unive.it (W.R.L.C.); barbante@unive.it (C.B.) * Author to whom correspondence should be addressed; E-Mail: barbara.zavan@unipd.it; Tel./Fax: +39-049-827-6096. Received: 23 December 2012; in revised form: 5 February 2013 / Accepted: 10 February 2013 / Published: 1 March 2013 Abstract: In this preliminary study, the silver nanoparticle (Ag NP)-based dressing, Acticoat™ Flex 3, has been applied to a 3D fibroblast cell culture in vitro and to a real partial thickness burn patient. The in vitro results show that Ag NPs greatly reduce mitochondrial activity, while cellular staining techniques show that nuclear integrity is maintained, with no signs of cell death. For the first time, transmission electron microscopy (TEM) and inductively coupled plasma mass spectrometry (ICP-MS) analyses were carried out on skin biopsies taken from a single patient during treatment. The results show that Ag NPs are released as aggregates and are localized in the cytoplasm of fibroblasts. No signs of cell death were observed, and the nanoparticles had different distributions within the cells of the upper and lower dermis. Depth profiles of the Ag concentrations were determined along the skin biopsies. In the healed sample, most of the silver remained in the surface layers, whereas in the unhealed sample, the silver penetrated more deeply. The Ag concentrations in the cell cultures were also determined. Clinical Int. J. Mol. Sci. Int. J. Mol. Sci. 2013, 14 4819 Considering the effective antibacterial properties of Ag NPs and the enormous interest in their application as coatings for medical devices and in wound therapy, their safety and biocompatibility need to be urgently clarified. We present here an in-depth study of a Ag NPs containing dressing. The product chosen is a flexible polyethylene cloth coated with nanocrystalline Ag particles and is one of the most widely used Ag based dressings in burns centers worldwide. The dressing was developed to guarantee a controlled and prolonged release of nanocrystalline silver [33] to the wound area; according to the manufacturer, the nanosilver particles also release silver ions. Physical vapor deposition was used to coat the polyethylene with nanocrystals that have a mean diameter of 10–15 nm [34–36]. Previously published in vitro studies regarding the safety of this product have been carried out on human derived skin cells [37,38] and are in contrast with in vivo studies [39,40] and with daily observation of patients treated with this dressing [41]. In an attempt to clarify matters, our research work was carried out in vitro on a three dimensional cell culture system of human skin fibroblasts [42,43], as well as in vivo on skin biopsies from burns patients. The dressing was applied onto the cells and was changed every three days to simulate dressing changes by a clinician. The Ag concentration in the culture medium and in the used dressings has been measured by inductively coupled plasma mass spectrometry (ICP-MS), and the Ag amount absorbed by the cells has been evaluated by difference. Biochemical and morphological techniques have been employed to determine both the viability and the spatial distribution of the cells during the application of the dressing. In order to compare in vitro with in vivo results, skin biopsies were taken from a patient at different times during the healing process. These samples were subjected to histological analysis, and transmission electron microscopy (TEM) analyses were performed to determine the distribution profile of Ag NPs and their subcellular localization. Our in vitro results confirm that Ag NPs alter mitochondrial functionality in human fibroblasts, but interestingly, this does not seem to lead to cell death. Despite the reduced metabolic activity, we demonstrate that the cells are still viable, as no features of apoptosis were detected. 1. Introduction Even though Ag NPs-containing dressings are declared to be safe for patients and non-cytotoxic [22–24], recent studies have shown possible toxic effects on human fibroblasts and keratinocytes [25,26]. The cytotoxic effects that have been observed in different cell lines in vitro include decreases in mitochondrial function [27], cellular shrinkage and irregular shape [28], as well as production of reactive oxygen species (ROS) [29,30]. Carlson et al. found that ROS production was particle size, as well as concentration dependent [31], whilst Hackenberg and co-workers found that Ag NPs induce DNA damage in human mesenchymal stem cells [32]. Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2013, 14 We retain that the apparent cytotoxicity observed by other authors is probably an artifact of the use of a mitochondrial specific activity assay that is an indirect measure of cells viability. Our results suggest that mitochondria are activated to protect the cell and, in particular, the nucleus, against the action of Ag NPs. In vivo results demonstrate that application of Ag NP-based dressings allows wound healing and recovery. For the first time, it has been demonstrated that during application of Ag NP-based dressings on real patients, Ag NPs are released and enter into the cells as agglomerates. The Ag NPs do not dissolve entirely, but remain in the fibroblasts’ cytoplasm during the whole healing process and change shape over time. This effectively demonstrates the safety of Acticoat™ Flex 3, a Ag NP-based product currently used in burn care. Although Ag NPs treatment reduces mitochondrial activity, it does not appear to affect cell viability. Hence the Ag NPs released could be defined as toxic to mitochondria, causing a temporary reduction in metabolic activity in the cell, without causing cell death. Cells remain viable and are able to re-proliferate once the silver is passivated, leading to reconstruction of the dermal tissue in vivo. Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2013, 14 4820 2. Results and Discussion 2.1. In Vitro Study 2.1.1. MTT Assay 2.1.1. MTT Assay To investigate if Ag NPs could negatively affect the healing process, we evaluated their toxicity on fibroblasts in vitro. A collagen-based scaffold was employed as a support for a 3D cell culture of fibroblasts to obtain a dermal-like tissue. At three, six and nine days, MTT assays were carried out to assess the mitochondrial function in cells treated with Ag NPs. As reported in Figure 1, a time-dependent decrease in metabolic activity was observed in the cells treated with the Ag NP-based dressing. This confirms the ability of Ag NPs to impair mitochondrial function, as reported by Burd [12] and Foldbjerg [27]. Our results demonstrate that the mitochondrial activity rapidly decreases over the first three days of treatment. After three days of exposure to Ag NPs, the cells had only 17% ± 0.54% of mitochondrial functionality normalized to the untreated sample value. In the following three days, the relative mitochondrial activity decreased to 7% ± 0.01%. In the final three days of the experiment, the mitochondrial functionality relative to the untreated control dropped to 5% ± 0.03%. This indicates that the Ag NPs and silver ions heavily impair mitochondrial functionality, and this is probably correlated to the generation of ROS, in agreement with the results found by AshaRani [28] and Hsin [30]. Figure 1. Mitochondrial activity in silver nanoparticle (Ag NP)-treated 3D fibroblast cultures, (one sample n = 2 readings ± SD versus time). The mitochondrial activity in the treated samples is expressed as a percentage of the activity of the untreated samples. For each time point, MTT values were obtained from duplicate readings of a single sample. 2.1.2. Morphological Analysis 2.1.2. Morphological Analysis 2.1.2. Morphological Analysis Morphological analyses were carried out to investigate nuclei morphology and cellular distribution within the scaffold. Hoechst dye was employed to stain the nuclei blue and was used to verify the nuclear integrity or the presence of any apoptotic features, such as chromatin condensation and fragmentation, as well as the presence of apoptotic bodies. Under UV light, the characteristic fluorescence at 461 nm allows the localization of the cells in the three dimensional matrix. To confirm the results obtained, YO-PRO®-1 staining was carried out. As reported in Figure 2a, in the untreated Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2013, 14 4821 3D fibroblast cultures (controls), the cells proliferated mainly on the surface, although some cells grew also inside. Therefore, the dermal-like tissue appears as a multilayer of cells, where the fibroblasts are able to proliferate during the course of the experiments (Figure 2b). No signs of apoptosis were detected (Figure 2c). The similar distribution of cells was seen in the Ag NP-treated samples (Figure 2d). Interestingly, despite the reduced mitochondrial functionality observed, the nuclei are still present and appear to be undamaged. There was no observable presence of apoptotic bodies or nuclear fragmentation (Figure 2e,f). These results are in agreement with those recently observed by Zanette et al. [26] in the HaCaT cell line, supporting the hypothesis that Ag NPs reduce mitochondrial functionality without seeming to cause genotoxicity and cell death. In Figure 3, a quantitative comparison of the number of live cells in the treated and untreated 3D cell cultures can be seen. The results show that the number of live cells increased with time at the same rate in both samples. The YO-PRO®-1 assay showed that there were no apoptotic cells visible in the sample treated with Ag NPs. Figure 2. Dermal-like tissue reconstructed in vitro. Cells, visible thanks to the Hoechst blue staining of the nuclei, can be seen inside the collagen-based scaffold and appear to be organized in layers. (a) Un-treated control after three days from the beginning of the experiments; (b) Untreated control at nine days; (c) Enlargement of selected area; (d) Ag NP-treated fibroblast at three days; (e) Ag NPs fibroblast at nine days; (f) Enlargement of selected area. Figure 2. Dermal-like tissue reconstructed in vitro. Cells, visible thanks to the Hoechst blue staining of the nuclei, can be seen inside the collagen-based scaffold and appear to be organized in layers. 2.1.3. Ag Release and Distribution 2.1.3. Ag Release and Distribution 2.1.3. Ag Release and Distribution The level of Ag in the new unused dressings was 821 ± 20 µg cm−2 (n = 9). This result is in agreement with the value of 827 ± 58 µg cm−2 previously estimated by Rigo et al. [44] and demonstrates that the inter-batch variability is negligible for Acticoat™ Flex 3. Table 1 summarizes the concentrations of Ag, and it is expressed as µg of Ag per cm2 (µg cm−2) of culture covered with the dressing. This is reported as per individual application of the dressing. During the experiment, three consecutive applications of Ag NP-based dressing were carried out. For the second and third application, the old dressing was substituted with a new one and deposited on the same sample of the cell culture. Concurrently, the medium (cDMEM) was removed and substituted with the same volume of fresh medium. The level of Ag applied to the culture at each step was derived from the average concentration of the unused dressing (see above), considering that all the pieces applied had the same surface area (0.283 cm2, 6 mm diameter). The fraction corresponding to the used dressing (called Dressing in Table 1) is comprised of Ag that was never released and Ag that was released, but re-adhered to the surface in another form, e.g., small fragments of scaffold containing Ag stuck to the surface. The fraction medium represents the Ag released into the liquid medium, and the fraction culture was calculated by difference and represents the Ag captured by the 3D culture. For each individual application of the dressing, most of the Ag (~94%) was revealed to remain in the dressing. The result is compatible with those previously obtained for the release of Ag in solution [44], where 94%–99% of Ag was found to still be present in the dressing after three days, depending on the composition of the solution. In this study, the fraction with the smallest silver concentration is the cell culture. Although the concentration is low, it is not irrelevant: the cumulative value reaches 68 ± 7 µg cm−2 (n = 3) after nine days of treatment. 2.1.2. Morphological Analysis (a) Un-treated control after three days from the beginning of the experiments; (b) Untreated control at nine days; (c) Enlargement of selected area; (d) Ag NP-treated fibroblast at three days; (e) Ag NPs fibroblast at nine days; (f) Enlargement of selected area. Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2013, 14 4822 Figure 3. Progression of cell growth in time in a 3D dermal-like tissue after Ag NPs treatment (dark grey) and in the control sample (light grey); mean value ± SD (n = 2) samples versus time. The count of the live cells in the sample is obtained as the sum of the live cells at various depths at each position. 2.1.3. Ag Release and Distribution 2.1.3. Ag Release and Distribution Although the cell culture was not changed at each three-days (unlike the dressing and the medium), the absorption rate of Ag increased from 18 ± 3 µg cm−2 (n = 9) in the first step to 25 ± 2 µg cm−2 (n = 6) in the second and 25 ± 3 µg cm−2 (n = 3) in the third steps. Such an inverse trend between Ag and relative mitochondrial activity (see Figure 1) levels in the culture seems to confirm their direct causal relationship. The fraction of Ag released in the medium follows an opposite trend with decreasing values from the first to the second Int. J. Mol. Sci. 2013, 14 4823 step. This can be explained by the fact that the dressing releases a constant quantity of Ag into the medium, but cellular uptake increases in the second and third steps. In order to validate the indirect estimates of Ag concentration in the cell cultures reported in Table 1, the Ag concentrations in the samples used for the MTT analyses were determined by ICP-MS by analyzing the MTT and iDMSO fractions. These direct measurements and their sum are reported below in Table 2 and are compared with the estimated results obtained above. Table 1. The results for three separate applications of Ag to the culture, expressed as µg of Ag per cm2 (µg cm−2) of culture covered dressing. Applied Ag levels are assumed to be equal, based on analysis of the new unused dressing. Silver concentrations in the dressings and the medium were determined by ICP-MS. The Ag concentration in the 3D culture was obtained by difference. Ag (µg cm−2) Days Sample size Total applied Dressing Medium Culture * 1–3 n = 9 821 767 ± 4 37 ± 5 18 ± 3 3–6 n = 6 821 765 ± 3 31 ± 3 25 ± 2 6–9 n = 3 821 765 ± 4 32 ± 2 25 ± 3 Note: * calculated as the mean of the differences. Note: * calculated as the mean of the differences. Table 2. Cumulative concentration of Ag (µg cm−2) in the cell culture (n = 1) and control experiment (n = 3) with a comparison of the values estimated by difference with the sum of the direct measurements in the MTT and dimethyl sulfoxide in isopropanol (iDMSO) fractions. TMAH: tetramethylammonium hydroxide. Table 2. 2.1.3. Ag Release and Distribution Cumulative concentration of Ag (µg cm−2) in the cell culture (n = 1) and control experiment (n = 3) with a comparison of the values estimated by difference with the sum of the direct measurements in the MTT and dimethyl sulfoxide in isopropanol (iDMSO) fractions. TMAH: tetramethylammonium hydroxide. Days Estimated MTT + iDMSO MTT iDMSO TMAH 3 16.5 16.3 5.8 10.5 - 6 40.9 30.9 8.0 22.9 - 9 62.9 46.5 10.3 36.2 - Control experiment in absence of cells n = 3 replicates ± 1 SD 3 - 12.5 ± 5.1 8.9 ± 3.7 3.3 ± 1.8 0.7 ± 0.3 The direct determination of Ag in the cell cultures resulted in levels lower than the indirect estimates, which are derived from the mass balance of the metal. It should be noted that the direct determinations are based on single replicates, so an uncertainty value cannot be provided. The underestimation is probably due to a non-quantitative recovery of Ag by the two extractions (in MTT buffer and iDMSO), so that part of Ag probably remains bound to the scaffold. We have shown that the accurate direct determination of Ag in the culture would require a separate replicate of the experiment. The indirect determination of Ag is a viable alternative to describe the kinetics of Ag distribution in the system, reducing the amount of time and materials required. The fraction extracted into iDMSO is always greater than that in MTT, and the proportion increases almost linearly between the third to the ninth day of treatment. In the control experiment, the Ag amount measured in the MTT fraction is 70% of the total and is similar to the Ag concentration found in the MTT fraction after six days, when fibroblasts were present. The amount of Ag measured in the iDMSO fraction is 25% of the Ag released and is much Int. J. Mol. Sci. 2013, 14 4824 less than found when cells are present, and the amount of Ag found after dissolution of the scaffold was found to be 5% of the total. This partitioning can be easily explained, because MatriDerm® is a dermal substitute made of elastin and collagen type I, III and V obtained from bovine nuchal ligaments (as stated by the manufacturer); these proteins have a limited number of cysteine residues. Therefore, Ag binds loosely to MatriDerm® and is easily extracted during the first extraction (MTT fraction). 2.2. In Vivo Study 2.2. In Vivo Study 2.1.3. Ag Release and Distribution The Ag concentration in the MTT fraction should represent the amount of Ag loosely bound to the scaffold or the external surfaces of the cell membranes. It is well known that Ag has a strong interaction with DMSO [45] and that it causes cell lysis. So, it can be reasonably assumed that the Ag concentration in the iDMSO fraction represents the major part of the Ag fraction inside the cells, as well as being strongly bound to the structural constituents of the cells. This is confirmed by the control experiment that shows that the amount of Ag extracted by the iDMSO solution from the scaffold is always much less the value found when cells are present, and that complete dissolution of the scaffold only releases 5% of the total Ag present. This demonstrates that most of the Ag released from the dressing over the nine days of the study is taken up by the cells or is adsorbed strongly to their surface. Considering that there is a linear increase of Ag in the iDMSO fraction during the experiment, we conclude that Ag uptake by the cells did not cease and that the cellular Ag binding sites either in or outside the cells had not been saturated. 2.2.1. Microscopy Optical microscopy observations of the skin biopsies after hematoxylin eosin staining show the main differences between the four samples. Figure 4a is burnt skin after surgical cleaning before silver application. Figure 4b is healed skin after seven days of treatment. Figure 4d is unhealed skin after seven days of treatment, and Figure 4f shows the same area healed after 17 days of treatment. Figure 4. Optical microscopy (OM) Images of the skin samples: burnt (a), healed after seven days (b and c), unhealed after seven days (d and e) and after complete re-epithelialization (f) after 10 more days of treatment. Healed and unhealed skin sections are shown with H/E and toluidine blue staining. Scale bar: 100 µm. Int. J. Mol. Sci. 2013, 14 4825 The burnt skin prior to Ag NP dressing application shows epidermal necrosis, diffuse perivascular infiltrate and important collagen degeneration at the level of the papillary dermis, characteristic of a deep dermal burn (Figure 4a). The burnt skin prior to Ag NP dressing application shows epidermal necrosis, diffuse perivascular infiltrate and important collagen degeneration at the level of the papillary dermis, characteristic of a deep dermal burn (Figure 4a). After seven days of treatment with Acticoat™ Flex 3, the structure of the biological tissue has been completely restored in the healed area (Figure 4b). Under staining with toluidine blue, it can be seen that the healed skin is composed of a well-stratified epidermis, complete with basal, spinous, granular and cornified layers (Figure 4c). Macroscopic evaluation shows more over the presence of a well vascularization at the dermal–subcutaneous interface. In the unhealed skin samples (Figure 4d,e), the tissue organization has not been re-established. The epidermis has not yet re-formed (Figure 4d) and the dermis still has a disorganized and irregular structure (Figure 4e). The wound was treated with Acticoat™ Flex 3 for 10 more days. When the dressing was removed, the wound appeared healed, and a new biopsy was taken to verify the re-establishment of the tissue architecture. Optical microscopy observations of the sample (Figure 4f) confirm the re-growth of the tissue structure, and in particular, it is possible to observe the restoration of the epidermis. This result of complete patient healing by the 17th day shows that, despite the presence of Ag NPs in the tissue and inside the cells, the healing process does not seem to be impeded. 2.2.1. Microscopy Our results agree with and support the results recently published by Gravante et al. [46]. They found that Acticoat™ Flex 3 was the dressing with the shortest healing times for deep partial thickness burns (16 days average; our result, 17 days). The average healing times for sodium carboxymethyl cellulose were longer than those of nanocrystalline silver (21 days), but were shorter than paraffin gauzes (26.5 days) and collagenase cream (29 days). This data confirms that the healing process is not impeded during the treatment with Ag NPs. TEM images of the healed skin sample after seven days are shown in (Figure 5a–g). The sample was observed from the epidermis to a point at which Ag NPs were no longer visible, which corresponded to a depth of ~3 mm. In Figure 5a, the presence of a great number of agglomerates of nanoparticles can be seen surrounding the fibroblasts in the upper part of the dermis. No Ag NPs or aggregates were visible in epidermis. A higher magnification of the same area shows that the aggregates are located in the extracellular matrix, close to the cell membrane. In Figure 5b, as described by Xu [47], a slight broadening of the intercellular space can occur and is probably due to a previous inflammatory phase. In Figure 5c, it can be seen that agglomerates of Ag NPs enter into the cells via endocytic vesicles in the form of agglomerates, as recently observed separately by Kim [48] and Greulich [49] in different cell lines. The individual nanoparticles that are visible were measured to have diameters of <10 nm (Figure 5d). After the agglomerates were released into the cytoplasm (Figure 5e), they typically had a round shape and were located close to the mitochondria (labeled M). No Ag NPs were detected inside the nucleus (labeled NM), and no fragmented nuclei were observed. Despite the presence of Ag NPs inside the cytoplasm, the nuclear membrane is intact and is round in shape. The nucleolus visible in Figure 5b (labeled N) confirms that the chromatin is not condensed, but that it has a structure that allows transcription to proceed. In Figure 5e, it was observed that the mitochondria are generally located in the proximity of the nuclear membrane. It was observed that the fibroblasts in the upper part of the dermis seem to possess a particular spatial distribution of mitochondria. Int. J. Mol. Sci. 2013, 14 4827 In the lower part of the dermis (Figure 5f,g), the Ag NP agglomerates have a different shape and location inside the fibroblast, compared to what has been found in the upper stratus of the dermis. The agglomerates appear elongated and are located very close to the nuclear membrane. The mitochondria (labeled M) are generally distant from the agglomerates, indicated with an arrow, and appear to be undamaged and healthy (Figure 5g). As Acticoat™ Flex 3 was applied to the surface of the burn, the cells in the wound bed (lower part of the dermis) were the first to receive the Ag NPs released from the dressing. These cells were exposed to Ag NP aggregates for a longer time with respect to fibroblasts of the upper part of the dermis. During this time period, the Ag NPs could have undergone chemical changes that passivated them or quenched their ROS generating ability. In light of this hypothesis, Ag NPs could remain inside the cells, without provoking toxic effects. This could explain the different spatial distribution of the Ag NP agglomerates in the fibroblasts of the upper and lower dermis, but further investigations are necessary. In the unhealed skin, the tissue structure is highly disorganized and the epidermis had not reformed. The TEM images in Figure 6a,b show vesicles containing electron-dense matter, but it was not possible to verify if this material contained Ag. Unlike in the healed skin, in the unhealed skin sample, it was not possible to identify nanoparticles. It is possible that the electron-dense matter is composed of Ag salts or Ag bound to proteins via thiol or selenide groups and then precipitated [56]. Figure 6. TEM images of the unhealed skin sample, DM (dark matter), contained in vesicle. Scale bar: 1 µm. The SEM analyses showed the presence of particles in the unhealed skin samples. As shown in Figure 7a, such particles have a diameter <10 µm. The EDS spectrum (Figure 7b) shows that the particles are composed of virtually pure Ag metal. In the healed skin sample, it was not possible to identify Ag in any form. This was probably due to the low sensitivity of the technique and the absence of Ag microparticles on the surface of the sample. 2.2.1. Microscopy We speculate that these organelles have been replicated and then moved by the microtubules [50,51] to surround the nucleus to protect the DNA from possible damage caused by Ag NPs. It has been already demonstrated in different cell lines that Ag NPs are capable of Int. J. Mol. Sci. 2013, 14 4826 generating ROS inside the cells [29,31] that could damage the genetic material [32]. We hypothesize that once the Ag NPs have been released into the cytoplasm, they generate ROS. This could result in an increase in the number of mitochondria as a response to oxidative stress, as demonstrated by Lee et al. [52], to try and compensate for the reduced activity, as seen in Figure 3. We speculate that mitochondria are moved by the microtubules around the nucleus to act as a physical, as well as “chemical”, barrier to prevent ROS and Ag NPs from reaching the nuclear membrane. If the mitochondrial membrane breaks down due to the action of ROS, antioxidative enzymes, such as mitochondrial superoxide dismutase (mtSOD), catalase, glutathione peroxidase and thioredoxin peroxidase [53], are released from the mitochondria in the cytoplasm to quench the ROS. It has been already demonstrated that Ag NPs and Ag ions cause increased levels of SOD in human [54] and yeast [55] cells and that in vitro, the administration of ROS scavengers (such as SOD, catalase, mannitol and sodium selenite) can partially block the genotoxic effects of Ag NPs in human bronchial epithelial cells [54]. Figure 5. TEM images of the healed skin sample. (a) epidermis and dermis; (b) detail of a fibroblast surrounded by Ag NPs, in the upper part of the dermis; (c) endocytic vesicle containing Ag NP agglomerates; (d) magnification of endocytic vesicle containing Ag NP agglomerates; (e) Ag NPs have been released into the cytoplasm of a fibroblast and are located near the mitochondria; (f) a fibroblast in the lower part of the dermis—Ag NP aggregates are near the nuclear membrane; (g) a healthy undamaged mitochondrion. Key: DE, dermis; EP, epidermis; EV, endocytic vesicle; IS, intercellular space; M, mitochondrion; N, nucleolus; NM, nuclear membrane. Arrows indicate Ag NP agglomerates. Scale bars: (a) 500 nm; (b) 1 µm; (c) 100 nm; (d) 20 nm; (e) 200 nm; (f) 1 µm; (g) 100 nm. Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2013, 14 The superficial concentration of Ag in the unhealed sample after seven days of treatment was lower (37.5 ± 0.6 ng mg−1), but the drop in the concentration profile was more gradual, as the second and third slices still had high concentrations of the metal (29.8 ± 0.5 and 9.6 ± 0.2 ng mg−1, respectively) compared to the healed sample. The cumulative amount of Ag in the unhealed tissue was 223 ng, which is slightly higher than the value in the healed tissue of 171 ng. The sample that was collected after an additional 10 days of treatment had a Ag profile (not shown) similar to that of the healed tissue after seven-days of treatment, but with approximately double the concentrations: 110 ± 2, 31.0 ± 0.7 and 13.2 ± 0.1 ng mg−1 in the first three slices, respectively. 2.2.2. Ag Release and Depth Profiles 2.2.2. Ag Release and Depth Profiles The depth profiles of Ag concentrations in the healed and unhealed biopsies taken after seven days of treatment are shown in Figure 8. This shows that the level of Ag in the healed sample decreases rapidly from 50.8 ± 0.8 ng mg−1 to 6.0 ± 0.2 ng mg−1 between the first and second slices. This trend continues between the third and deepest slices, to reach a range between 0.04 and 0.1 ng mg−1. The superficial concentration of Ag in the unhealed sample after seven days of treatment was lower (37.5 ± 0.6 ng mg−1), but the drop in the concentration profile was more gradual, as the second and third slices still had high concentrations of the metal (29.8 ± 0.5 and 9.6 ± 0.2 ng mg−1, respectively) compared to the healed sample. The cumulative amount of Ag in the unhealed tissue was 223 ng, which is slightly higher than the value in the healed tissue of 171 ng. The sample that was collected after an additional 10 days of treatment had a Ag profile (not shown) similar to that of the healed tissue after seven-days of treatment, but with approximately double the concentrations: 110 ± 2, 31.0 ± 0.7 and 13.2 ± 0.1 ng mg−1 in the first three slices, respectively. The comparison between the Ag profiles and the corresponding OM images of the biopsies is also shown in Figure 8. This allows us to correlate the penetration of the metal into the tissue with its structural organization. Int. J. Mol. Sci. 2013, 14 However, Ag microparticles were identified by SEM-EDS in the skin sample collected after 17 days of treatment, when the previously unhealed part of the wound had returned to a healthy condition. DM DM DM The SEM analyses showed the presence of particles in the unhealed skin samples. As shown in Figure 7a, such particles have a diameter <10 µm. The EDS spectrum (Figure 7b) shows that the particles are composed of virtually pure Ag metal. In the healed skin sample, it was not possible to identify Ag in any form. This was probably due to the low sensitivity of the technique and the absence of Ag microparticles on the surface of the sample. However, Ag microparticles were identified by SEM-EDS in the skin sample collected after 17 days of treatment, when the previously unhealed part of the wound had returned to a healthy condition. The SEM analyses showed the presence of particles in the unhealed skin samples. As shown in Figure 7a, such particles have a diameter <10 µm. The EDS spectrum (Figure 7b) shows that the particles are composed of virtually pure Ag metal. In the healed skin sample, it was not possible to identify Ag in any form. This was probably due to the low sensitivity of the technique and the absence of Ag microparticles on the surface of the sample. However, Ag microparticles were identified by SEM-EDS in the skin sample collected after 17 days of treatment, when the previously unhealed part of the wound had returned to a healthy condition. Int. J. Mol. Sci. 2013, 14 4828 Figure 7. SEM images of the unhealed sample (a) and energy-dispersive X-ray spectroscopy (EDS) spectrum (b) of the particle indicated by the arrow. Scale bar: 50 µm. Figure 7. SEM images of the unhealed sample (a) and energy-dispersive X-ray spectroscopy (EDS) spectrum (b) of the particle indicated by the arrow. Scale bar: 50 µm. 2.2.2. Ag Release and Depth Profiles The depth profiles of Ag concentrations in the healed and unhealed biopsies taken after seven days of treatment are shown in Figure 8. This shows that the level of Ag in the healed sample decreases rapidly from 50.8 ± 0.8 ng mg−1 to 6.0 ± 0.2 ng mg−1 between the first and second slices. This trend continues between the third and deepest slices, to reach a range between 0.04 and 0.1 ng mg−1. Int. J. Mol. Sci. 2013, 14 The healed skin sample has the organized structure of a well-reconstructed tissue. The Ag NP aggregates released from the dressing were not able to penetrate deeply into the tissue, so they remained immobilized, mainly in the upper stratum of dermis, where they were covered by newly grown keratinocytes. The unhealed skin, instead, has a messy and disorderly structure and the normal connective tissue organization has not yet been re-established. Fibroblasts are low in number and the connective tissue poorly formed. This means that the Ag NPs released by Acticoat™ Flex 3 are able to pass through the connective tissue to reach the lower strata of the dermis. As observed in the quantification of Ag in the in vitro MTT and iDMSO fractions (see above, Section 2.1.3), the Ag concentration in the skin samples increases linearly with the number of dressings applied on the wound. The same is seen in vivo; a skin sample treated with two pieces of Acticoat™ Flex 3 has double the Ag concentration of skin samples after only one application. The slightly higher cumulative Ag concentration measured in the unhealed skin sample after seven days of treatment can be explained by considering that the presence of body fluids and exudates can increase Ag release from dressings [56]. Int. J. Mol. Sci. 2013, 14 4829 Figure 8. Depth profiles of Ag concentration (ng mg−1) after seven days of treatment. In this pilot study, the biopsies were obtained from the same patient. One biopsy for silver analysis was taken from the healed part of the wound (top) and one from the unhealed (bottom) tissue. Panoramic images of the other duplicate sample were obtained by optical microscopy, and zoomed in areas of representative portions are also shown for comparison. 2.2.3. Clinical Observations Our results are reinforced by the clinical observations that were collected between January 2011 to September 2012 in the Plastic Reconstructive Surgery Division. In this time interval, 98 patients were treated with Acticoat™ Flex 3 for coverage of burn wounds. A total of 102 applications, corresponding to 58,400 cm2 of dressing containing a total of nearly 48 g of high purity silver, were used. The annual use in 2011, which was the year of introduction of Acticoat™ Flex 3, was around 33,000 cm2. Data from average monthly use in 2012 are comparable to 2011, and we foresee a stable annual use. Int. J. Mol. Sci. 2013, 14 The patients were 63 males and 45 females, with an average age of 49 years. Ninety-five patients received a single silver dressing application; three patients, two. Burning causes comprised of fire (55 patients), hot liquids (23, of which three were boiling oil) and others (six contact burns, four 3. Clinical Observations 2.2.3. Clinical Observations Our results are reinforced by the clinical observations that were collected between January 2011 to September 2012 in the Plastic Reconstructive Surgery Division. In this time interval, 98 patients were treated with Acticoat™ Flex 3 for coverage of burn wounds. A total of 102 applications, corresponding to 58,400 cm2 of dressing containing a total of nearly 48 g of high purity silver, were used. The annual use in 2011, which was the year of introduction of Acticoat™ Flex 3, was around 33,000 cm2. Data from average monthly use in 2012 are comparable to 2011, and we foresee a stable annual use. The patients were 63 males and 45 females, with an average age of 49 years. Ninety-five patients received a single silver dressing application; three patients, two. Burning causes comprised of fire (55 patients), hot liquids (23, of which three were boiling oil) and others (six contact burns, four Int. J. Mol. Sci. 2013, 14 4830 chemical burns). The average total body surface area (TBSA) burn percent was 23.6% ± 17.1%. Out of 98 cases, 100% reached re-epithelialization. The average amount of dressing used per patient was 602.4 ± 163.5 cm² (minimum 200 cm²; maximum 1100 cm²). Re-epithelialization was spontaneous in 80 cases, and lesions were recorded as healed within 32 ± 18 days from the last silver dressing application. None of the patients in this study complained of pain or had any symptoms or signs suggesting argyria. None of the 98 patients treated with Acticoat™ Flex 3 between January 2011 to September 2012 in the Plastic Reconstructive Surgery Division was recorded to have staining of skin due to silver accumulation. Eighteen patients reached healing after autologous skin grafting. Autologous grafting was performed at 23 ± 9 days after the last silver dressing application. Healing times recorded for burn wounds are consistent with the data previously reported by other authors [34,57,58]. Khundkar et al. 2.2.4. Limits of This Study and Future Perspectives A major limit of this preliminary work is the number of in vitro and in vivo samples that were made available. In particular, it would be useful to verify the results of the MTT and YO-PRO®-1 assays on a larger number of samples and recruit more patients for the study. Another limit of this work is that the mitochondrial activity was only monitored for nine days, which was not long enough to verify the revival of mitochondrial activity that is suggested by our in vivo observations. To clarify this aspect, the in vitro experiments should be carried out for a prolonged time period (>17 days, time for complete healing in patients). The side effects and fate of Ag NPs remaining deposited in the healed skin after treatment are still unknown and should be investigated by patient follow-up in any future work. Int. J. Mol. Sci. 2013, 14 [20] demonstrated that Ag NP-based dressings are effective in reducing the time for re-epithelialization and the requirement for grafting in comparison to other treatment widely used in burn centers, such as silver sulfadiazine and 0.01% neomycin and polymyxin solution. Strand et al. [59] compared the length of stay for hospitalized patients treated with Acticoat™ Flex 3 with those treated with Mepitel, a flexible polyamide net coated with soft silicone. The authors found the mean in-patient stay was 12.5 days when treated with Mepitel, whilst the mean stay was 4.5 days when the patients were treated with nanocrystalline silver dressing. In our study, the percentage of patients (18%) that required skin grafting is limited and comparable to the results (8%) obtained by Strand et al. [59]. Literature data, clinical observations collected by the Plastic Reconstructive Surgery Division of Padua and the results obtained in this work on a limited number of samples support each other in confirming the safety of Acticoat™ Flex 3. 2.2.4. Limits of This Study and Future Perspectives 3.1. Dressing Acticoat™ Flex 3 (Smith & Nephew, Milan, Italy) dressing is a flexible polyethylene cloth coated with nanocrystalline Ag particles at a concentration between 0.69 and 1.64 mg/cm2. At the Plastic Reconstructive Surgery Division of the University Hospital of Padova, Acticoat™ Flex 3 is used for burn wounds with the indication of partial-thickness burn, which needs improvement of wound-bed quality for faster recovery. Acticoat™ Flex 3 is fixed with metallic sutures after performance of surgical toilette of the wounds. The gauze dressing over the wound is moistened with Int. J. Mol. Sci. 2013, 14 4831 distilled water and changed weekly. Patients are discharged from the hospital after taking into account their general condition; this is usually 7–10 days post-operation, and further, out-patient care is provided on a regular weekly basis. The Ag NP dressing is removed at around day 14. If the wound is recalcitrant, the patient is then subjected to further wound cleaning and autologous skin grafting. If reactivation of re-epithelialization from the edges is observed, the wound is treated with Vaseline® gauze plus Amukine Med 0.05% (a sodium hypochlorite cutaneous solution). 3.2. In Vitro Study 3.2.2. MTT Assay To determine the kinetics of cell growth with or without Ag NPs, the MTT-based (methyl-thiazolyl-tetrazolium) cytotoxicity assay was performed according to the method of Denizot and Lang with minor modifications [62]. This colorimetric assay is an indirect method for assessing cell growth and proliferation. MTT gives a yellowish aqueous solution, which, on reduction with dehydrogenases or reducing agents present in metabolically active cells, yields a violet-blue water insoluble dye compound, formazan. The lipid soluble formazan is extracted with organic solvents and quantified spectrophotometrically. The amount of MTT formazan produced is directly proportional to the metabolic activity of cells. After harvesting the culture medium, the cells were incubated for 3 h at 37 °C in 1 mL of 0.5 mg/mL MTT solution prepared in phosphate buffer saline solution (PBS). After removal of the MTT solution by pipette, 0.5 mL of 10% dimethyl sulfoxide in isopropanol (iDMSO) was added to extract the formazan in the samples for 30 min at 37 °C. For each sample, absorbance values at 570 nm were recorded in duplicate on 200 µL aliquots deposited in microwell plates using a multilabel plate reader (Victor 3 Perkin Elmer, Milano, Italy). The mitochondrial functionality in the Ag NP-treated cells is calculated as the ratio between the absorbance at 570 nm of the treated sample and the absorbance of a control sample expressed as a percentage. 3.2.1. Cell Cultures Human dermal fibroblasts were prepared according to a modified version of the Rheinwald and Green protocol [60]. After epithelial sheet dispase removal, the dermis was cut into small pieces (2–3 mm2), and fibroblasts were isolated by sequential digestion with 0.25% w/v trypsin for 20 min and 0.25% w/v collagenase for 4 h. These cells were then cultured with Dulbecco’s Modified Eagle Medium (DMEM), (Lonza S.r.l., Milano, Italy) supplemented with 10% Fetal Bovine Serum (FBS) (Bidachem S.p.A., Milano, Italy) and 100 units/mL penicillin and 100 µg/mL streptomycin to form complete DMEM (cDMEM). The medium was changed twice a week, and the cells were harvested by trypsin treatment. After detachment from culture plates, fibroblasts were cultured in 3D collagen-based scaffolds (MatriDerm®, Dr. Suwelack Skin & Health Care AG, Billerbeck, Germany) at a density of 1.2 × 105 cells/cm2, obtaining a reconstructed dermal-like tissue in vitro. The number of the cells used to seed was derived from previous studies by Zavan and co-workers [42,43,61]. Cells were grown in the 3D scaffold for 10 days in 800 µL of cDMEM. The Ag NP-based dressing was applied above the 3D cell cultures. To guarantee experimental reproducibility, all the experiments were carried out using subsamples taken always from the same dressing and scaffold. Both the dressing and scaffold was cut with skin biopsy punches. For the dressing, a 6 mm internal diameter punch was used to obtain 40 subsamples, whereas for the scaffold, an 8 mm internal diameter punch was employed to obtain 15 subsamples. The flow chart of the experimental plan is reported in Figure 9. Every 3 days, the dressing was removed from the culture and substituted with a new one. Concurrently with dressing change, the culture medium was removed and replaced with fresh medium. As reported in Figure 9, one set of 9 control and 9 treated cultures was created. At fixed time points (3, 6, 9 days), an MTT assay was carried out on a control and treated sample to verify mitochondrial activity. Histological analyses were performed in duplicate to verify cell distribution within the collagen-based scaffold, in the control and Ag NP treated samples. Cell culture medium and the used dressings have been collected to quantify the Ag release by ICP-MS analysis, as reported in Section 3.4. Int. J. Mol. Sci. 2013, 14 4832 Figure 9. Flow chart of the experimental plan adopted for the in vitro study. 3.2.1. Cell Cultures The cycle of applications is repeated to carry out the chemical, toxicological and mitochondrial functionality determinations for 3, 6 and 9 days of cumulative duration of the treatment. 3.2.2. MTT Assay Ag level MTT and Ag level Histochemistry Cell culture Medium (residual dressing) Treated cultures: starting n=9 n=1 n=2 After 3 days: removal of the  dressing and medium Application of the  dressing New dressing and  medium Control cultures: starting n=9 n=1 n=2 After 3 days:  removal of the  medium New medium Control cultures: starting n=9 Control cultures: starting n=9 Treated cultures: starting n=9 Application of the  dressing Medium (residual dressing) After 3 days:  removal of the  medium After 3 days: removal of the  dressing and medium Ag level n=1 n=2 New dressing and  medium 3.2.2. MTT Assay 3.2.3. Morphological Analysis Concurrently with the MTT assay, morphological analyses were carried out on the duplicate samples. After removing the Ag NP-based dressing and the culture medium, the remaining dermal-like tissue was embedded in Optimal Cutting Temperature (OCT) compound, frozen in liquid nitrogen and preserved at −80 °C until cutting. Tissue sections (>7 µm thickness) were obtained using a cryostat (CM1950, Leica, Milano, Italy) and deposited onto gelatin-coated glass slides. They were fixed with absolute acetone for 10 min at room temperature and cryopreserved at −20 °C until use. In order to visualize the cell distribution inside the scaffold and to investigate the possibility of nuclear Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2013, 14 4833 fragmentation, the fibroblasts nuclei were stained with Hoechst H33342 fluorochrome (Sigma Aldrich, Milano, Italy, final concentration of 2 µg/mL). The samples were observed using a Zeiss Axioplan fluorescence microscope equipped with a digital camera (DC500, Leica, Milano, Italy). In order to quantify the number of live cells and highlight the presence of apoptotic cells, a parallel set of in vitro experiments were carried out. Hoechst H33342 dye was added to the 3D dermal-like cell culture simultaneously with YO-PRO®-1 iodide dye (excitation wavelength 491 nm/emission wavelength 509 nm, Molecular Probes). Hoechst H33342 dye stains the nuclei in the whole population of cells, while YO-PRO®-1 stains specifically the apoptotic cells. YO-PRO®-1 is a green fluorescent probe, which can enter cells once their plasma membrane has reached a certain degree of permeability. The cell membrane during apoptosis becomes slightly permeable and YO-PRO®-1 can freely enter the cell and bind to its nucleic acids, enhancing its fluorescence intensity. The number of different cells was counted, and live cells are calculated as the difference between the number of cells stained with Hoechst H33342 and the number of apoptotic cells stained with YO-PRO®-1. Immediately after the removal of the Ag NP-based dressing from the 3D cell cultures, Hoechst 33342 and YO-PRO®-1 were added to the cell cultures. The cells were incubated at 37 °C for one hour, and then, the culture multiwell plate containing the cells was transferred to a confocal laser scanning microscope to monitor YO-PRO®-1 and Hoechst fluorescence. A fluorescence confocal laser scanning microscope (Axiovert 100M, Zeiss, Germany) with a 10× magnification objective was used for the detection of Hoechst H33342 and YO-PRO®-1 stained cells. 3.2.3. Morphological Analysis The fluorescent dye, YO-PRO®-1, was excited with a 25 mW Argon laser at 488 nm. Emission was recorded above 510 nm. The Hoechst H33342 fluorescence was detected at 460 nm after excitation at 346 nm. The microscope was equipped with a motorized stage, and the LSM 510 (Zeiss) software enabled memorization of stage positions. For each sample, images were taken at the preset stage positions at various depths. The count of the live cells in the sample is obtained as the sum of the live cells at various depths at each position. 3.3. In Vivo Study 3.3.1. Human Skin Samples Patients were eligible for the study if recruited <24 h post-burn injury and were affected by partial-thickness burns. Patients were excluded if they were affected by full thickness burns or had a compromised immune system or were known to be hypersensitive to silver and its compounds. Patients were also excluded in case of comorbidity (e.g., diabetes, cardiac or renal disease), chemical or electrical burns, multiple trauma or were aged <5 or >60. Skin biopsies were obtained from a set of eligible patients who gave consent for taking biopsy materials for scientific purposes, and the study was performed in compliance with the Declaration of Helsinki ethical guidelines. Biopsies were collected by using punches of 4 mm inner diameter × 7 mm depth, according to the experimental plan represented in Figure 10, from 1 patient. Duplicate samples were collected from the same patient before application of the dressing (time zero). After seven days of treatment at dressing removal, two more duplicates were taken, one from the healed area and another from an unhealed zone. After 10 more days of treatment with a new dressing, another duplicate sample was taken from Int. J. Mol. Sci. 2013, 14 4834 the newly healed area. For each sample, the first duplicate was preserved in formalin and then divided vertically into two portions: one was used directly for histological analysis after hematoxylin and eosin staining, while the other part was transferred into a 2.5% glutaraldehyde solution and, subsequently, prepared for analysis by transmission electron microscopy (TEM). The second duplicate was immediately frozen and then used for the environmental scanning electron microscopy (ESEM) and chemical analysis, as reported in Section 3.4. Figure 10. Flow chart of the experimental plan adopted for the in vivo study. Figure 10. Flow chart of the experimental plan adopted for the in vivo study. g p p p y 3.3.2. Optical Microscopy, TEM and ESEM Time zero Application of the dressing 7 days treatment Healed Unhealed  Application of new dressing 10 days treatment Healed Patient 2nd duplicate 1st duplicate OM and TEM Histochemistry  SEM and Ag levels Patient Histochemistry 1st duplicate Healed 3.3.2. Optical Microscopy, TEM and ESEM The samples preserved in formalin were used for histological analyses to evaluate the tissue structure during the course of the healing process. They were then paraffin-embedded, stained with hematoxylin and eosin and observed using optical microscopy (OM). 3.4. Determination of Ag Levels by ICP-MS The concentration of Ag in all samples (dressing, culture medium, cell culture extracts and in vivo collected skin) were determined by inductively coupled plasma-quadrupole-mass spectrometry (ICP-QMS) using a model 7500cx instrument from Agilent Technologies (Tokyo, Japan). The main instrumental parameters are reported in Table 3, whilst sample preparation is briefly outlined below. All reagents were purchased from Sigma-Aldrich, Milan, Italy. Table 3. Inductively coupled plasma-quadrupole-mass spectrometry (ICP-QMS) operating conditions. RF power 1500 W Plasma gas flow rate 15 L min−1 Carrier gas flow rate 1.2 mL min−1 Nebulizer V-groove Ions lens setting Optimized daily for best sensitivity using a 10 ng mL−1 Ag in NH4OH 2.8% w/w solution Monitored masses 107, 109 Points per peak 3 Acquisition time per point 1 s Replicates 5 New (unused) and residual dressings (collected after the in vitro application) were mineralized by acid digestion in an Ethos1 (Milestone) microwave oven. The digestion was carried out in Teflon vessels following a two step program by adding to ~2 mg of the samples 10 mL of concentrated HNO3 (1st step at 200 °C, 50 min) and then 2 mL H2O2 30% w/w (2nd step at 200 °C, 10 min). The digests of the residual dressings were centrifuged at 3000 rpm for 10 min to separate the AgCl precipitate formed, due to the presence of Cl− in the cDMEM. The supernatant was collected and directly diluted in NH4OH 2.8% w/w, while the precipitate was dissolved in 1.5 mL of concentrated NH4OH (28% w/w) and then diluted in NH4OH 2.8% w/w for ICP-QMS analysis. The Ag concentration was also determined in cDMEM (~400 µL) collected from the cell culture (both untreated and treated with Ag NPs) used for MTT and histological analyses. The solutions were directly diluted in NH4OH 2.8%, tetramethylammonium hydroxide (TMAH) 0.1% and Triton-X 0.1% w/w and analyzed for total Ag by ICP-QMS, as above. The concentration of Ag in the cell culture (and the scaffold) was indirectly estimated by the difference between the level in the new dressing and the combined levels found in the residual dressing and in the medium. To validate the estimates, they were compared with the direct measurement of the Ag concentration in the cell cultures (plus scaffold) previously used for the MTT assay (the only non-destructive analysis). 3.3.1. Human Skin Samples To select the areas with the most altered cellular morphology and, hence, potential Ag localization, a preliminary morphological characterization of the other vertical portion was carried out on semi-thin sections after toluidine blue staining (1% toluidine blue 1% borax for OM). The selected areas were then re-sampled to provide information on the distribution of Ag NPs at the sub-cellular level. This is done by fixing in a 2.5% glutaraldehyde/0.1 M sodium cacodylate buffer overnight at 4 °C. The samples were then treated with 1% OsO4/0.1 M sodium cacodylate buffer and dehydrated using ethanol solutions of increasing concentrations before embedding in EPON™ epoxy resins. Ultrathin sections (ultramicrotome, LKB, Stockholm, Sweden) were obtained and treated with 1% uranyl acetate and 1% lead citrate. The samples were analyzed by TEM (Electronic Microscopy Service, Department of Biology, University of Padova, Padua, Italy) using a Tecnai G12 electron microscope (FEI, acceleration voltage 100 kV). The image acquisition system consisted of a video camera, TIETZ (Tietz Video and Image Processing Systems GmbH, Gauting, Germany), and the TIA FEI Imaging Software (FEI Company, Hillsboro, OR, USA). The ESEM analysis was carried out at the Interdepartmental Service Center C.U.G.A.S. (University of Padova) using a Quanta 200 (FEI company, Hillsboro, OR, USA) instrument equipped with an energy dispersive X-ray (EDAX Inc; Mahwah, NJ, USA) detector. Samples were dried in a thermostatted oven at 37 °C for 24 h prior to analysis. The samples were analyzed directly after placing on a vitreous carbon planchet, without metallization. Backscattered electron images were collected at 25 kV. Representative energy-dispersive X-ray spectroscopy (EDS) spectra were collected Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2013, 14 4835 in potentially interesting sites to obtain a qualitative indication of the elemental composition of the surface and particles identified in the samples. in potentially interesting sites to obtain a qualitative indication of the elemental composition of the surface and particles identified in the samples. 3.4. Determination of Ag Levels by ICP-MS The Ag concentration in the cell culture and underlying scaffold was obtained as the sum of the concentrations measured in the fraction collected after MTT incubation and in the reunified iDMSO fractions after absorbance measurement. The iDMSO fraction was treated with Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2013, 14 4836 1.5 mL of NH4OH 28% w/w to solubilize any Ag attached to the scaffold; the sample was then centrifuged at 3000 rpm for 10 min. The supernatant was diluted to a final volume of 15 mL in TMAH 0.1% and Triton-X 0.1%. The MTT fraction was first diluted (1 + 14) in a solution containing NH4OH 2.8% TMAH 0.1% and Triton-X 0.1%. This solution was then used to dilute both fractions (200 times) prior to ICP-QMS analysis. A parallel control experiment at the 3 day time point in the absence of cells was carried out to quantify the partitioning of silver between the fractions described above and the scaffold. In this experiment, after extraction with iDMSO, 500 µL of concentrated ammonium hydroxide solution (28%, as sold by Sigma Aldrich) was added to the vial containing the collagen-based matrix and the iDMSO solution. To ensure that all the silver attached to the surface of the scaffold was removed, the vial was vigorously shaken for 1 min at room temperature. The solution was removed by pipette, and the collagen-based matrix was washed twice with 500 µL concentrated ammonium hydroxide solution to recover the amount of surface bound Ag from the scaffold and to solubilize any insoluble silver forms present (in particular, AgCl or silver bound to insoluble proteins, as reported in Rigo et al. [47]). All the collected fractions were reunified within the iDMSO fraction and analyzed by ICP-MS. The remaining scaffold was mineralized in 500 µL of concentrated TMAH (25% w/v in water) and was then analyzed by ICP-MS to quantify the amount of Ag trapped in the scaffold. Once the ESEM-EDS analysis was completed, the biopsies collected in vivo were cut into transverse sections of approximately 2 mg weight. Concentrated TMAH solution (100 µL) was added to each section, and alkaline digestion was carried out overnight in a thermostatic bath at 60 °C. The digests were diluted 200-fold in 0.1% Triton X-100 and NH4OH 2.8% w/w solution and analyzed for total Ag concentration by ICP-QMS, as reported above. 4. Conclusions In this pilot study, in vitro results indicate the safety of Acticoat™ Flex 3. It seems that Ag NPs may reduce mitochondrial functionality, but this probably occurs temporarily. As indicated by our results, the reduction in mitochondrial activity does not affect cell viability. The in vivo study, although limited in scope, since it was conducted on a single patient, by taking a biopsy from an unhealed area and a healed area of the same wound, seems to support the absence of toxicity: in the healed skin, no signs of apoptosis or necrosis were observed, despite the presence of a great quantity of Ag NPs in the cytoplasm of the fibroblasts. We observed that a Ag NP-based dressing does not create an obstacle to the recovery of severe partial thickness burns. After application for an extended period (17 days), the organized skin structure (dermis and epidermis) was re-established in a previously unhealed part of the wound. In this pilot study, we have also demonstrated that the application of Ag NP-based dressings, even for a prolonged time, does not seem to negatively affect the proliferation of fibroblasts and keratinocytes, leading to the restoration of normal skin. References 1. Klasen, H.J. Historical review of the use of silver in the treatment of burns. I. Early uses. Burns 2000, 26, 117–130. 1. Klasen, H.J. Historical review of the use of silver in the treatment of burns. I. Early uses. Burns 2000, 26, 117–130. 2. Klasen, H.J. A historical review of the use of silver in the treatment of burns. II. Renewed interest for silver. Burns 2000, 26, 131–138. 2. Klasen, H.J. A historical review of the use of silver in the treatment of burns. II. Renewed interest for silver. Burns 2000, 26, 131–138. 3. Hussain, S.; Ferguson, C. 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References Early healing events in a porcine model of contaminated wounds: Effects of nanocrystalline silver on matrix metalloproteinases, cell apoptosis, and healing. Wound Repair Regen. 2002, 10, 141–151. 41. Huang, Y.; Li, X.; Liao, Z.; Zhang, G.; Liu, Q.; Tang, J.; Peng, Y.; Liu, X.; Luo, Q. A randomized comparative trial between Acticoat and SD-Ag in the treatment of residual burn wounds, including safety analysis. Burns 2007, 33, 161–166. 42. Tonello, C.; Zavan, B.; Cortivo, R.; Brun, P.; Panfilo, S.; Abatangelo, G. In vitro reconstruction of human dermal equivalent enriched with endothelial cells. Biomaterials 2003, 24, 1205–1211. 43. Tonello, C.; Vindigni, V.; Zavan, B.; Abatangelo, S.; Abatangelo, G.; Brun, P.; Cortivo, R. In vitro reconstruction of an endothelialized skin substitute provided with a microcapillary network using biopolymer scaffolds. FASEB J. 2005, 19, 1546–1548. 44. Rigo, C.; Roman, M.; Munivrana, I.; Vindigni, V.; Azzena, B.; Barbante, C.; Cairns, W.R.L. 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Methods 1986, 89, 271–277. © 2013 by the authors; licensee MDPI, Basel, Switzerland. References This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). © 2013 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 license (http://creativecommons.org/licenses/by/3.0/). © 2013 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 license (http://creativecommons.org/licenses/by/3.0/).
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Optimisation of medications used in residential aged care facilities: a systematic review and meta-analysis of randomised controlled trials
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Almutairi et al. BMC Geriatrics (2020) 20:236 https://doi.org/10.1186/s12877-020-01634-4 Almutairi et al. BMC Geriatrics (2020) 20:236 https://doi.org/10.1186/s12877-020-01634-4 Open Access Optimisation of medications used in residential aged care facilities: a systematic review and meta-analysis of randomised controlled trials Hend Almutairi1* , Andrew Stafford1, Christopher Etherton-Beer2 and Leon Flicker3 © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: Frail older adults living in residential aged care facilities (RACFs) usually experience comorbidities and are frequently prescribed multiple medications. This increases the potential risk of inappropriate prescribing and its negative consequences. Thus, optimising prescribed medications in RACFs is a challenge for healthcare providers. Objective: Our aim was to systematically review interventions that increase the appropriateness of medications used in RACFs and the outcomes of these interventions. Methods: Systematic review and meta-analysis of randomised control trials (RCTs) and cluster randomised control trials (cRCTs) were performed by searching specified databases (MEDLINE, PubMed, Google scholar, PsycINFO) for publications from inception to May 2019 based on defined inclusion criteria. Data were extracted, study quality was assessed and statistically analysed using RevMan v5.3. Medication appropriateness, hospital admissions, mortality, falls, quality of life (QoL), Behavioural and Psychological Symptoms of Dementia (BPSD), adverse drug events (ADEs) and cognitive function could be meta-analysed. Results: A total of 25 RCTs and cRCTs comprising 19,576 participants met the inclusion criteria. The studies tested various interventions including medication review (n = 13), staff education (n = 9), multi-disciplinary case conferencing (n = 4) and computerised clinical decision support systems (n = 2). There was an effect of interventions on medication appropriateness (RR 0.71; 95% confidence interval (CI): 0.60,0.84) (10 studies), and on medication appropriateness scales (standardised mean difference = −0.67; 95% CI: −0.97, −0.36) (2 studies). There were no apparent effects on hospital admission (RR 1.00; 95% CI: 0.93, 1.06), mortality (RR 0.98; 95% CI: 0.86, 1.11), falls (RR 1.06; 95% CI: 0.89,1.26), ADEs (RR 1.04; 95% CI: 0.96,1.13), QoL (standardised mean difference = 0.16; 95% CI:-0.13, 0.45), cognitive function (weighted mean difference = 0.69; 95% CI: −1.25, 2.64) and BPSD (RR 0.68; 95% CI: 0.44, 1.06) (2 studies). Conclusion: Modest improvements in medication appropriateness were observed in the studies included in this systematic review. However, the effect on clinical measures was limited to drive strong conclusions. edication optimisation, Intervention, Elderly, Systematic review, Aged care facility, Clinical outcomes Keywords: Medication optimisation, Intervention, Elderly, Systematic review, Aged care facility, Clinical outcomes, Meta-analysis * Correspondence: phhend@hotmail.com 1Medical school, University of Western Australia, Perth, Australia Full list of author information is available at the end of the article Correspondence: phhend@hotmail.com 1Medical school, University of Western Australia, Perth, Australia Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 19 Almutairi et al. BMC Geriatrics (2020) 20:236 Table 1 Study characteristics of the included studies Study ID Author Year Country Design Setting Sample size Age Intervention Follow- up Outcomes Summary of results 1 Avorn et al. [14] 1992 USA Cluster RCT Nursing homes NHs = 12 NHR = 823 65 and older - physicians who is there prescribing of psychoactive drugs was above threshold at the baseline evaluation were invited by pharmacists for separate sessions (3 interactive visits). - All physicians of NHR received 6 literature summaries (insomnia, behavioural problems) in 3 mailings. - 4 training sessions were delivered to nurses/ nursing assistants on geriatric pharma psychology, alternatives to psychoactive drugs 5 months -psychoactive drug use scores -proportion of residents using antipsychotics. -psychoactive drug use mean in intervention 27% compared to 80% in control group (P = 0.02). -antipsychotics ceased in intervention 32% versus 14% in control. - no of days/patient/ month greatly reduced in intervention than control. -no of non-recommended hypnotics ceased and substituted with alternative drugs/discontinued completely were 45% in intervention versus 21% control. 2 Rovner et al. [15] 1996 USA RCT Nursing homes 1 NH (250 bed community NH) NHR = 89 65 and older -As ap art of a dementia care program: implementation of new prescribing guidelines based on protocol for psychotropic drug management -Educational rounds weekly for 1 h to discuss patient’s behavioural disorders, and medical status 6 months - antipsychotic drug - behaviour disorders -restraint use, - and cognitive/ functional status. -Statistically significant 71% reduction in agitation with intervention versus 49% with control 3 Meador et al. [16] 1997 USA RCT Nursing homes NHs = 12 NHR = 1311 65 and older -geropsychiatrist delivered educational visit to physicians (45-60 min) -NH staff received 5–6 1-h programmes over 1 week delivered by nurse educator. -after 1 month, follow up sessions - when requested, evening meetings for families. 6 months -Proportion of APs drug use in days/ 100 /days of stay. -severity and presence of behavioural symptoms. -APs use per 100 days at baseline in intervention gp decreased from 25.3 days to 19.7 per 100. -Aps reduction is 23% in intervention gp to control gp. −33% NHR in intervention gp had their antipsychotics ceased. 4 Schmidt et al. 1&2 [17, 18] 1998 Sweden Cluster RCT Nursing homes NH = 33 NHR = 1854 65 and older -Monthly multidisciplinary meetings led by pharmacist for 12 months 12 months -Proportion of residents with psychotropics -non recommended hypnotics, antidepressants. −19% of residents in the intervention gp ceased APs (p = 0.007). −37% of residents ceased non- recommended hypnotics in intervention gp (p < 0.001). Schmidt et al. [19] 2000 Sweden Cluster RCT Nursing homes NH = 36 NHR = 1549 65 and older Nursing homes participated in 1995 were followed up. three- year follow- up Medication appropriateness -proportion of residents prescribed non- recommended hypnotics were lower (14.0%) compared to previous study 1995 (19.0%). - in1998 5% of residents were prescribed non recommended hypnotics compared to control gp (10.1%). Claesson et al. [20] 1998 Sweden Cluster- RCT Nursing homes NH = 33 NHR = 1854 65 and older - regular multidisciplinary meetings (physician, pharmacist, NH nurses/ assistant) reviewed resident’s drug use on a monthly basis over 12 months. 14 months Medication- related problems -NH residents were prescribed on average 7.7o (range: 6–11) medications. - laxatives (70%) Page 3 of 19 Almutairi et al. BMC Geriatrics (2020) 20:236 Table 1 Study characteristics of the included studies (Continued) Study ID Author Year Country Design Setting Sample size Age Intervention Follow- up Outcomes Summary of results -education for selected pharmacists (5 occasions = 65.5 h), topics were drug use in elderly, geriatrics. -psychotropic drugs (77%, range: 50– 95%). 5 Furniss et al. [21] 2000 England. Cluster RCT Nursing homes NH = 14 NHR = 330 residents: (172 ctrl, 158 Int) 65 and older -Medication review led by pharmacist. - pharmacist review the medications at NH, GPs surgery, or over phone. -Pharmacist collected details of current medication, medical history and current problem identified by nursing home staff. - 3 weeks post-medicine review, NH were revisited to identify any prob- lems and to ascertain on whether changes had been implemented. 8 months -no of prescribed medications -Types of medications, reason for using neuroleptic medications. -hospital admission (in- patient days) -MMSE -GDS -BASDEC -CRBRS -Falls and death - 239 of recommendations accepted by GP (91.6%). -change of medications =144 -In total MMSE were declined. - Mean CRBRS scores increased in Int compared to ctrl - deaths in ctrl were higher than Int NHs. 6 Stein et al. [22] 2001 USA Cluster RCT Nursing homes NH = 20 NHR = 147 65 and older -Staff training sessions (30 min) -Study physician visited/telephone to all primary care physicians -physicians received messages about NSAIDs risks and benefits , algorithm for stopping NSAIDs, or aternatives such as paracetamol or topical agents and non-pharmacological management for pain. 3 months NSAIDs and paracetamol Use in the past week -Mean number of days of NSAIDs use deceased in Int gp from 7.0–1.9 days compared to ctrl gp (7.0–6.2 days), P = 0.0001 - paracetamol use in Int gp increased (3.1 days) compared to ctrl (0.31 days), P = 0.0001. 7 Roberts et al. [23] 2001 Australia Cluster RCT Nursing homes NH = 52 NHR = 3230 65 and older -nurse education (6–9 problem-based education sessions) including geriatric medications and common problems in long care such as depression & pain. -supported by bulletins, wall charts and clinical pharmacist visits. - clinical pharmacist average contact 26 h/NH -clinical pharmacist reviewed drug regimen for 500 residents selected by home staff. 12 months -Mortality rate -hospital admission -Drug use -ADEs -Medication- related problems -mean no of psycholeptics administered /resident in Int gp decreased (−0.14, 95% CI −0.28-0.0, p = 0.044) - in the intervention group mean number of benzodiazepines Administered/ resident reduced (−0.06, 95% CI −0.06 to 0.04, p = 0.29). 8 Crotty et al. (a) [24] 2004 Australia Cluster RCT Aged care facility NH = 10 NHR = 154 65 and older −2 multidisciplinary case conferences were conducted 6–12 weeks. -pharmacists, geriatrician, residential care staff, GP, and a representative of the Alzheimer’s Association of South Australia. -medication review prepared by the 7 months -MAI score -Mean MAI score in Int gp 4.1 (2.1–6.1) versus 0.4 (0.4–1.2) in ctrl gp. - benzodiazepines: mean MAI score in int.gp 0.73 (0.16–1.30) versus −0.38 (−1.02 to 0.27) in ctrl gp. Falls and death 6 Stein et al. [22] 2001 USA Cluster RCT Nursing homes NH = 20 NHR = 147 65 and older -Staff training sessions (30 min) -Study physician visited/telephone to all primary care physicians -physicians received messages about NSAIDs risks and benefits , algorithm for stopping NSAIDs, or aternatives such as paracetamol or topical agents and non-pharmacological management for pain. 3 months NSAIDs and paracetamol Use in the past week -Mean number of days of NSAIDs use deceased in Int gp from 7.0–1.9 days compared to ctrl gp (7.0–6.2 days), P = 0.0001 - paracetamol use in Int gp increased (3.1 days) compared to ctrl (0.31 days), P = 0.0001. 7 Roberts et al. [23] 2001 Australia Cluster RCT Nursing homes NH = 52 NHR = 3230 65 and older -nurse education (6–9 problem-based education sessions) including geriatric medications and common problems in long care such as depression & pain. -supported by bulletins, wall charts and clinical pharmacist visits. - clinical pharmacist average contact 26 h/NH -clinical pharmacist reviewed drug regimen for 500 residents selected by home staff. 12 months -Mortality rate -hospital admission -Drug use -ADEs -Medication- related problems -mean no of psycholeptics administered /resident in Int gp decreased (−0.14, 95% CI −0.28-0.0, p = 0.044) - in the intervention group mean number of benzodiazepines Administered/ resident reduced (−0.06, 95% CI −0.06 to 0.04, p = 0.29). 8 Crotty et al. (a) [24] 2004 Australia Cluster RCT Aged care facility NH = 10 NHR = 154 65 and older −2 multidisciplinary case conferences were conducted 6–12 weeks. -pharmacists, geriatrician, residential care staff, GP, and a representative of the Alzheimer’s Association of South Australia. -medication review prepared by the 7 months -MAI score -Mean MAI score in Int gp 4.1 (2.1–6.1) versus 0.4 (0.4–1.2) in ctrl gp. - benzodiazepines: mean MAI score in int.gp 0.73 (0.16–1.30) versus −0.38 (−1.02 to 0.27) in ctrl gp. Page 4 of 19 Almutairi et al. BMC Geriatrics (2020) 20:236 Table 1 Study characteristics of the included studies (Continued) Study ID Author Year Country Design Setting Sample size Age Intervention Follow- up Outcomes Summary of results resident’s GP before case conference. 9 Crotty et al. (b) [25] 2004 Australia RCT long-term care facility/ hospital discharge NH = 85 NHR = 110 Discharged from 3 hospitals 65 and older -pharmacist transition coordinator transfers the medication-related infor- mation to the family physician and community pharmacist. -case conference at facility within month of transfer include pharmacist, nurse, family physician, community pharmacist, 8 weeks -MAI score -Hospital admission -Medication related problems -ADEs, -falls -No change in MAI score in Int gp 2.5, 95% CI1.4–3.7) -In ctrl gp MAI score had worsened 6.5, 95%CI 3.9–9.1) 10 Crotty et al. (c) [26] 2004 Australia RCT Residential care facilities NH = 20 NHR = 715 65 and older -Educational intervention: two (30 min) outreach visits of pharmacists to doctors. - presenting detailed audit information on psychotropic use, stroke risk reduction, and fall rates. −4 (2 h training sessions) for link nurse in each facility. 7 months -MAI score -Hospital admissions - MRP -No significant difference in psychotropic drug use before &after intervention (0.89,95%CI 0.69–1.15). -PRN of antipsychotics drug use increased in Int gp compared to ctrl gp (4.95,95%CI 1.69–14.50). - No significant difference in BZD drug use before & after intervention (0.89, 95%CI 0.69–1.15). - No significant difference in falls (1.17, 95%CI 0.86–1.58). 11 Fossey et al. [27] 2006 UK Cluster RCT Nursing homes NH = 12 NHR = 349 65 and older -Training and support to care staff on non-pharmacological interventions, al- ternatives to neuroleptic use. -Medication review by Led by old age psychiatrist, senior nurse every 3 months -contact between psychiatrist and prescribers to provide and wrote prescribing recommendations 12 months -Proportion of residents receiving neuroleptics. -CMAI - QoL - reduction in neuroleptic use/resident (19.1, 95% CI 0.5–37.7%, P = 0.045) --Neuroleptic use decrease 24% in exp. (47 to 23%) but increased in ctrl 7.6% (49.7 to 42.1%). -No significant changes in CMAI 12 Zermansky et al. [28] 2006 UK RCT Nursing homes and residential homes NH = 65 NHR = 661 65 and older - Pharmacist medication review by using the resident’s medical record. - consultation with the resident’s and carer. -pharmacist forward written recommendations to GP. 6 months -no. of changes in medication/ patient -Hospital admissions -Medication- related problems -Medicine costs -Number of medicines per participant - Mortality - Falls - SMMSE -Barthel index -GP consultations - Increase in mean number of drug changes/patient ctrl: 2.4 versus 3.1 in Int (P < 0.01) -no of falls reduced significantly - pharmacist recommendations accepted (75.6%), and 76.6% of these recommendations were implemented. 13 Gurwitz 2008 USA Cluster Two large Facility = 2 65 -Computer program (order entry with 1 year in -Number of -None Table 1 Study characteristics of the included studies (Continued) Study ID Author Year Country Design Setting Sample size Age Intervention Follow- up Outcomes Summary of results resident’s GP before case conference. 9 Crotty et al. (b) [25] 2004 Australia RCT long-term care facility/ hospital discharge NH = 85 NHR = 110 Discharged from 3 hospitals 65 and older -pharmacist transition coordinator transfers the medication-related infor- mation to the family physician and community pharmacist. -case conference at facility within month of transfer include pharmacist, nurse, family physician, community pharmacist, 8 weeks -MAI score -Hospital admission -Medication related problems -ADEs, -falls -No change in MAI score in Int gp 2.5, 95% CI1.4–3.7) -In ctrl gp MAI score had worsened 6.5, 95%CI 3.9–9.1) 10 Crotty et al. (c) [26] 2004 Australia RCT Residential care facilities NH = 20 NHR = 715 65 and older -Educational intervention: two (30 min) outreach visits of pharmacists to doctors. - presenting detailed audit information on psychotropic use, stroke risk reduction, and fall rates. −4 (2 h training sessions) for link nurse in each facility. 7 months -MAI score -Hospital admissions - MRP -No significant difference in psychotropic drug use before &after intervention (0.89,95%CI 0.69–1.15). -PRN of antipsychotics drug use increased in Int gp compared to ctrl gp (4.95,95%CI 1.69–14.50). - No significant difference in BZD drug use before & after intervention (0.89, 95%CI 0.69–1.15). - No significant difference in falls (1.17, 95%CI 0 86 1 58) 95%CI 0.86–1.58). 11 Fossey et al. [27] 2006 UK Cluster RCT Nursing homes NH = 12 NHR = 349 65 and older -Training and support to care staff on non-pharmacological interventions, al- ternatives to neuroleptic use. -Medication review by Led by old age psychiatrist, senior nurse every 3 months -contact between psychiatrist and prescribers to provide and wrote prescribing recommendations 12 months -Proportion of residents receiving neuroleptics. -CMAI - QoL - reduction in neuroleptic use/resident (19.1, 95% CI 0.5–37.7%, P = 0.045) --Neuroleptic use decrease 24% in exp. (47 to 23%) but increased in ctrl 7.6% (49.7 to 42.1%). -No significant changes in CMAI 12 Zermansky et al. [28] 2006 UK RCT Nursing homes and residential homes NH = 65 NHR = 661 65 and older - Pharmacist medication review by using the resident’s medical record. - consultation with the resident’s and carer. -pharmacist forward written recommendations to GP. 6 months -no. of changes in medication/ patient -Hospital admissions -Medication- related problems -Medicine costs -Number of medicines per participant - Mortality - Falls - SMMSE -Barthel index -GP consultations - Increase in mean number of drug changes/patient ctrl: 2.4 versus 3.1 in Int (P < 0.01) -no of falls reduced significantly - pharmacist recommendations accepted (75.6%), and 76.6% of these recommendations were implemented. 13 Gurwitz 2008 USA Cluster Two large Facility = 2 65 -Computer program (order entry with 1 year in -Number of -None Page 5 of 19 Almutairi et al. BMC Geriatrics (2020) 20:236 Table 1 Study characteristics of the included studies (Continued) Study ID Author Year Country Design Setting Sample size Age Intervention Follow- up Outcomes Summary of results et al. [29] and Canada RCT long-term care facilities. Residents = 1118 and older clinical decision support system). - more than 600 potentially serious drug-drug interac- tions alerts were reviewed. -no of ADEs were identified (preventable events including errors and drug-drug interactions were determined). -alerts included in the CDSSs were assessed to determine if any of them could have prevented the prescribing of these drugs. one facility and 6 months in the other preventable ADEs - ADEs severity - ADEs preventability ADEs = (1.06,95% CI 0.92–1.23) Preventable ADEs = (1.02,95% CI 0.81–1.30) 14 Field et al. [30] 2009 Canada Cluster RCT long-term care facility -One long- term care Facility - 22 long- stay units Residents= 833 65 and older The 22 long-stay units were randomly assigned - for Intervention units’ prescriber: Alerts related to medication prescribing for residents with renal insufficiency were displayed. -Control units: Alerts hidden and tracked - The types alerts were: maximum recommended daily dose/frequency of administration, medication to be avoided, and missing information. 12 months -Proportion of final drug orders alert that were appropriate -Appropriate final drug orders proportion were high in Int (1.2, 95% CI 1.0–1.4) for frequency. -for drugs that should be avoided (2.6, 95% CI 1.4–5.0). for missing information (1.8, 95% CI 1.1 to 3.4). -Appropriate final drug orders Significant in Int (1.2 95% CI 1.0–1.4). 15 Patterson et al. [31] 2010 Ireland Cluster RCT Nursing homes NH = 11 NHR = 334 65 and older -intervention homes were visited monthly by trained pharmacists for 1 year. Resident’s information was collected from records, GP and community pharmacist. Interviews were conducted with the residents and next of kin to assess the need for medicines. - applied an algorithm to assess appropriateness of psychoactive medication and worked with GPs to improve the prescribing of these medications. Monthly for 12 months -Proportion of residents prescribed inappropriate psychoactive medications. -no of falls - At 12 months, residents taking inappropriate psychoactive medications in Int gp (19.5%) decreased compared to ctrl gp (50%) intervention homes (0.26, 95% CI 0.14–0.49) -No change the falls rate 16 Testad et al. [32] 2010 Norway Cluster RCT Nursing homes NH = 4 NHR = 211 65 and older -Education and training program (2 days seminar and monthly group guidance for six months). 12 months -% of residents using antipsychotic drugs - Restraint use -No statically significant difference in antipsychotic use. - Significant reduction in Aggression in Int gp at 6 & 12 month follow-up. -Significant reduction in proportion of residents Page 6 of 19 Page 6 of 19 Almutairi et al. BMC Geriatrics (2020) 20:236 Table 1 Study characteristics of the included studies (Continued) Study ID Author Year Country Design Setting Sample size Age Intervention Follow- up Outcomes Summary of results 17 Lapane et al. [33] 2011 United States Cluster RCT Nursing homes NH = 25 NHR = 3321 65 and older - GRAM is automatically generated to assist consultant pharmacists identify residents at risk for delirium/ falls -Detailed instruction of consultant pharmacists providing targeted medication review for all residents at high-risk. - Reports within 24 h of admission and used during monthly review. 12 months −Mortality −Hospital admission potentially due to ADEs. -Mortality rate /1000 resident-months, HR: 0.90 (adjusted HR 0.89, 95% CI 0.73– 1.08) -Hospital admission/1000 resident-months, HR: 1.13 (adjusted HR 1.11, 95% CI 0.94– 1.31). 18 Pope et al. [34] 2011 UK, Ireland RCT Nurse- managed continuing- care NHR = 10 nurse- managed continuing- care Residents = 225 65 and older -medical assessment by a geriatrician, and using Beer’s criteria for multidisciplinary panel medication review. - recommendations forwarded to the GP. - after 6 months, reassessment occurred 6 months -no of drugs prescribed -mortality -medication cost −92.7% of patients received medication recommendations and 80.1% accepted. - total number of medications/ patient/d reduced in Int gp (11.64–11.09 compared to ctrl 11.07–11.5). 19 Kersten et al. [35] 2013 Norway RCT Nursing homes NH = 22 NHR = 87 65 and older -A paper-based review with a view to reduce ADS scores were conducted by clinical pharmacist. -clinical pharmacist discuss discontinue or replace an anticholinergic drug with the physician before changes were implemented. 8 weeks - Cognitive function - anti-cholinergic side-effects - cognitive function not improved - anti-cholinergic side-effects not improved 20 Milos et al. [36] 2013 Switzerland RCT Nursing homes or community NHR = 279 75 years or older Pharmacists-led medication review that included assessment of relevant parts of (EMRs) and collection of patient’s blood sample data. - clinical pharmacist-initiated medication reviews based on the background information to identify DRPs. 2 months - no of PIMs. - DRPs −6% decreased in PIM in Int gp -Total no of DRPs in the intervention group was 431 [mean 2.5 (1.5) / patient (range 0–9) - No significant difference between the no of DRPs in nursing home patients [mean 2.53 (1.33)] and community- dwelling patients [mean 2.55 (1.29)] Significant in changes in the actions taken by the physician were for lowered dosage. 21 Frankenthal et al. -HR QoL decreased in Int gp (−0.038, 95% CI −0.054 to −0.022) compared to ctrl gp (−0.072,95% CI −0.089 to − 0.055) (P = .005). -hospital admission decreased significantly in int gp (1.4 days/person/ year, 95% CI 1.2 to −1.6) compared to Table 1 Study characteristics of the included studies (Continued) Study ID Author Year Country Design Setting Sample size Age Intervention Follow- up Outcomes Summary of results START workshop, and adverse drug reactions in older people. -participants also received educational material and references - on-demand support (via phone) for 6 months provided by the educator. admissions Medication appropriateness (STOPP-START) -Falls 19.3–28% and not significantly change in the intervention group from 25.3– 23.9%. 23 Pitkala et al. [39] 2014 Finland Cluster- RCT Assisted living facilities Facility = 20 Residents = 227 65 and older -two 4-h interactive training sessions for nursing staff aimed to enable nurses to recognize potentially harmful medica- tions and corresponding adverse drug events. -the second 4-h sessions: case-study- based. - nurses in this intervention were asked to identify potential MDR and highlight these to the consulting doctor. 12 months -Medication appropriateness -Hospital admissions -Mortality -QoL -MMSE -mean number of potentially harmful drugs lowered in int gp (−0.43, 95% CI- 0.71 to −0.15) and not changed in ctrl gp (+ 0.11, 95% CI −0.09 to + 0.31) (P = .004). -HR QoL decreased in Int gp (−0.038, 95% CI −0.054 to −0.022) compared to ctrl gp (−0.072,95% CI −0.089 to − 0.055) (P = .005). -hospital admission decreased significantly in int gp (1.4 days/person/ year, 95% CI 1.2 to −1.6) compared to ctrl gp (2.3 days/person/year; 95% CI 2.1to −2.7), RR = 0.60, 95% CI 0.49 to − 0.75, P < .001). 24 Connolly et al. [40] 2015 New Zealand Cluster- RCT RACFs NH = 36 NHR = 1998 65 and older - Gerontology nurse specialist delivered staff education and clinical coaching. - benchmarking of resident indicators including restraint use, falls, etc.). - multidisciplinary team meeting (1 h) monthly for the first 3 months. 14 months - Hospital admissions (ambulatory sensitive hospitalisations, total acute admissions). -Mortality -no differences between Int and ctrl gp in rates of ambulatory sensitive hospitalisations admission (1.07; 95% CI 0.85–1.36; P = 0.59). -no difference in mortality (1.11; 95% CI 0.76–1.61; P = 0.62). 25 Potter et al. [41] 2016 Australia RCT RACFS Facility = 4 Residents = 95 65 and older -medication review followed by discontinuing non-beneficial medica- tions conducted by a GP and a geriatri- cian/clinical Pharmacologist - During deprescribing, the GP reviewed participants weekly. 12 months -no of falls -mortality -no of fallers -cognitive function -QoL -mortality 26% in int gp and 40% in ctrl gp (HR 0.60, 95%CI 0.30 to 1.22). -QoL Changes in Int gp (−1.0 ± 4.3) compared to ctrl gp (−1.0 ± 4.7). -Falls -Patients with one or more falls in int gp (0.56, 95% CI 0.42–0.69) compared to ctrl gp(0.65, 95% CI 0.50– 0.77), (p = 0.40) Abbreviations: RCT Randomised Controlled Trials, NHR Nursing Home Residents, NHs Nursing Homes, CDSSs Computerised Clinical Decision Support Systems, CI Confidence Interval, GP General Practitioner, no number, min minutes, hr. hour, APs Antipsychotics, gp group, ctrl control, Int intervention, MMSE Mini-Mental State Exam, GDS Geriatric Depression Scale, BASDEC Brief Assessment Schedule Depression Cards, CRBRS Crichton- Royal Behaviour Rating Scale, P, p value, NSAIDs Non-Steroidal Anti-Inflammatory Drugs, ADEs Adverse Drug Events, MAI Medication Appropriateness Index, MRP Medication-related Problem, PRN pro re nata (when necessary), BZD Benzodiazepine, CMAI Cohen-Mansfield Agitation Inventory, QoL Quality of Life, exp experiment group, % percentage, GRAM Geriatric Risk Assessment Med Guide, HR Hazard Ratio, ADS Anticholinergic Drug Scale, EMRs electronic medical records, PIMs Potential Inappropriate medications, PPOs potential prescription omissions, STOPP/START Screening Tool of Older Person’s potentially inappropriate Prescriptions and Screening Tool to Alert doctors to Right Treatment, FIM Functional Independence Measure based. - nurses in this intervention were asked to identify potential MDR and highlight these to the consulting doctor. -MMSE 95% CI −0.054 to −0.022) compared to ctrl gp (−0.072,95% CI −0.089 to − 0.055) (P = .005). -hospital admission decreased significantly in int gp (1.4 days/person/ year, 95% CI 1.2 to −1.6) compared to ctrl gp (2.3 days/person/year; 95% CI 2.1to −2.7), RR = 0.60, 95% CI 0.49 to − 0.75, P < .001). 24 Connolly et al. [40] 2015 New Zealand Cluster- RCT RACFs NH = 36 NHR = 1998 65 and older - Gerontology nurse specialist delivered staff education and clinical coaching. - benchmarking of resident indicators including restraint use, falls, etc.). - multidisciplinary team meeting (1 h) monthly for the first 3 months. 14 months - Hospital admissions (ambulatory sensitive hospitalisations, total acute admissions). [37] 2014 Israel RCT chronic care geriatric facility NH = 1 NHR = 359 65 and older -medication review conducted by pharmacist -to identify PIMs and PPOs medications screened with STOPP/ START criteria then followed up with recommendations to the chief physician. - chief physician decided to accept or not. 12 Months -medication appropriateness -mortality -hospital admission -QoL -MRP -medication cost -significant decreased in the average number of drugs prescribed in Int gp (P < .001). - significant decreased in the average number of falls in Int gp (P = .006). -decrease in the average drug costs in Int gp by US$29. - hospitalization, FIM scores, and QoL were same in both groups. 22 García- Gollarte, et al. [38] 2014 Spain Cluster- RCT Nursing homes NH = 36 NHR = 716 65 and older −30 doctors received educational intervention. - The educational intervention included general drug use in elderly, STOPP 6 Months - Medication appropriateness (STOPP-START) -Hospital - The mean number of inappropriate drugs was higher in ctrl gp (1.29–1.56) compared to Int gp (0.81–1.13). -no of falls increased in the ctrl gp from Page 7 of 19 Almutairi et al. BMC Geriatrics (2020) 20:236 Table 1 Study characteristics of the included studies (Continued) Study ID Author Year Country Design Setting Sample size Age Intervention Follow- up Outcomes Summary of results START workshop, and adverse drug reactions in older people. -participants also received educational material and references - on-demand support (via phone) for 6 months provided by the educator. admissions Medication appropriateness (STOPP-START) -Falls 19.3–28% and not significantly change in the intervention group from 25.3– 23.9%. 23 Pitkala et al. [39] 2014 Finland Cluster- RCT Assisted living facilities Facility = 20 Residents = 227 65 and older -two 4-h interactive training sessions for nursing staff aimed to enable nurses to recognize potentially harmful medica- tions and corresponding adverse drug events. -the second 4-h sessions: case-study- based. - nurses in this intervention were asked to identify potential MDR and highlight these to the consulting doctor. 12 months -Medication appropriateness -Hospital admissions -Mortality -QoL -MMSE -mean number of potentially harmful drugs lowered in int gp (−0.43, 95% CI- 0.71 to −0.15) and not changed in ctrl gp (+ 0.11, 95% CI −0.09 to + 0.31) (P = .004). -Mortality -no differences between Int and ctrl gp in rates of ambulatory sensitive hospitalisations admission (1.07; 95% CI 0.85–1.36; P = 0.59). -no difference in mortality (1.11; 95% CI 0.76–1.61; P = 0.62). 25 Potter et al. [41] 2016 Australia RCT RACFS Facility = 4 Residents = 95 65 and older -medication review followed by discontinuing non-beneficial medica- tions conducted by a GP and a geriatri- cian/clinical Pharmacologist - During deprescribing, the GP reviewed participants weekly. 12 months -no of falls -mortality -no of fallers -cognitive function -QoL -mortality 26% in int gp and 40% in ctrl gp (HR 0.60, 95%CI 0.30 to 1.22). -QoL Changes in Int gp (−1.0 ± 4.3) compared to ctrl gp (−1.0 ± 4.7). -Falls -Patients with one or more falls in int gp (0.56, 95% CI 0.42–0.69) compared to ctrl gp(0.65, 95% CI 0.50– 0.77), (p = 0.40) Abbreviations: RCT Randomised Controlled Trials, NHR Nursing Home Residents, NHs Nursing Homes, CDSSs Computerised Clinical Decision Support Systems, CI Confidence Interval, GP General Practitioner, no number, min minutes, hr. hour, APs Antipsychotics, gp group, ctrl control, Int intervention, MMSE Mini-Mental State Exam, GDS Geriatric Depression Scale, BASDEC Brief Assessment Schedule Depression Cards, CRBRS Crichton- Royal Behaviour Rating Scale, P, p value, NSAIDs Non-Steroidal Anti-Inflammatory Drugs, ADEs Adverse Drug Events, MAI Medication Appropriateness Index, MRP Medication-related Problem, PRN pro re nata (when necessary), BZD Benzodiazepine, CMAI Cohen-Mansfield Agitation Inventory, QoL Quality of Life, exp experiment group, % percentage, GRAM Geriatric Risk Assessment Med Guide, HR Hazard Ratio, ADS Anticholinergic Drug Scale, EMRs electronic medical records, PIMs Potential Inappropriate medications, PPOs potential prescription omissions, STOPP/START Screening Tool of Older Person’s potentially inappropriate Prescriptions and Screening Tool to Alert doctors to Right Treatment, FIM Functional Independence Measure viations: RCT Randomised Controlled Trials, NHR Nursing Home Residents, NHs Nursing Homes, CDSSs Computerised Clinical Decision Support Systems, CI Confidence Interval, GP General Practitioner, no number, minutes, hr. Background evidence, and the impact of these interventions on demen- tia patients’ outcomes required more research [10]. Inappropriate medication prescription encompasses mis- prescribing, overprescribing, and underprescribing. Mis- prescribing involves the use of medication that significantly increases the risk of adverse drug events (ADEs) and involves incorrect dose, frequency, adminis- tration and duration. Use of medications that are likely to cause drug-drug interactions or drug-disease interac- tions is also an aspect of misprescribing. Overprescribing involves the use of medications without clear indica- tions. Underprescribing is the omission of clinically- indicated medication that may have potential benefit for treatment of the disease [1]. We therefore aimed to systematically review the avail- able interventions conducted by a health professional that aimed to increase the appropriateness of medica- tions used in residential aged care facilities and to evalu- ate their effects on medication appropriateness and residents’ clinical outcomes. hour, APs Antipsychotics, gp group, ctrl control, Int intervention, MMSE Mini-Mental State Exam, GDS Geriatric Depression Scale, BASDEC Brief Assessment Schedule Depression Cards, CRBRS Crichton- Behaviour Rating Scale, P, p value, NSAIDs Non-Steroidal Anti-Inflammatory Drugs, ADEs Adverse Drug Events, MAI Medication Appropriateness Index, MRP Medication-related Problem, PRN pro re nata (when sary), BZD Benzodiazepine, CMAI Cohen-Mansfield Agitation Inventory, QoL Quality of Life, exp experiment group, % percentage, GRAM Geriatric Risk Assessment Med Guide, HR Hazard Ratio, ADS Anticholinergic Scale, EMRs electronic medical records, PIMs Potential Inappropriate medications, PPOs potential prescription omissions, STOPP/START Screening Tool of Older Person’s potentially inappropriate Prescriptions and ning Tool to Alert doctors to Right Treatment, FIM Functional Independence Measure 25 Almutairi et al. BMC Geriatrics (2020) 20:236 Almutairi et al. BMC Geriatrics (2020) 20:236 Page 8 of 19 Data sources and search strategy An electronic search of the literature was conducted from inception to May 2019 using the following data- bases — MEDLINE, PubMed, Google scholar, Psy- cINFO. A combination of the following keywords and MeSH terms were used: “Optimize OR improve OR maximize OR optimization AND medication OR drugs OR medicines AND side effects OR safety OR adminis- tration OR review AND nursing homes OR residential OR aged care”. The reference lists of the relevant articles and reviews were hand-searched to further identify any additional studies. The complete search strategy is pre- sented in Additional file 2. Previous studies indicate that about 40% of prescrip- tions for RACF residents may be suboptimal or inappro- priate [5]. Consequently, there is a heightened risk of adverse drug reactions (ADRs), hospitalisations, and medical expense [3, 6]. Therefore, there is an urgent need to improve prescribing and to optimise drug ther- apy for older people living in care homes [5, 7]. Medication optimisation is a person-centred approach designed to ensure medication safety and improved clin- ical outcomes via effective use of medicine [8, 9]. A range of interventions for optimisation of prescribed medica- tions in RACFs have been developed to potentially opti- mise prescribing. These include medication review, education programs, the use of clinical decision support technology, and multidisciplinary case-conferencing. These interventions have been evaluated to determine the effect of optimising prescribing in nursing homes and in older people with dementia, but the results were not pooled statistically [10–12], and the nursing home specific data require updating. The 2011 review concluded that in nursing homes, educational interventions including aca- demic detailing seems to show most promise [12]. The other 2011 review found that education and pharmacist drug review may reduce inappropriate drug use under cer- tain circumstances [11]. The other 2018 review of 18 ex- perimental studies specific to older people living with dementia in any setting concluded that the improvement of medication appropriateness is supported by emerging Methods This systematic review was conducted and reported in com- pliance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [13]. A PRISMA checklist can be found in Additional file 1. The re- view was registered with the international prospective regis- ter of systematic reviews PROSPERO CRD42020148669. Residents of aged care facilities (RACFs) are often frail and have multiple comorbidities. On average RACF resi- dents take more medications than younger age groups, and more than community-dwelling elderly with similar disease complications [2]. They are frequently prescribed multiple medications that can increase the risk of ADEs, morbidity and mortality [3, 4]. Moreover, the majority of these residents have dementia and the use of psycho- tropic drugs is typically high. Age-related changes in pharmacodynamics and pharmacokinetics, multiple co- morbidities, and the presence of polypharmacy are the main factors often associated with ageing that makes op- timisation of drug therapy a complex task. Furthermore, Study selection Th i l d The title and abstract of all retrieved articles were ini- tially reviewed to find those potentially relevant to the study area. The abstract of the selected papers was assessed against five inclusion criteria: (i) randomised controlled trials (RCTs) and cluster randomised con- trolled trials (cRCTs); (ii) residents’ age 60 and older; settings are residential aged care facility or nursing homes or residential continuing care hospitals; (iii) inter- ventions to increase the appropriateness of medications used in nursing homes (iv) reported in English; (v) pub- lished between 1980 and 2019. Data extraction and quality assessment Data extraction and quality assessment Details of the included articles were independently ex- tracted by two authors (H.A, and L.F). Data included de- tails of the authors, publication year, country, study design, age, setting, sample size, intervention, follow-up, outcomes, and summary of results. Table 1 depicts the study characteristics of the included studies. Almutairi et al. BMC Geriatrics (2020) 20:236 Page 9 of 19 Assessment of risk of Bias The quality of each article and risk of bias were assessed independently by the two reviewers (H.A, and L.F). For assessing risk of bias we used the Cochrane Collabora- tion’s tool [42]. The studies assessed based on standard criteria: adequate sequence generation, allocation con- cealment, blinding of participants and personnel, blind- ing of outcome assessment, incomplete outcome data, selective reporting, and other bias. Risk of bias tables provided in RevMan v5.3 was used to assess reporting Fig. 1 PRISMA flow diagram Fig. 1 PRISMA flow diagram Study design The design consisted of 15 studies [14, 17, 19–24, 27, 29–33, 38–40] comprising five cRCTs and 10 RCTs [15, 16, 25, 26, 28, 34–37, 41]. Interventions Various interventions applied by pharmacist, physician or a multidisciplinary team (physicians, pharmacists, and nurses) in the included studies were evaluated. Methods to review residents’ medications were presented in 12 studies [21, 23, 25, 28, 31, 33–38, 41]; nine studies [14– 16, 22, 26, 27, 32, 39, 40] investigated the impact of staff education, four studies [17, 20, 24, 25, 40] evaluated the implementation of multi-disciplinary case conferencing, and two studies [29, 30] evaluated computerised clinical decision support systems. Other outcomes Hospital admission Eleven studies [21, 23, 25, 28, 33, 34, 37–41] specified hospital admission as an outcome measure. Furniss et al. [21] reported in-patient days as hospital admission. Rob- erts et al. [23] investigated the proportion of hospitalised residents. Crotty et al. [25] reported hospital usage based on unplanned visits to emergency department and hos- pital readmission. Zermansky et al. [28] reported hospi- talisation rate during a 6-month period per resident. Lapane et al. [33] investigated any hospitalisation and potential ADE-related hospitalisation in a randomised cluster trial. Pope et al. [34] reported the number of admissions to acute hospital. Frankenthal et al. [37] reported hospital admissions. Garcia-Gollarte et al. [38] reported the total number of days spent in hos- pital. Pitkala et al. [39] reported hospital days/resi- dent/year. Connolly et al. [40] reported all acute admissions and ambulatory sensitive hospitalisations. Potter et al. [41] reported hospital admission as the Statistical analysis Outcome measures such as medication appropriateness, hospitalisation, mortality and other outcomes were assessed for heterogeneity and were pooled for meta- analysis using fixed effects methods if little heterogeneity was found or using random effects methods if hetero- geneity was present (P value < .05). The software Review Manager (RevMan) version 5.3 (The Cochrane Collabor- ation, The Nordic Cochrane Centre, Copenhagen, Denmark) was used. The risk ratios (RR) for dichotom- ous outcomes with 95% confidence intervals (CIs) were calculated. Heterogeneity was measured by I2. Continu- ous outcomes were expressed as mean difference (MD) and standardized mean difference (SMD) between groups with a 95% confidence interval (95% CI). Funnel plots were used to assess possible publication bias (Add- itional file 3: Figs. S1 to S9). Effect estimates were con- sidered statistically significant of the p value was less than 0.05 (2 tailed). Country and settings Trials in residential aged care settings or residential con- tinuing care hospitals (long-term care) were conducted in the USA (n = 5) [14–16, 22, 33], Australia (n = 5) [23– 26, 41], UK (n = 3) [21, 27, 28], Norway (n = 2) [32, 35], and one each in Canada [30], Israel [37], Sweden (4 pa- pers reported results of one) [17–20], Finland [39], Spain [38], Switzerland [36], Ireland [31], New Zealand [40] and one combined between USA and Canada [29] and one combined between UK and Ireland [34]. Outcomes Most outcome measures in the reviewed studies were re- ported as (a) medication appropriateness (n = 16) [14– 17, 19, 23–27, 30, 31, 35–39], hospital admission (n = 11) [21, 23, 25, 28, 33, 34, 37–41], mortality (n = 9) [21– 23, 28, 33, 34, 37, 39, 40], medication-related problems (n = 7) [18, 20, 21, 23, 25, 28, 36, 37], falls (n = 7) [26– 28, 31, 33, 38, 41], quality of life (n = 5) [27, 34, 37, 39, 41], Behavioural and Psychological Symptoms of De- mentia BPSD (n = 4) [15, 16, 27, 32], ADEs (n = 2) [25, 29], and cognitive function (n = 2) [15, 35]. Assessment of risk of Bias criteria: adequate sequence generation, allocation con- cealment, blinding of participants and personnel, blind- ing of outcome assessment, incomplete outcome data, selective reporting, and other bias. Risk of bias tables provided in RevMan v5.3 was used to assess reporting The quality of each article and risk of bias were assessed independently by the two reviewers (H.A, and L.F). For assessing risk of bias we used the Cochrane Collabora- tion’s tool [42]. The studies assessed based on standard Page 10 of 19 Page 10 of 19 Almutairi et al. BMC Geriatrics (2020) 20:236 Almutairi et al. BMC Geriatrics (2020) 20:236 bias. See Risk of Bias in Included Studies Section (Figs. 2 and 3). Results Medication appropriateness was assessed in 11,470 resi- dents encompassing 16 RCTs [14–17, 19, 23–27, 30, 31, 35–39] by different tools including Medication Appropri- ateness Index (MAI) [24–26], STOPP-START criteria [37, 38], indicators of appropriate neuroleptic prescribing in nursing homes [27], Beers criteria, Anticholinergic Drug Score (ADS), number of psychotropic medications and non-steroidal anti-inflammatory drugs (NSAIDs( [39]. The literature search provided a total of 6024 potentially relevant publications. Following independent screening for eligibility, 106 articles were assessed for eligibility of which 25 RCTs and cRCTs were included in this system- atic review. The flowchart of the literature search is rep- resented in Fig. 1. Falls Seven studies [26–28, 31, 33, 38, 41] included falls as an outcome measure. Crotty et al. [26] calculated the per- centage of residents who fell in 3 months prior. Fossey et al. [27] reported the proportion of residents who had at least one fall over a 12-month period. Zermansky et al. [28] reported number of falls over 6 months. Pat- terson et al. [31] calculated falls rate per 100 resident- months. Lapane et al. [33] reported the number of people falling over 12 months. Garcia-Gollarte et al. [38] reported the number of falls and fallers post- intervention. Potter et al. [41] reported the proportion of patients with one or more falls. Participants Studies included involved older people living in residen- tial aged care facilities aged 60 years and older with a mean age range of 81.2 to 87.2 years. Almutairi et al. BMC Geriatrics (2020) 20:236 Page 11 of 19 Fig. 2 Risk of bias graph proportion of residents experiencing an unplanned hospital admission. pharmacist, and the accepted recommendations by the general physician (GP) and the actual changes in medica- tions. Roberts et al. [23] calculated the number of drug changes. Crotty et al. [25] categorised medication-related problems to different categories such as high dose, admin- istration time and no indication. Zermansky et al. [28] measured the recommendations made by the pharmacist against the number of accepted/rejected recommenda- tions of the doctor. Milos et al. [36] measured the percent- age of medications changed. Frankenthal et al. [37] measured the number of recommendations accepted by the GP according to the STOPP-START criteria. There was no extractable data for this outcome and therefore meta-analysis was not performed. Mortality y Nine studies [21–23, 28, 33, 34, 37, 39, 40] included mortality as an outcome measure. Furniss et al. [21] re- ported mortality as a number of deaths over 8-months, by Zermansky et al. [28] over 6-months. The number of deaths was reported by Stein et al. [22] over a 3-month evaluation period, by Pope et al. [34] over a 6-month period, and by Frankenthal et al. [37] over 1 year. Rob- erts et al. [23] reported residents’ cumulative survival and death proportion for 1 year. Lapane et al. [33] calcu- lated the average percentage of mortality per 1000 person-months. Pitkala et al. [39] used a Cox propor- tional hazard model to calculate hazard ratios. Connolly et al. [40] reported death risk ratio over 14 months. Quality of Life (QoL) Five studies [27, 34, 37, 39, 41] reported patient Quality of Life (QoL). Fossey et al. [27] reported rating for well- being in residents. Pope et al. [34] measured QoL by asking patients with Abbreviated Mental Test Score (AMTS ≥8) or staff who were familiar with the patient about whether the intervention had been of benefit. Frankenthal et al. [37] used the Medical Outcomes Study 12-item Short-form Health survey (SF-12). Pitkala et al. [39] used the 15-dimensional instrument of health- related QoL (15D). Potter et al. [41] used self-reported QoL assessed with Quality of Life in Alzheimer’s De- mentia (QOLAD). Behavioural and Psychological Symptoms of Dementia (BPSD) Four studies [15, 16, 27, 32] assessed Behavioural and Psychological Symptoms of Dementia (BPSD). Rovner et al. [15] reported behaviour disorder. Fossey et al. [27] reported aggression events in past 12 months and Testad et al. [32] used the Cohen-Mansfield agitation inventory tool (CMAI) to measure agitated behaviour of residents. Meador et al. [16] used Nursing Home Behaviour Prob- lem Scale (NHBPS). Cognitive function Two studies [15, 35] included cognitive function as an outcome measure. Rovner et al. [15] used the Norwegian version of the global cognitive test Mini-Mental Sate Examination (MMSE) to assess cognition. Kersten et al. [35] used the Consortium to Establish a Registry for Alz- heimer’s Disease (CERAD)‘s 10-word list test for delayed recall and recognition and MMSE. Effectiveness of the interventions Medication appropriateness Meta-analysis of medication appropriateness (Fig. 4) in- cluding 6754 residents [15, 17, 23–27, 30, 31, 37–39] showed a significant improvement on medication appro- priateness (RR 0.71; 95% confidence interval (CI): 0.60, 0.84, despite high heterogeneity (P < 0.00001; I2 = 91%). This outcome was assessed for the intervention subtypes of staff education (RR 0.66, 95% CI:0.43, 1.01), imple- mentation of multi-disciplinary case conferencing (RR 0.97,95% CI:0.92, 1.03) computerised clinical decision support systems (RR 0.78, 95% CI:0.64, 0.95) and medi- cation review (RR 0.62 95% CI:0.41, 0.93) (See Fig. 5). The standardised mean difference of medication ap- propriateness scales for the remaining two Crotty et al. studies [24, 25] was calculated separately (standardised mean difference = −0.67; 95% CI: −0.97, −0.36) with a heterogeneity of I2 = 3%. ADEs Fig. 3 Risk of bias summary Two studies [25, 29] reported ADEs. One study defined ADE as an injury resulting from the use of a drug [29]. Crotty et al. [25] investigated number of ADEs during the 8-week follow-up period. Medication - related problems Medication - related problems Seven studies [18, 20, 21, 23, 25, 28, 36, 37] included medication - related problems as an outcome measure. Claesson and Schmidt et al. [18, 20] reported the type and frequency of drug-related problems discussed by clinical teams and their recommendations. Furniss et al. [21] re- ported the total number of recommendations made by the Page 12 of 19 Page 12 of 19 Almutairi et al. BMC Geriatrics (2020) 20:236 Risk of bias in included studies Assessment of the risk of bias is summarised in (Figs. 2 and 3). Except for nine studies with unclear risk [14, 16, 17, 19, 20, 22, 32, 35, 37], the remaining 16 studies had low risk of selection bias. Performance bias was high in 21 (77.8%) studies [15, 19–21, 23–26, 28–34, 36–41]; de- tection bias was high in eight (29.6%) studies [15, 19, 21, 23, 26, 31, 33, 35], and allocation concealment was found in four (14.8%) studies [15, 26, 27, 35]. In most of the studies, blinding of participants and staff was not possible due to the nature of the intervention. Medication- related problems Claesson and Schmidt et al. [18, 20] found 819 medica- tion -related problems in 395 participants resulting in an action taken in 90% (737) with withdrawal of a drug in 368 (45%) and change of medications in 162 (20%). Fur- niss et al. [21] reported that 239 (92%) of 261 recom- mendations were accepted by the GP resulting in change in therapy in 144 patients. The most common reasons for recommendations (33%) were the medication indica- tion was no longer present. Roberts et al. [23] found that medication reviews resulted in changes to medications in 54 (39%) of residents. Crotty et al. [25] reported that the most common medication-related problem identified in control and intervention groups was that the residents were allocated to a new family physician when transfer to long-term care facility (n = 35, 62.5% intervention; Hospital admission Meta-analysis of hospital admission (Fig. 6) as an out- come measure investigated in 11,272 residents resulted in the analysis of eight studies [25, 28, 33, 34, 37, 38, 40, 41] (10,610 residents), which showed that interventions have no effect on hospital admission RR = 1.00, 95% CI: 0.93,1.06) with a heterogeneity of I2 = 0%. Almutairi et al. BMC Geriatrics (2020) 20:236 Page 13 of 19 a b Fig. 4 a: A meta-analysis of the effect of interventions on medication appropriateness. b: Standardised mean difference in the change of MAI score comparing experimental (intervention) group and control group a b Fig. 4 a: A meta-analysis of the effect of interventions on medication appropriateness. b: Standardised mean difference in the change of MAI score comparing experimental (intervention) group and control group a a b Fig. 4 a: A meta-analysis of the effect of interventions on medication appropriateness. b: Standardised mean difference in the change of MAI score comparing experimental (intervention) group and control group Fig. 4 a: A meta-analysis of the effect of interventions on medication appropriateness. b: Standardised mean difference in the change of MAI score comparing experimental (intervention) group and control group n = 41, 75.9% control). Zermansky et al. [28] found a sig- nificant difference in the mean number of drug changes per resident (mean 3.1, SD = 2.7 for intervention and mean 2.4, SD = 2.6 for control) (P < 0.0001). Milos et al. [36] found similar number of drug-related problems be- tween community-dwelling patients (mean 2.55, SD = 1.29) and nursing home residents (mean 2.53, SD = 1.33) (p = 0.767). 56% of drug-related problems resulted in an action taken and change of medications (mean 1.44, SD = 1.33) with no difference between the community dwelling and the nursing home patients (p = 0.946). Frankenthal et al. [37] made 327 recommendations - 245 in 129 residents based on STOPP and 82 in 65 residents based on START. The physician accepted 82.4% of STOPP recommendations and 92.6% of START recommendations. Falls Meta-analysis of falls (Fig. 8) as an outcome measure in- vestigated in 9382 residents [26–28, 31, 33, 38, 41] showed that interventions had no effect on falls (RR = 1.06; 95%CI: 0.89,1.26) with a heterogeneity of I2 = 87%. Mortality Meta-analysis of mortality (Fig. 7) as an outcome meas- ure investigated in 13,675 residents [21–23, 28, 33, 34, 37, 39, 40] showed no significant difference between the intervention group and control group (RR 0.98, 95% CI: 0.86,1.11, P = 0.07) with a heterogeneity of I2 = 43%. Quality of Life (QoL) Meta-analysis of quality of life (Fig. 9) outcomes in 570 residents, of a total of 1141 residents that included QoL as an outcome measure, found that the interventions had no effect on residents’ QoL (standardised mean dif- ference = 0.16 95% CI:-0.13, 0.45) with a heterogeneity of I2 = 57% between trials [37, 39, 41]. Pitkala et al. [39] re- ported that health-related QoL in the intervention group (−0.038, 95% CI: −0.054, −0.022) declined more slowly than in control group during 12-month follow-up (− 0.072, 95% CI: −0.089, −0.055). Frankenthal et al. [37] found no significant difference between groups in the Almutairi et al. BMC Geriatrics (2020) 20:236 Page 14 of 19 Fig. 5 Subgroup analysis of the effect of interventions medication review, multi-disciplinary team meetings, staff education and computerised clinical decision Fig. 5 Subgroup analysis of the effect of interventions medication review, multi-disciplinary team meetings, staff education and computerised clinical decision physical average score (intervention mean 33.1 ± 8.1, control mean 33 ± 8.3, p = 0.09) and mental components (intervention mean 37.7 ± 1.7, control mean 39.6 ± 11.3 p 0 70) of the SF 12 questionnaire BPSD Of 1941 residents examined, pooled analysis of BPSD of two studies [15, 27] comprising 419 residents showed no physical average score (intervention mean 33.1 ± 8.1, control mean 33 ± 8.3, p = 0.09) and mental components (intervention mean 37.7 ± 1.7, control mean 39.6 ± 11.3 p = 0.70) of the SF-12 questionnaire. BPSD Of 1941 residents examined, pooled analysis of BPSD of two studies [15, 27] comprising 419 residents showed no Of 1941 residents examined, pooled analysis of BPSD of two studies [15, 27] comprising 419 residents showed no Fig. 6 A meta-analysis of the effect of interventions on hospital admission Almutairi et al. BMC Geriatrics (2020) 20:236 Page 15 of 19 Fig. 7 A meta-analysis of the effect of interventions on mortality significant change after the intervention (RR 0.68, 95% CI: 0.44,1.06; I2 =0%) (Fig. 10). homes, when applied either individually or through multi-faceted approaches. Our meta-analysis of available data showed that the interventions implemented in the included studies can improve medication appropriate- ness in older residents, although heterogeneity was high among included studies. Whilst these results were prom- ising, the impact on the residents’ clinical outcomes was undetectable. Cognitive function Meta-analysis of 145 residents [15, 35] indicated that the interventions had no effect on cognitive function (weighted mean difference = 0.69, 95%CI: −1.25, 2.64) (Fig. 12). No heterogeneity was detected between trials (I2 =0). ADEs Of the 1206 residents [25, 29] examined for ADEs after the intervention. Neither of the two RCTs reported any statistically significant differences in ADEs between the intervention and control groups. The RR for all ADEs was 1.04 (95% CI: 0.96,1.13; I2 =0%) (Fig. 11). Of the 1206 residents [25, 29] examined for ADEs after the intervention. Neither of the two RCTs reported any statistically significant differences in ADEs between the intervention and control groups. The RR for all ADEs was 1.04 (95% CI: 0.96,1.13; I2 =0%) (Fig. 11). There are several published systematic reviews on clin- ical outcomes of different interventions conducted in aged care homes. In a review by Forsetlund and colleagues [11], the authors found that both educational outreach/educa- tional interventions and medication review by pharmacists under certain situations could reduce inappropriate drug use in nursing homes. However, they reported that the evidence was of poor quality and too low to assess the ef- fect of the interventions on health outcomes. A review by Loganathan et al. [12] grouped the interventions into four Quality of Life (QoL) There was limited evidence for effective- ness of interventions in reducing hospitalisation, all- cause mortality, falls, ADEs, cognitive function or BPSD. Discussion This systematic review examined how a wide variety of interventions optimise medications prescribed in nursing Fig. 8 A meta-analysis of the effect of interventions on falls Almutairi et al. BMC Geriatrics (2020) 20:236 Page 16 of 19 Fig. 9 A meta-analysis of the effect of interventions on quality of life (QoL) demonstrated that the lack of effect was attributed to staff attrition, short study duration, and not all the phy- sicians in the recruited homes participated in the study. groups (staff education including academic detailing, multi-disciplinary team meetings, medication review, and computerised clinical decision support system). No one interventional strategy was found to be effective. However, the most promising intervention seems to be education including academic detailing. That review reported that multifaceted interventions are likely to be required to im- prove prescribing in care homes [12]. A narrative review by Shafiee et al. [10], which included 18 studies, seven of them RCTs, found that the interventions may improve medication appropriateness in people with dementia in any settings, but the evidence for the effect of the inter- ventions on health outcomes remained uncertain. Very few of the interventions were based on strong theoretical foundations. An exception was the educa- tional study by Pitkälä et al. [39], who suggested that the use of constructive learning theory to recognise poten- tially harmful medications was more likely to change practice in healthcare than using lectures alone. Deficits in the education of health careworkers were thought to be important. Forsetlund et al. [11] suggested that health care providers receive inadequate training in geriatric medications in their education. Therefore, any interven- tion for minimising medications usually requires some form of education. Our findings on clinical outcomes are in line with that of previous reviews [43, 44], which found no evidence for the interventions impacting resident’s clinical out- comes such as ADEs, mortality, QoL and hospital admis- sion. Since the elderly often exhibit non-specific clinical symptoms such as depression, constipation, falls and confusion, it is difficult to detect ADEs as opposed to the general condition of the residents. Another possible reason for the lack of significant effect of interventions on falls and ADEs may be the potential for underreport- ing of incidents that were obtained from nursing re- cords. Discussion The lack of effect of interventions on QoL in the treatment group compared to the control may be attrib- uted to the wide variation in the length of the follow up period (3–12 months) [43]. Although our primary focus was on clinical outcomes, some interventions demonstrated a decrease in medication-related costs. Frunsis et al. [21] reported a reduction in the cost of medicine per resident over 4 months period by 27.47 GB Pounds in the intervention group. Roberts et al. [23] reported savings in drug cost (64 AUD/ resident/year in intervention group) in the clinical pharmacy program. Frankenthal et al. [37] found a significant reduction in the average monthly costs of medications in the intervention group ILS 279 ± 171.9 compared to baseline ILS 382.7 ± 279 (P < 0.001) at 12 months follow-up period. Pope et al. [34] reported a net reduction in medication cost in intervention group over a 6-month period. While, Crotty et al. [24] reported similar Pharmaceutical Benefit Scheme (PBS) monthly drug costs of regular medications between groups (mean AUD 359 in intervention versus AUD 303 in control (P = 0.837). These interventions require resources and Interventions that focus on individual team members may had limited effectiveness in busy clinical environ- ment. For example, the educational intervention deliv- ered by a pharmacist [26] failed to have any significant effect on major outcomes. The investigators Fig. 10 A meta-analysis of the effect of interventions on Behavioural and Psychological Symptoms of Dementia (BPSD) Fig. 10 A meta-analysis of the effect of interventions on Behavioural and Psychological Symptoms of Dementia (BPSD) Almutairi et al. BMC Geriatrics (2020) 20:236 Page 17 of 19 Fig. 11 A meta-analysis of the effect of interventions on adverse drug events (ADEs) therefore, evaluating these interventions economically and their cost-effectiveness should be considered in fu- ture research. measurement of specific outcomes. Certain outcomes, such as cognitive function, were examined in a lim- ited number of articles, reducing the power of the analysis. Further, certain studies were small or had short study periods, which may potentially limit the effect of an intervention on the outcomes. We attempted to evaluate medication-related problems such as drug interactions, number of pharmacist rec- ommendations etc. but these outcomes were not con- sistently reported in the studies and this our ability to draw any robust conclusions was limited. Strengths and limitations This systematic review was based on a comprehensive search of the literature that was limited to 25 studies with robust design (RCT, cRCT) and compared to previ- ous reviews on related topics [12, 44], our sample size may be regarded as sufficiently powered. Another strength of this review was a focus only on residents in care homes. The nursing home population is at heightened risk of receiving multiple drugs because of their comorbidities. Therefore, evaluating specific available interventions optimising medications in this setting is required. We were able to complete meta- analyses to pooled the overall effects. Heterogeneity was notable among some studies in- cluded in the meta-analysis. The factors that caused this heterogeneity were difficult to discern. Due to differ- ences in training, the characteristics of the nursing home residents, healthcare culture, the number of physicians’ visits and their usual practices the ability to generalise findings from one country to another is difficult. This study is not without limitations. We included only English language publications, which may lead to potential omission of other interventions. Although MEDLINE, PubMed, Google scholar, and PsycINFO da- tabases were searched for relevant articles, some studies indexed in other databases may have been missed. We identified several additional articles manually which may indicate poor indexing of older studies and a lack of consistent terminology. Not applicable. 10. Shafiee Hanjani L, Long D, Peel NM, Peeters G, Freeman CR, Hubbard RE. Interventions to optimise Prescribing in older people with dementia: a systematic review. Drugs Aging. 2019;36(3):247–67. https://doi.org/10.1007/ s40266-018-0620-9. Funding Hend Almutairi is supported by University of Western Australia International Fee Scholarship (Uifs) And University Postgraduate Award (Upa). Leon Flicker is supported by a Medical Research Future Fund Next Generation Clinical Researchers Practitioner Fellowship (1155669). The actual review received no specific funding. The funding sources had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. The actual review received no specific funding. The funding sources had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Implications for research and practice f h d bl In view of the considerable investment in strategies aimed at improving medication appropriateness in RACFs worldwide, our findings question the value of such interventions based on the apparent lack of out- comes that may be meaningful to RACF residents. Large, high quality RCT studies are required to identify effective interventions to optimise medications used in RACFs. Regarding physicians or staff accept- ance of the intervention, only limited information was provided in the studies. Further qualitative study Due to the nature of the interventions, performance and detection biases may have resulted from the diffi- culty in maintaining blinding. Meta-analysis of some studies was difficult due to the variations in the Fig. 12 A meta-analysis of the effect of interventions on cognitive function Almutairi et al. BMC Geriatrics (2020) 20:236 Almutairi et al. BMC Geriatrics (2020) 20:236 Page 18 of 19 Page 18 of 19 and AS. All authors have read and approved the final version of this manuscript. and AS. All authors have read and approved the final version of this manuscript. utilising semi-structured interviews may provide use- ful information to obtain the opinion of healthcare professionals with regard to process outcomes, such as whether the intervention was perceived to be suc- cessful or not, and to identify the potential means to overcome the barriers to changing professional behav- iour by this method. More intensive interventions on medical and care staff with more stringent monitoring may be required. References 1. O’Connor MN, Gallagher P, O’Mahony D. Inappropriate prescribing. Drugs Aging. 2012;29(6):437–52. https://doi.org/10.2165/11632610-000000000- 00000. 2. Burns E, McQuillan N. Prescribing in care homes: the role of the geriatrician. TACD. 2011;2(6):353–8. https://doi.org/10.1177/2040622311421438. 3. Hamilton HJ, Gallagher PF, O'Mahony D. Inappropriate prescribing and adverse drug events in older people. BMC Geriatr. 2009;9(1):5–3. https://doi. org/10.1186/1471-2318-9-5. 4. Spinewine A, Schmader KE, Barber N, Hughes C, Lapane KL, Swine C, et al. Appropriate prescribing in elderly people: how well can it be measured and optimised? Lancet. 2007;370(9582):173–84. https://doi.org/10.1016/ S01406736(07)61091-5. 4. Spinewine A, Schmader KE, Barber N, Hughes C, Lapane KL, Swine C, et al. Appropriate prescribing in elderly people: how well can it be measured and optimised? Lancet. 2007;370(9582):173–84. https://doi.org/10.1016/ S01406736(07)61091-5. Supplementary information y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12877-020-01634-4. Supplementary informatio 1186/s12877-020-01634-4. Additional file 1. PRISMA checklist. Additional file 2. Search Strategy. Additional file 3 Funnel plots (pdf). Figure S1. Medication appropriateness funnel plot (10studies). Figure S2. Medication appropriateness funnel plot (2 studies). Figure S3. Hospital admission funnel plot. Figure S4. Mortality funnel plot. Figure S5. Falls funnel plot. Figure S6. Quality of life (QoL) funnel plot. Figure S7. Behavioural and Psychological Symptoms of Dementia (BPSD) funnel plot. Figure S8. Adverse drug events (ADEs) funnel plot. Figure S9. Cognitive function funnel plot. Received: 2 March 2020 Accepted: 29 June 2020 Received: 2 March 2020 Accepted: 29 June 2020 Author details 1 1Medical school, University of Western Australia, Perth, Australia. 2School of Medicine and Pharmacology, Western Australian Centre for Health and Ageing, University of Western Australia , Perth, Australia. 3Medical School, University of Western Australia, Western Australian Centre for Health and Ageing University of Western Australia, Perth, Australia. Consent for publication Not applicable. Consent for publication Not applicable. Consent for publication Not applicable. The authors declare that they have no competing interests. The authors declare that they have no competing interests. Abbreviations RACFs: Residential aged care facilities; RCTs: Randomised control trials; cRCTs: Cluster randomised control trials; QoL: Quality of life; BPSD: Behavioural and Psychological Symptoms of Dementia; ADEs: Adverse drug events; CI: Confidence interval; RR: Risk ratio; MD: Mean difference; SMD: Standardized mean difference; ADRs: Adverse drug reactions; PRIS MA: Preferred reporting items for systematic reviews and meta-analyses; MAI: Medication appropriateness index; ADS: Anticholinergic drug score; NSAIDs: Non-steroidal anti-inflammatory drugs; AMTS: Abbreviated mental test score; 15 D: 15-dimensional instrument of health-related QoL; QOLAD: Quality of Life in Alzheimer’s Dementia; CMAI: Cohen-Mansfield agitation inventory tool; NHBPS: Nursing Home Behaviour Problem Scale; MMSE: Mini-Mental Sate Examination; CERAD: Consortium to Establish a Registry for Alzheimer’s Disease; PIP: Potentially inappropriate prescriptions; PBS: Pharmaceutical Benefit Scheme 5. Gallagher P, Barry P, O'Mahony D. Inappropriate prescribing in the elderly. J Clin Pharm Ther. 2007;32(2):113–21. https://doi.org/10.1111/j.1365-2710.2007. 00793.x. 5. Gallagher P, Barry P, O'Mahony D. Inappropriate prescribing in the elderly. J Clin Pharm Ther. 2007;32(2):113–21. https://doi.org/10.1111/j.1365-2710.2007. 00793.x. 6. Jiwa M. Optimising medication in frail older people. Prescriber. 2018;29(3): 18–24. https://doi.org/10.1002/psb.1655. 6. Jiwa M. Optimising medication in frail older people. Prescriber. 2018;29(3): 18–24. https://doi.org/10.1002/psb.1655. 7. Anrys P, Strauven G, Boland B, Dalleur O, Declercq A, Degryse J-M, et al. Collaborative approach to optimise medication use for older people in nursing homes (COME-ON): study protocol of a cluster controlled trial. Implement Sci. 2015;11(1):35. https://doi.org/10.1186/s13012-016-0394-6. 8. Cadogan CA, Ryan C, Hughes CM. Appropriate polypharmacy and medicine safety: when many is not too many. Drug Saf. 2016;39(2):109–16. https://doi. org/10.1007/s40264-015-0378-5. 8. Cadogan CA, Ryan C, Hughes CM. Appropriate polypharmacy and medicine safety: when many is not too many. Drug Saf. 2016;39(2):109–16. https://doi. org/10.1007/s40264-015-0378-5. 9. NICE M, Prescribing CU. Medicines optimisation: the safe and effective use of medicines to enable the best possible outcomes. 2015. 9. NICE M, Prescribing CU. Medicines optimisation: the safe and effective use of medicines to enable the best possible outcomes. 2015. Ethics approval and consent to participate Not applicable Ethics approval and consent to participate Not applicable. Additional file 1. PRISMA checklist. Additional file 2. Search Strategy. Additional file 3 Funnel plots (pdf). Figure S1. Medication appropriateness funnel plot (10studies). Figure S2. Medication appropriateness funnel plot (2 studies). Figure S3. Hospital admission funnel plot. Figure S4. Mortality funnel plot. Figure S5. Falls funnel plot. Figure S6. Quality of life (QoL) funnel plot. Figure S7. Behavioural and Psychological Symptoms of Dementia (BPSD) funnel plot. Figure S8. Adverse drug events (ADEs) funnel plot. Figure S9. Cognitive function funnel plot. Conclusion h The datasets supporting the conclusions of this article are included within the article and its additional files. This systematic review found that multifaceted interven- tions including medication review, staff education/train- ing, multi-disciplinary case-conferencing and clinical decision support technology could improve the appro- priateness of medications at RACFs. However, evidence for the effect of these interventions on residents’ clinical outcomes was scarce and no conclusion could be drawn. More robust clinical studies are required to ascertain the health outcomes benefits. Authors’ contributions Meador KG, Taylor JA, Thapa PB, Fought RL, Ray WA. 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J Am Geriatr Soc. 2010;58(1):44–53. https://doi.org/ 10.1111/j.1532-5415.2009.02617.x. 14. Avorn J, Soumerai SB, Everitt DE, Ross-Degnan D, Beers MH, Sherman D, et al. A randomized trial of a program to reduce the use of psychoactive drugs in nursing homes. N Engl J Med. 1992;327(3):168–73. https://doi.org/ 10.1056/NEJM199207163270306. 32. Testad I, Ballard C, Bronnick K, Aarsland D. The effect of staff training on agitation and use of restraint in nursing home residents with dementia: a single-blind, randomized controlled trial. J Clin Psychiatry. 2010;71(1):80–6. https://doi.org/10.4088/JCP.09m05486oli. 15. Rovner BW, Steele CD, Shmuely Y, Folstein MF. A randomized trial of dementia care in nursing homes. J Am Geriatr Soc. 1996;44(1):7–13. https:// doi.org/10.1111/j.1532-5415.1996.tb05631.x. 33. Lapane KL, Hughes CM, Daiello LA, Cameron KA, Feinberg J. Effect of a pharmacist-led multicomponent intervention focusing on the medication monitoring phase to prevent potential adverse drug events in nursing homes. J Am Geriatr Soc. 2011;59(7):1238–45. https://doi.org/10.1111/j.1532- 5415.2011.03418.x. 16. Authors’ contributions Pitkala KH, Juola A-L, Kautiainen H, Soini H, Finne-Soveri UH, Bell JS, et al. Education to reduce potentially harmful medication use among residents of assisted living facilities: a randomized controlled trial. J Am Med Dir Assoc. 2014;15(12):892–8. https://doi.org/10.1016/j.jamda.2014.04.002. 23. Roberts MS, Stokes JA, King MA, Lynne TA, Purdie DM, Glasziou PP, et al. Outcomes of a randomized controlled trial of a clinical pharmacy intervention in 52 nursing homes. Br J Clin Pharmacol. 2001;51(3):257–65. https://doi.org/10.1046/j.1365-2125.2001.00347.x. 40. Connolly MJ, Boyd M, Broad JB, Kerse N, Lumley T, Whitehead N, et al. The aged residential care healthcare utilization study (ARCHUS): a multidisciplinary, cluster randomized controlled trial designed to reduce acute avoidable hospitalizations from long-term care facilities. J Am Med Dir Assoc. 2015;16(1):49–55. https://doi.org/10.1016/j.jamda.2014.07.008. 24. Crotty M, Halbert J, Rowett D, Giles L, Birks R, Williams H, et al. An outreach geriatric medication advisory service in residential aged care: a randomised controlled trial of case conferencing. Age Ageing. 2004;33(6):612–7. https:// doi.org/10.1093/ageing/afh213. 25. Crotty M, Rowett D, Spurling L, Giles LC, Phillips PA. Does the addition of a pharmacist transition coordinator improve evidence-based medication management and health outcomes in older adults moving from the hospital to a long-term care facility? Results of a randomized, controlled trial. Am J Geriatr Pharmacother. 2004;2(4):257–64. https://doi.org/10.1016/j. amjopharm.2005.01.001. 41. Potter K, Flicker L, Page A, Etherton-Beer C. Deprescribing in frail older people: a randomised controlled trial. PLoS One. 2016;11(3):e0149984. https://doi.org/10.1371/journal.pone.0149984. 42. Higgins JP, Altman DG, Gøtzsche PC, Jüni P, Moher D, Oxman AD, et al. The Cochrane Collaboration’s tool for assessing risk of bias in randomised trials. Bmj. 2011;34(3):5423–928. https://doi.org/10.1136/bmj.d5928. 26. Crotty M, Whitehead C, Rowett D, Halbert J, Weller D, Finucane P, et al. An outreach intervention to implement evidence based practice in residential care: a randomized controlled trial. BMC Health Serv Res. 2004;4(1):6–2. https://doi.org/10.1186/1472-6963-4-6. 43. Patterson SM, Cadogan CA, Kerse N, Cardwell CR, Bradley MC, Ryan C, et al. Interventions to improve the appropriate use of polypharmacy for older people. Cochrane Database Syst Rev. 2014(10). DOI:org/https://doi.org/10. 1002/14651858.CD008165.pub4. 27. Fossey J, Ballard C, Juszczak E, James I, Alder N, Jacoby R, et al. Effect of enhanced psychosocial care on antipsychotic use in nursing home residents with severe dementia: cluster randomised trial. Bmj. 2006; 332(7544):756–61. https://doi.org/10.1136/bmj.38782.575868.7C. 44. Alldred DP, Kennedy MC, Hughes C, Chen TF, Miller P. Interventions to optimise prescribing for older people in care homes. Cochrane Database Syst Rev. 2016;2:CD009095. https://doi.org/10.1002/14651858.CD009095. pub3. 28. Authors’ contributions Zermansky AG, Alldred DP, Petty DR, Raynor DK, Freemantle N, Eastaugh J, et al. Clinical medication review by a pharmacist of elderly people living in care homes--randomised controlled trial. Age Ageing. 2006;35(6):586–91. https://doi.org/10.1093/ageing/afl075. 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. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 29. Gurwitz JH, Field TS, Rochon P, Judge J, Harrold LR, Bell CM, et al. Effect of computerized provider order entry with clinical decision support on adverse drug events in the long-term care setting. J Am Geriatr Soc. 2008;56(12): 2225–33. https://doi.org/10.1111/j.1532-5415.2008.02004.x.
https://openalex.org/W2912514528
http://www.scielo.br/pdf/rcbc/v46n1/en_0100-6991-rcbc-46-01-e2011.pdf
English
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Simulador de dreno de tórax: desenvolvimento de modelo de baixo custo para capacitação de médicos e estudantes de medicina.
Revista do Colégio Brasileiro de Cirurgiões
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Simulador de dreno de tórax: desenvolvimento de modelo de baixo custo para capacitação de médicos e estudantes de medicina. Ana Luísa Bettega, AcCBC-PR1; Luis Fernando Spagnuolo Brunello, AcCBC-PR1; Guilherme Augusto Nazar1; Giovanni Yuji Enomoto De-Luca1; Lucas Mansano Sarquis, AsCBC-PR2; Henrique de Aguiar Wiederkehr, AcCBC-PR2; José Aguiomar Foggiatto3; Silvania Klug Pimentel, TCBC-PR2 A B S T R A C T Objective: by using a 3D printer, to create a low-cost human chest cavity simulator that allows the reproduction of the closed chest drainage technique (CCD), comparing its effectiveness with that of the animal model. Methods: it was made a 3D printing of the bony framework of a human thorax from a chest computerized tomography scan. After printing the ribs, we performed tests with several materials that contributed to form the simulation of the thoracic cavity and pleura. An experimental, randomized, and controlled study, comparing the efficacy of the simulator to the efficacy of the animal model, was then carried out in the teaching of CCD technique for medical students, who were divided into two groups: animal model group and simulator model group, that trained CCD technique in animals and in the simulator model, respectively. Results: the chest reconstruction required anatomical knowledge for tomography analysis and for faithful 3D surface editing. There was no significant difference in the safety of performing the procedure in both groups (7.61 vs. 7.73; p=0.398). A higher score was observed in the simulator model group for “use as didactic material” and “learning of the chest drainage technique”, when compared to the animal model group (p<0.05). Conclusion: the final cost for producing the model was lower than that of a commercial simulator, what demonstrates the feasibility of using 3D printing for this purpose. In addition, the developed simulator was shown to be equivalent to the animal model in relation to the simulation of the drainage technique for practical learning, and there was preference for the simulator model as didactic material. Keywords: Simulation. Models, Animal. Simulation Training. Training. Education, Medical. Drainage. Ch Keywords: Simulation. Models, Animal. Simulation Training. Training. Education, Medical. Drainage. Chest Tubes. 1 - Federal University of Parana, Faculty of Medicine, Curitiba, PR, Brazil. 2 - Hospital do Trabalhador, General Surgery and Trauma Service, Curitiba, PR, Brazil. 3 - Federal Technological University of Parana, Academic Department of Mechanics, Curitiba, PR, Brazil. Original Article Original Article DOI: 10.1590/0100-6991e-20192011 INTRODUCTION activities become predictable, standardized, secure, and reproducible3. S imulation consists of a tool that aims to reproduce, interactively and safely, aspects of real life1,2. In the health area, simulation is seen as an effective form of teaching, which improves patient and safety care, since the individual undergoing training ― student or physician ― goes through experiences very close to those of real life and consistent with decision-making in daily medical practice1. Simulation in the medical field can also provide the ability to recognize limitations and technical failures in procedures performed by health professionals. A well-conducted simulation program can create a favorable scenario in which S Simulators can be classified in different ways, ranging from computerized systems, dolls that play the role of patients, environments that simulate work situations, and even real people playing roles following a previously elaborated script. Another important aspect is the fidelity of the simulator. Here, “fidelity” is understood as the simulator’s ability to approach the aspect and activities of the system to be simulated in real life2. Simple simulators can be highly effective when used to simulate cognitive tasks and review procedure steps. On the other hand, more complex simulators are needed to train and develop fine motor coordination skills. Rev Col Bras Cir 46(1):e2011 Bettega d 2 Bettega Chest tube simulator: development of low-cost model for training of physicians and medical students. However, one of the major problems and limitations that simulation centers present is the cost of most equipment, mannequins, partial models used, instructors, and maintenance1-8. Nevertheless, it is fully possible to develop low-cost simulators that prioritize the learning of tasks and routines in which repetitive practice is more important1. For simulations involving teams, detailed environments, high-tech mannequins, high-value softwares, integrated simulators, instructors, and real patients, the cost will be higher, but there will be the development of more complex simulations, so as to fit the specific purpose of each station1,2,4. students to evaluate its effectiveness and the satisfaction of the participants in relation to the simulator model compared to the animal model. METHODS An experimental, randomized, and controlled study was carried out through the development of a 3D printing model that allowed the simulated training of the closed chest drainage procedure for medical students at the emergency room of Hospital do Trabalhador, Curitiba, Parana, Brazil. A 3D printing of the bony framework of a human thorax was made using a chest tomography, without the need of a funder and through a partnership with Federal Technological University of Parana (UTFPR) with a total cost of approximately 133 dollars (Figure 1). After printing the ribs, tests were performed with various materials that contributed to form the simulation of the thoracic cavity and pleura (Figure 2). The use of porcine rib hemithorax for training of the chest drain insertion technique by physicians and Brazilian medical students has already been described, with a high anatomical correlation between the porcine ribs and the human thorax9. The main advantage of the method is its low cost and easy obtainment of the material, since animal segments are relatively easier to acquire than a complete corpse. However, the model also presents limitations: because it is biological and perishable material, it cannot be reused and requires cooling care, so that the material does not deteriorate and the work does not become unfeasible. In addition, it is necessary that the skin and the subcutaneous tissue are intact and with optimum consistency and thickness, and, although the porcine rib is relatively easy to find in conventional supermarkets, the “ideal material”, under the mentioned conditions, is not. Figure 1. 3D printing of a thorax. The objective of this work was to create a low-cost simulator using 3D printing, with anatomical conditions similar to a segment of the human thoracic cavity, in order to reproduce the closed chest drainage technique and apply the model developed in the training of medical Figure 1. 3D printing of a thorax. Figure 1. 3D printing of a thorax. Rev Col Bras Cir 46(1):e2011 Bettega 3 3 Bettega Chest tube simulator: development of low-cost model for training of physicians and medical students. Figure 2. Chest drain simulator model (SIM drain). Figure 2 Chest drain simulator model (SIM drain) Figure 2. Chest drain simulator model (SIM drain). METHODS The students were randomly divided into two groups: AMG, animal model group, the traditional simulation model, which performed drainage in the porcine model (porcine ribs), and SMG, simulator model group, which performed drainage in the simulator model developed by the authors. After completion, this model was used for the training of medical students in internship period at the emergency room of Hospital do Trabalhador. The academics, from the fifth to the eighth period, were invited to participate in the research on a voluntary basis. They were students from the five medical schools of Curitiba: Federal University of Parana (UFPR), Universidade Positivo (UP), Pontifical Catholic University of Parana (PUCPR), Evangelical Faculty of Parana (FEPAR), and Faculdades Pequeno Príncipe (FPP). A brief lecture was presented, exposing the chest drainage technique with the visual aid of a video. After this first part, the students answered part of the questionnaire evaluating the technique of the model. They answered questions about their safety in performing chest drainages after the presentation of its theoretical content. These technical questions were taken from a checklist of the chest drainage procedure developed by professors of the Training and Simulation discipline at Federal University of Parana. After the practical activity, the students answered the questionnaire composed of two parts. The first part was about the students’ experience in simulation and their safety in performing chest drainages in patients after the practical activity with the simulator. The second part was related to the content of the research. The students answered questions about the importance of this procedure in the undergraduation program, as well as if the model was useful as didactic material and allowed the learning of the technique, if they would like to use other simulators developed with the same concept, if the practical activity was adequate, and if the learning of the procedure was Rev Col Bras Cir 46(1):e2011 Bettega Chest tube simulator: development of low-cost model for training of physicians and medical students. Bettega Chest tube simulator: development of low-cost model for training of physicians and medical students. Bettega students Bettega t d t Bettega Chest tube simulator: development of low-cost model for training of physicians and medical students. 4 appropriate. All questions in the questionnaire should be answered through a scale ranging from 0 to 10. At the end, the academics answered questions to identify the sample, such as age, gender, university, and period. METHODS They had space to write comments and suggestions. was 7.60; in the SMG, 7.73 ― p=0.398). The score of the participants of this study (n=49) ranged from 5.44 to 7.67 (p<0.001). In the AMG, it ranged from 5.47 to 7.60, reflecting an increase of 38.9% (p<0.0001). In the SMG, the score ranged from 5.42 to 7.73, reflecting an increase of 42.6% (p<0.0001) (Table 1). The results obtained were tabulated by Microsoft Excel. The normal distribution variables were described using mean and standard deviation. Epidemiological data were calculated with simple statistical percentages. The continuous variables were analyzed using Student's t-test. The rejection level of the null hypothesis was set at 5%. The present study was approved by Research Ethics Committee Involving Human Beings/Secretary of Health of Parana State under the number 2199465. In relation to the experience in the simulation, the students were asked to punctuate some items about the safety they would have to perform the following techniques in a patient: approximately 1.5cm skin incision, divulsion of the planes with penetration into the pleural cavity, introduction of the index finger into the pleural orifice, drain insertion (posterosuperiorly) until the mark is reached. When comparing the average of the grades of the two groups after the practical activity, totals of 8.95 for the AMG and 9.11 for the SMG (p=0.335) were found for the first item evaluated; for the second item, 7.95 for the AMG and 8.57 for the SMG (p=0.146); for the third item, 8.69 for the AMG and 9.07 for the SMG (p=0.194); and, for the fourth item, 8.26 for the AMG and 8.73 for the SMG (p=0.143) (Table 2). DISCUSSION the average of the grades was 8,0 for the AMG and 8.88 (p=0.016) for the SMG. When asked if the model allowed the learning of the chest drainage techniques, the average of the grades was 8.17 for the AMG; 8.88 for the SMG (p=0.022) (Table 4). Medical education has been undergoing a paradigm shift, placing simulation as an effective tool to meet the new educational and social Table 1. Safety of performing a chest drainage procedure on a patient. Total (n=49) AMG* (n=23) SMG** (n=26) p After theoretical presentation 5.44 (±1.93) 5.47 (±2.04) 5.42 (±1.87) 0.460 After practical training 7.67 (±1.63) 7.61 (±1.43) 7.73 (±1.82) 0.398 * AMG: animal model group; ** SMG: simulator model group. Table 1. Safety of performing a chest drainage procedure on a patient. Table 2. Technical experience undergone through practical training. Total (n=49) AMG*(n=23) SMG**(n=26) p Approximately 1.5cm skin incision 9.04 (±1.29) 8.95 (±1.49) 9.11 (±1.17) 0.335 Divulsion of the planes with penetration into the pleural cavity 8.28 (±2.04) 7.95 (±2.34) 8.57 (±1.72) 0.146 Introduction of the index finger into the pleural orifice 8.89 (±1.53) 8.69 (±1.89) 9.07 (±1.12) 0.194 Drain insertion (posterosuperiorly) until the mark is reached 8.51 (±1.52) 8.26 (±1.81) 8.73 (±1.22) 0.143 * AMG: animal model group; ** SMG: simulator model group. able 2. Technical experience undergone through practical training. Table 3. Range of the score, before and after the practical activity. Rev Col Bras Cir 46(1):e2011 Table 3. Range of the score, before and after the practical activity. After theoretical presentation After practical activity Range (%)   AMG* SMG** AMG* SMG** AMG* SMG** Approximately 1.5cm skin incision 8.21 8.03 8.95 9.11 9,0 13.4 Divulsion of the planes with penetration into the pleural cavity 6.82 6.84 7.95 8.57 16.5 25.2 Introduction of the index finger into the pleural orifice 7.34 8.34 8.69 9.07 18.3 8.7 Drain insertion (posterosuperiorly) until the mark is reached 5.91 6.23 8.26 8.73 39.7 40.1 * AMG: animal model group; ** SMG: simulator model group. Table 4. Didactic content. Total (n=49) AMG*(n=23) SMG**(n=26) p Was the model useful as didactic material for the undergraduation program? 8.46 (±1.45) 8.0 (±1.56) 8.88 (±1.24) 0.016*** Did the model allow the learning of the chest drainage techniques? 8.55 (±1.24) 8.17 (±1.26) 8.88 (±1.14) 0.022*** Was the practical activity adequate? 9.02 (±1.42) 8.91 (±1.78) 9.11 (±1.03) 0.312 Was the learning of the procedure adequate? Bettega Ch b Bettega Chest tube simulator: development of low-cost model for training of physicians and medical students. 5 5 RESULTS A total of 49 medical students participated in this study. The mean age was of 22.4 years and 30 students (61.2%) were female. Only 15 academics (30.6%) stated that they did not have experience with simulators. Twenty-three (46.9%) were submitted to the practical activity with the animal model (porcine ribs) and 26 (53%) with the simulator model, developed by the researchers. The range of the average of the grades (in percentage) after the theoretical presentation and the practical activity was described in table 3. In both groups, there was an increase in the average of the grades in all evaluated items of the checklist. Regarding the content, the participants considered of great relevance the practice of the chest drainage procedure for medical undergraduate students, attributing an average score of 9.81. When the SMG students were asked if they would like to use other simulators developed with the same concept, the average of the grades was 9.69. When asked whether the model was useful as didactic material for the undergraduation program, After the theoretical presentation on the chest drainage technique and the video showing the procedure, the 49 medical students answered this question: “How safe would you feel to perform the procedure on a patient?". The average of the grades was 5.44 (in the AMG, the average was 5.47; in the SMG, 5.42 ― p=0.460). After the practical activity, the 49 academics were asked to answer the same question, and, on this occasion, the average of the grades was 7.67 (in the AMG, the average Rev Col Bras Cir 46(1):e2011 Rev Col Bras Cir 46(1):e2011 Bettega Chest tube simulator: development of low-cost model for training of physicians and medical students. Bettega Ch t t b DISCUSSION 8.51 (±1.67) 8.30 (±1.89) 8.69 (±1.46) 0.211 * AMG: animal model group; ** SMG: simulator model group; *** statistically significant result. Bettega Chest tube simulator: development of low-cost model for training of physicians and medical students. Bettega Chest tube simulator: development of low-cost model for training of physicians and medical students. Bettega t d t Bettega Chest tube simulator: development of low-cost model for training of physicians and medical students. 6 challenges of our time10. The high cost of simulators is still the main limitation of their use by universities in the teaching of medical students11. Thus, the role of the practical, effective, and low-cost simulator developed by the authors of the article is highlighted. Nowadays, the “3 R’s Program” is known and well-established. Its objective is of reducing, refining, and replacing the number of animals used in research and improving the conduction of the studies in order to reduce to the possible minimum the suffering and to increase the search for alternative methods that, in the end, replace the in vivo tests14. Thus, the use of the simulator is an excellent option for training, since, besides having the consistency similar to that of a real model, it does not allow the sacrifice of an animal model, in the initial phase of the academic learning15. When questioned about the safety in performing the chest drainage procedure in a patient after the theoretical class, very similar scores were observed between the SMG and the AMG, what confirms the homogeneity of the groups in which they were separated. By relating the scores the students gave to the same questioning after the theoretical presentation and after the practical activity, the results were statistically significant. There was an increase in the score for both the AMG and the SMG, what reveals that the practical activity has a superior benefit in developing confidence and safety to perform the procedure when compared to just theoretical presentation and illustrative videos, and, in this respect, both models that were used fulfilled the goal. Literature is extensive in stating that the observation-based learning model is debatable because it does not stimulate the student’s total involvement and does not produce effective training. Thus, for skill acquisition, especially in the surgical and interventional procedure areas, sustained practice is necessary12. Bettega Ch b 7 7 model, with statistically significant difference. This result was already expected, since satisfaction with simulation as a teaching method has been consistently proven19. The use of technological advancements for teaching is imperative, primarily for younger generations of medical students, and educational formats must adapt to learning styles and preferences20. that poor positioning can result in non-adequate functioning and complications16,17. Although the checklist item called “marking the drainage site” was not evaluated, it should be considered that only the simulator model can guide the student to fulfill this requirement, since it preserves the anatomical reference points of the human chest, unlike the animal model, in which a piece of porcine rib is used for the same purpose. As limitation to the study, we can mention the subjectivity of the evaluated items in the form of a questionnaire response (using a graduated scale), since the experience of each one with the simulation depends on several personal factors, such as previous experience with simulators, perception about the use of animals for teaching, and the student’s personal experience in the offered practical activity. As didactic material, the simulator model was preferred by the students, with statistically significant difference. This can be explained by the students’ perception of being more comfortable using the simulator in relation to the animal model. The use of animals remains as a training alternative, but with increasing respect for ethical norms for their utilization and respecting their use for increasingly specific situations in which complexity is high18. The search for alternative resources to the use of animals leads to the ethical valorization in the educational environment, besides preserving the ethical, moral, psychological, and social integrity of the students13. In addition, the students stated that, for the learning of the chest drainage technique, the simulator model was also superior to the animal With our work, we could conclude that the final cost for producing the model was lower than that of a commercial simulator, what demonstrates the feasibility of using 3D printing for this purpose. In addition, the developed simulator was shown to be equivalent to the animal model in relation to the simulation of the drainage technique for practical learning, and there was preference for the simulator model as didactic material. Objetivo: criar, em impressora 3D, um simulador de baixo custo de caixa torácica humana que permita a reprodução da técnica de drenagem fechada de tórax (DFT) comparando sua eficácia com a do modelo animal. Métodos: foi realizada impressão 3D do arcabouço ósseo de um tórax humano a partir de uma tomografia de tórax. Após a impressão das costelas, foram realizados testes com diversos materiais que contribuíram para formar a simulação da caixa torácica e da pleura. Foi, então, realizado um estudo experimental, randomizado e controlado comparando sua eficácia ao modelo animal no ensino da DFT para estudantes de medicina, que foram divididos em dois grupos: Grupo Modelo Animal e Grupo Modelo Simulador, que treinaram DFT em animais e no modelo simulador, respectivamente. Resultados: a reconstrução do tórax exigiu o conhecimento anatômico para análise da tomografia e para edição fiel da superfície 3D. Não houve diferença significativa quanto à segurança de realizar o procedimento entre os grupos (7,61 vs. 7,73; p=0,398). Foi observada maior pontuação no grupo modelo simulador para uso como material didático e aprendizado da técnica de drenagem torácica quando comparado ao grupo modelo animal (p<0,05). Conclusão: o custo final para a confecção do modelo foi inferior ao de um simulador comercial, o que demonstra a viabilidade do uso da impressão 3D para esse fim. Além disso, o simulador desenvolvido se mostrou equivalente ao modelo animal quanto à simulação da técnica de drenagem para aprendizado prático e houve preferência pelo modelo simulador como material didático. Descritores: Simulação. Modelos Animais. Treinamento por Simulação. Capacitação. Educação Médica. Drenagem. Tubos Torácicos. DISCUSSION In addition, the use of some synthetic model is essential so that the student can gain confidence and, later, carry out clinical procedures on patients13. For comparison between groups, the researchers chose the checklist steps that depended on the practice in a simulator model to be performed. They were the following: approximately 1.5cm skin incision, divulsion of the planes with penetration into the pleural cavity, introduction of the index finger into the pleural orifice, drain insertion (posterosuperiorly) until the mark is reached. The results showed that there was an increase in the average of the grades in all the evaluated steps of the checklist, reinforcing the fact that, after the practical training activity, the learning is greater. There was no statistically significant difference in the average of the grades between the groups after the practical activity, what shows that the models are similar in relation to the objective of teaching the procedure for academics. However, it must be considered that the increase in the averages was higher for the SMG in three of the four evaluated parameters. It was also observed that the item called “drain insertion (posterosuperiorly) until the mark is reached” was the one which obtained the highest score increment after practical activity, in the AMG and SMG, with an increase of approximately 40% in both groups. This data is of great relevance for the teaching in medical schools, since it is known that one of the great difficulties of the apprentice doctor is the correct placement of the drain in the patient (posterosuperiorly) and In the question about how secure they felt in performing the procedure after the practical activity with the simulator, the scores were also similar for both groups, and, although the SMG presented a greater difference between the pre- and post- practice grades, there was no statistic significance between them, revealing that the simulator model may not be superior to the traditional animal model in the learning gain of the students, but it is equivalent. 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Maluf Junior I, Silva ABD, Groth AK, Lopes MAC, Kurogi AS, Freitas RDS, et al. An alternative experimental model for training in microsurgery. Rev Col Bras Cir. 2014;41(1):72-4. 4. Good ML. Patient simulation for training basic and advanced clinical skills. Med Educ. 2003;37 Suppl 1:14-21. 16. Ball CG, Lord J, Laupland KB, Gmora S, Mulloy RH, Ng AK, et al. Chest tube complications: how well are we training our residents? Can J Surg. 2007;50(6):450-8. 5. Heitz C, Eyck RT, Smith M, Fitch M. Simulation in medical student education: survey of clerkship directors in emergency medicine. West J Emerg Med. 2011;12(4):455-60. 17. Hashmi U, Nadeem M, Aleem A, Khan FUHH, Gull R, Ullah K, et al. Dysfunctional closed chest drainage - common causative factors and recommendations for prevention. Cureus. 2018;10(3):e2295. 6. Flores CD, Bez MR, Bruno RM. O uso de simuladores no ensino da medicina. Rev Bras Inform Educ. 2014;22(2):98-108. 18. Motta EV, Baracat EC. Treinamento de habilidades cirúrgicas para estudantes de medicina - papel da simulação. Rev Med (São Paulo). 2018;97(1):18-23. 7. Flato UAP, Guimarães HP. Educação baseada em simulação em medicina de urgência e emergência: a arte imita a vida. Rev Soc Bras Clin Med. 2011;9(5):360-4. 19. Lorello GR, Cook DA, Johnson RL, Brydges R. Simulation-based training in anaesthesiology: a systematic review and meta-analysis. Br J Anaesth. 2014;112(2):231-45. 8. Pezzi L, Pessanha Neto S. O Laboratório de habilidades na formação médica. Cad ABEM. 2008;4:16-22. 20. Troncon LEA, Maffei CML. A incorporação de recursos de simulação no curso de graduação em medicina da Faculdade de Medicina de Ribeirão Preto - USP. Medicina (Ribeirão Preto). 2007;40(2):153-61. 9. Netto FA, Sommer CG, Constantino MM, Cardoso M, Cipriani RF, Pereira RA. R E S U M O Objetivo: criar, em impressora 3D, um simulador de baixo custo de caixa torácica humana que permita a reprodução da técnica de drenagem fechada de tórax (DFT) comparando sua eficácia com a do modelo animal. Métodos: foi realizada impressão 3D do arcabouço ósseo de um tórax humano a partir de uma tomografia de tórax. Após a impressão das costelas, foram realizados testes com diversos materiais que contribuíram para formar a simulação da caixa torácica e da pleura. Foi, então, realizado um estudo experimental, randomizado e controlado comparando sua eficácia ao modelo animal no ensino da DFT para estudantes de medicina, que foram divididos em dois grupos: Grupo Modelo Animal e Grupo Modelo Simulador, que treinaram DFT em animais e no modelo simulador, respectivamente. Resultados: a reconstrução do tórax exigiu o conhecimento anatômico para análise da tomografia e para edição fiel da superfície 3D. Não houve diferença significativa quanto à segurança de realizar o procedimento entre os grupos (7,61 vs. 7,73; p=0,398). Foi observada maior pontuação no grupo modelo simulador para uso como material didático e aprendizado da técnica de drenagem torácica quando comparado ao grupo modelo animal (p<0,05). Conclusão: o custo final para a confecção do modelo foi inferior ao de um simulador comercial, o que demonstra a viabilidade do uso da impressão 3D para esse fim. Além disso, o simulador desenvolvido se mostrou equivalente ao modelo animal quanto à simulação da técnica de drenagem para aprendizado prático e houve preferência pelo modelo simulador como material didático. Rev Col Bras Cir 46(1):e2011 Bettega st dents Bettega t d t 8 Bettega Chest tube simulator: development of low-cost model for training of physicians and medical students. REFERENCES Teaching project: a low- cost swine model for chest tube insertion training. Rev Col Bras Cir. 2016;43(1):60-3. 10. Luna RA, Spight D. Simulação em educação médica: uma mudança necessária. Rev Hosp Univ Pedro Ernesto. 2014;13(4):57-61. Received in: 09/19/2018 Accepted for publication: 10/25/2018 Conflict of interest: none. Received in: 09/19/2018 Accepted for publication: 10/25/2018 Conflict of interest: none. Received in: 09/19/2018 Accepted for publication: 10/25/2018 Conflict of interest: none. Source of funding: none. 11. Ferreira C, Carvalho JM, Carvalho FL de Q. Impacto da metodologia de simulação realística, enquanto tecnologia aplicada à educação nos cursos de saúde. In: Seminário de Tecnologias Aplicadas a Educação e Saúde, 2, 2015 Out 26-27; Salvador (BA). Salvador: UNEB; 2015. p.32-40. Source of funding: none. Mailing address: Ana Luísa Bettega E-mail: bettega.ana@gmail.com silvaniaklug@gmail.com Rev Col Bras Cir 46(1):e2011
https://openalex.org/W4241482324
https://storage.gmth.de/zgmth/pdf/511
German
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Editorial
Zeitschrift der Gesellschaft für Musiktheorie
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ZGMTH Zeitschrift der Gesellschaft für Musiktheorie ebe, Folker (2010): Editorial. ZGMTH 7/2, 115–117. https://doi.org/10.31751/511 Editorial Die in dieser Varia-Ausgabe der ZGMTH versammelten Beiträge spiegeln die zunehmen- de Ausdifferenzierung der jüngeren Musiktheorie: Einen roten Faden auszumachen, der sie inhaltlich verbindet, bedürfte einer gewissen Mutwilligkeit. Gleichwohl wird im Lich- te eines traurigen Ereignisses sichtbar, inwiefern sie – in je unterschiedlicher Weise – von einem Paradigmenwechsel zeugen, der die musiktheoretische Landschaft so nachhaltig verändert hat, dass sich – in den Worten von Clemens Kühn – »kaum jemand vorstellen kann, es sei einmal anders gewesen«: Mit dem Tod Diether de la Mottes verliert die deutschsprachige Musiktheorie eine Persönlichkeit, deren maßgebliche Leistung es war, dass sich, so Kühn, »Analyse und Historie inzwischen mit rasanter Geschwindigkeit als Leitlinien durchgesetzt« haben. Von diesen »Leitlinien« sind auch die die deutschsprachi- gen Beiträge der vorliegenden Ausgabe bestimmt: vom Willen zur historisch differenzier- ten Perspektivierung der musikalischen Werke und ›Sprachen‹ und vom Anspruch, The- orien, Methoden und Begriffen in den Dienst »individueller Analyse« (Kühn) zu stellen. Den Anfang macht ein Artikel, der sich durch eine wohltuende Gelassenheit gegen- über den Geltungsansprüchen unterschiedlicher Denktraditionen und Analyseansätze auszeichnet: Markus Roth unterzieht François Couperins enigmatisches Cembalostück Les Ombres Errantes einer multiperspektivischen Analyse, in der ›historische‹, modell- basierte, schenkerianische und motivisch-thematische Sichtweisen einander ergänzen und relativieren. Am Ende steht ein »›Mosaik‹ aus Beobachtungen«, dass, so Roth, der Vielschichtigkeit musikalischer Kunstwerke, »die unterschiedlichste analytische Sichtwei- sen und damit Hörperspektiven zulässt«, eher gerecht werde als das Streben nach einem geschlossenen Gesamtbild. Einen konsequent produktionsästhetischen Ansatz verfolgt Jörn Arnecke. Ausgehend von dem Versuch, einen »hypothetischen Entstehungsprozess« der ersten vier Fugenthe- men aus Bachs Wohltemperiertem Klavier von 1722 zu rekonstruieren, möchte er den Modelldiskurs für ein Konzept ›nachschöpferischer Analyse‹ fruchtbar machen, Erkennt- nisse der jüngeren Forschung zu Thesen verdichten, die für die Fugenkomposition und -improvisation handlungsleitend sein können, und nicht zuletzt einen Beitrag leisten, die pädagogische Intention des Wohltemperierten Klaviers zu entschlüsseln. Analytische Befunde in ästhetische Werturteile zu überführen, ist ein problemati- sches Unterfangen: Die meisten in jüngerer Zeit ausgebildeten Musiktheoretiker können sich vermutlich kaum vorstellen, wie gegenwärtig der ›postadornitische‹ Diskurs noch in den frühen 90er Jahren an manchen deutschen Musikhochschulen war und wie ri- gide mit Verweis auf den »Stand des Materials« geurteilt wurde. Mittlerweile freilich scheinen selbst die hinter solchen Urteilen stehenden (und keineswegs von der Hand zu weisenden) Fragen weithin in Vergessenheit geraten zu sein. Die Frage, wie der »Proble- matik stilistischer Kohärenz« zu begegnen sei, sucht Jörg-Peter Mittmann am konkreten Gegenstand zu beantworten. © 2010 Folker Froebe Dieser Text erscheint im Open Access und ist lizenziert unter einer Creative Commons Namensnennung 4.0 International Lizenz. This is an open access article licensed under a Creative Commons Attribution 4.0 International License. veröffentlicht / first published: 05/07/2010 zuletzt geändert / last updated: 06/07/2010 Dieser Text erscheint im Open Access und ist lizenziert unter einer Creative Commons Namensnennung 4.0 International Lizenz. veröffentlicht / first published: 05/07/2010 zuletzt geändert / last updated: 06/07/2010 Editorial Anlass hierzu gibt die ›hybride‹ Piccola musica notturna (1954) von Luigi Dallapiccola (1904–1975), eine Zwölftonkomposition, die idiomatisch ZGMTH 7/2 (2010) | 115 EditOrial EditOrial Debussy und Ravel nahesteht. Die scheinbare Inkongruenz von Technik und Idiom, so Mittmann, bedeute nicht zwangsläufig eine »Regression gegenüber dem technischen Stand der Zeit«; zu fragen sei vielmehr nach der »sinnstiftende[n] Wechselwirkung« zwi- schen lyrischer Idee und strukturellem Konzept. Auch Lilo Kunkels Interesse gilt – nunmehr von unterrichtspraktischer Seite – dem musikalischen Impressionismus. Sie beklagt, dass die Klangsprache um 1900 in Gehör- bildung und Höranalyse, ungeachtet der Tendenz, auch diese Disziplinen historisch zu differenzieren, nach wie vor kaum eine Rolle spielt. Es bedürfe einer weitaus stärkeren Vernetzung von Theorie- und Gehörbildungsunterricht; die musikalischen Strukturen seien für die meisten Studierenden schlicht zu komplex, »um ohne fundierte Tonsatz- kenntnisse hörend erschlossen und begrifflich angemessen erfasst zu werden«. Ausge- hend von der Systematik ihres Lehrers Zsolt Gárdonyi und gestützt auf knappe Analysen charakteristischer Abschnitte der Préludes von Debussy entwickelt sie stilbezogene und methodisch vielfältige Aufgabenstellungen für den Gehörbildungsunterricht, die auch in- strumentale und vokale Übungen einbeziehen. Ian Quinn, seines Zeichens Herausgeber des amerikanischen Journal of Music Theo- ry, beschreibt ein von ihm entwickeltes, selbstlernendes Computerprogramms zur Ton- artbestimmung und erörtert dessen Leistungsfährigkeit. Obgleich der ›Mathematischen Musiktheorie‹ zugehörig, ist der Beitrag nicht nur für Spezialisten von Interesse: Zum ei- nen bezieht Quinn sich mit dem Bach-Choral auf einen Gegenstand, der zum Kernreper- toire des hiesigen ›Tonsatz‹-Unterrichts gehört; zum anderen wirft seine Fragestellung, welche Informationen ein Programm zur Tonartbestimmung benötigt, welche Fähigkei- ten es auf Grundlage automatisiert erhobener statistischer Werte erwerben kann und welche Vorgaben zu welchen Ergebnissen führen, ein Licht auch auf die elementaren, oft unzureichend reflektierten Voraussetzungen basaler Musikanalyse. Quinn als Autor für die ZGMTH gewonnen zu haben, sei Anja Volk an dieser Stelle herzlich gedankt. Drei weitere Beiträge widmen sich – wie jene Roths und Arneckes – wiederum der Musik des 18. Jahrhunderts: Die Organistin und Cembalistin Nicoleta Paraschivescu hat sich die »Wiederentdeckung der Partimento-Praxis als Kunstform« auf die Fahnen ge- schrieben. Ihr Bezugspunkt ist eines der wenigen aus dem 18. Jahrhundert überkom- menen Beispiele für die vollständige Ausarbeitung eines Partimento-Basses, Francesco Durantes Perfidia-Sonate. Paraschivescu erkennt Parallelen zwischen dem didaktischen Konzept, den zur Übung mitgeteilten Partimento-Bässen kurze Aussetzungsmodelle bei- zugeben, und der Anlage der Sonate: Diese gleiche einer kunstvollen Aneinanderreihung exemplarischer ›Modi‹ oder ›Pensieri‹. ZGMTH 7/2 (2010) | 117 Editorial Folker Froebe nimmt seine Rezension der von Ludwig Holtmeier, Johannes Menke und Felix Diergarten herausgegebenen und instruktiv kommentierten Regole per bene accompagnare il partimento o sia il basso fondamentale sopra il Cembalo des Haydn- Zeitgenossen Giovanni Paisiello zum Anlass, die Wiederentdeckung der Partimento-Tra- dition in grundsätzlicher Form zu würdigen. Felix Diergarten bespricht Markus Walduras Habilitationsschrift Von Rameau und Riepel zu Koch. Zum Zusammenhang zwischen theoretischem Ansatz, Kadenzlehre und Periodenbegriff in der Musiktheorie des 18. Jahrhunderts. Diergartens Kritik an einer Musiktheorie, der das historische Kolorit ihrer Begriffe bisweilen zum Selbstzweck ge- 116 | ZGMTH 7/2 (2010) EditOrial worden sei, mahnt, nicht aus den Augen zu verlieren, was der Zweck jeder historischen Differenzierung sein sollte und was vor 34 Jahren de la Mottes Reform der Harmonie- lehre motivierte: der Vielfalt musikalischer ›Sprachen‹ gerecht zu werden und – in den Worten de la Mottes – »Faszination« für »die Entwicklung und Wandlung der musikali- schen Sprache« zu vermitteln. Clemens Kühn schließlich findet seinem Lehrer und Freund Diether de la Motte zum Gedenken sehr persönliche Worte, die dessen Besonderheit und Bedeutung nicht nur als Musiktheoretiker umfassend würdigen. Folker Froebe ZGMTH 7/2 (2010) | 117 ZGMTH 7/2 (2010) | 117 ZGMTH 7/2 (2010) | 117
https://openalex.org/W4205357747
http://ray.yorksj.ac.uk/id/eprint/5892/1/2021_DNSoverHTTP_Detection_to%20submit-2.pdf
English
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An Explainable AI-based Intrusion Detection System for DNS over HTTPS (DoH) Attacks
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Downloaded from: https://ray.yorksj.ac.uk/id/eprint/5892/ The version presented here may differ from the published version or version of record. If you intend to cite from the work you are advised to consult the publisher's version: https://www.techrxiv.org/articles/preprint/An_Explainable_AI- based_Intrusion_Detection_System_for_DNS_over_HTTPS_DoH_Attacks/17696972 Research at York St John (RaY) is an institutional repository. It supports the principles of open access by making the research outputs of the University available in digital form. Copyright of the items stored in RaY reside with the authors and/or other copyright owners. Users may access full text items free of charge, and may download a copy for private study or non-commercial research. For further reuse terms, see licence terms governing individual outputs. Institutional Repository Policy Statement Tahmina, Zebin, Rezvy, Shahadate ORCID logoORCID: https://orcid.org/0000-0002-2684-7117 and Yuan, Luo (2022) An Explainable AI-based Intrusion Detection System for DNS over HTTPS (DoH) Attacks. Techrxiv. (Unpublished) Downloaded from: https://ray.yorksj.ac.uk/id/eprint/5892/ This paper was downloaded from TechRxiv (https://www.techrxiv.org). LICENSE An Explainable AI-based Intrusion Detection System for DNS over HTTPS (DoH) Attacks Tahmina Zebin,Shahadate Rezvy , Yuan Luo DoH encrypts DNS queries, which are disguised as regular HTTPS traffic, hence the DNS-over-HTTPS name. These DoH queries are sent to special DoH-capable DNS servers (called DoH resolvers), which resolve the DNS query inside a DoH request and reply to the user, also in an encrypted manner. DoH can be used instead of traditional DNS for domain name translation with encryption as a benefit [2]. Abstract—Over the past few years, Domain Name Service (DNS) remained a prime target for hackers as it enables them to gain first entry into networks and gain access to data for exfiltration. Although the DNS over HTTPS (DoH) protocol has desirable properties for internet users such as privacy and security, it also causes a problem in that network administrators are prevented from detecting suspicious network traffic generated by malware and malicious tools. To support their efforts in maintaining a secure network, in this paper, we have implemented an explainable AI solution using a novel machine learning framework. We have used the publicly available CIRA-CIC- DoHBrw-2020 dataset for developing an accurate solution to detect and classify the DNS over HTTPS attacks. Our proposed balanced and stacked Random Forest achieved very high pre- cision (99.91%), recall (99.92%) and F1 score (99.91%) for the classification task in hand. Using explainable AI methods, we have additionally highlighted the underlying feature contributions in an attempt to provide transparent and explainable results from the model. The companies and organizations that have DoH-capable products have been advertising DoH as a way to prevent ISPs from tracking users’ web traffic and as a way to bypass cen- sorship in oppressive countries. The readability of translated domain names in the traffic is exploited in application firewalls to check security policies, and intrusion detection systems to detect suspicious connections. Therefore, this paper focuses on the possibilities of encrypted traffic analysis, especially for the purpose of accurate detection of DoH attacks. We have implemented one of the very first explainable AI solutions to provide an accurate solution to detect and classify the DNS over HTTPS attacks. We have applied further explainable AI methods to make the solution reliable and interpretable to the human user. The remainder of this paper is organized as follows. CITATION Zebin, Tahmina; Rezvy,, Shahadate; Luo, Yuan (2022): An Explainable AI-based Intrusion Detection System for DNS over HTTPS (DoH) Attacks. TechRxiv. Preprint. https://doi.org/10.36227/techrxiv.17696972.v1 10.36227/techrxiv.17696972.v1 TRANSACTIONS ON INFORMATION FORENSICS AND SECURITY, VOL. 0, NO. 0, DECEMBER 2021 2 An Explainable AI-based Intrusion Detection System for DNS over HTTPS (DoH) Attacks Section II introduces the background literature in the recent development in DoH attack detection methods used in recent years, we introduced the concept of explainable AI in this section. We then introduced our dataset and the associated pre-processing stages in Section III. Section IV provides details on the workflow, model architecture and pa- rameter settings for the implemented model. The performance of the developed model for attack classification is evaluated in Section V. We presented the explanations from the model using a model dashboard we have deployed from the task in Section VI. Finally, the paper is concluded along with ideas for future work in the very last section. Index Terms—Secure Computing, Machine Learning, Intru- sion Detection System, Explainable AI. { } S. Rezvy is a lecturer at the Department of Computer Science, School of Science, Technology and Health, York St John University, UK. Email: {s.rezvy}@yorksj.ac.uk. I. INTRODUCTION D D OMAIN Name System (DNS) traffic is crucial for many existing security systems. Since an application must translate a domain name before a connection can be established, DNS traffic can identify many observable security threats in the network traffic. As per the EfficientIP and IDC 2021 Global DNS Threat Report, around 87% of the surveyed organizations have experienced DNS attacks in 2021 which is 8% more than the statistics in 2020 [1]. With the pandemic in recent times, a rapidly increasing number of people remotely working and using various cloud services on a daily basis, an increasing amount of cyber-attacks are disrupting the online services. The impact and cost of attacks remain extremely high and it affects company finances but also brand image and data confidentiality. Organizations have suffered more diverse types of attacks than ever before, showing that cyber-criminals are using all the tools at their disposal to exploit both the DNS protocol and misconfigurations. T. Zebin is a lecturer at the School of Computing science, The University of East Anglia, Norwich, UK. Email: {t.zebin}@uea.ac.uk. { } Y. Luo is a Senior Lecturer at the Faculty of Science and Technology, Middlesex University,London, UK. Email: {y.luo}@mdx.ac.uk. T. Zebin is a lecturer at the School of Computing science, The University of East Anglia, Norwich, UK. Email: {t.zebin}@uea.ac.uk. S. Rezvy is a lecturer at the Department of Computer Science, School of Science, Technology and Health, York St John University, UK. Email: {s.rezvy}@yorksj.ac.uk. Y. Luo is a Senior Lecturer at the Faculty of Science and Technology, Middlesex University,London, UK. Email: {y.luo}@mdx.ac.uk. II. BACKGROUND LITERATURE This section discusses the literature relevant for DoH de- tection or the detection of malicious use of DoH. The related work is divided into multiple parts. It starts with the broad scope of detecting malicious DNS traffic in general including available datasets and the learning techniques used for the classification of encrypted data. The literature then gradually narrows down to the recent related work that has specifically used the dataset similar to this research for the detection of malicious DoH traffic. We then presented some background concepts necessary for the use of explainable AI in DoH intrusion detection systems. Previously, DNS queries were made in plaintext, from an app to a DNS server, using the DNS settings of the local operating system received from its network provider, usually an Internet Service Provider (ISP). In recent times, a new protocol DNS over HTTPS (DoH) has been created to improve users’ privacy on the internet. DoH changes this paradigm. A. DoH attack detection : Learning Techniques A. DoH attack detection : Learning Techniques Numerous organizations have less active monitoring plans in terms of security checking on DNS as it is not used for TRANSACTIONS ON INFORMATION FORENSICS AND SECURITY, VOL. 0, NO. 0, DECEMBER 2021 3 Fig. 1. The technical framework of DNS tunnels for generating Malicious DoH traffic. Fig. 1. The technical framework of DNS tunnels for generating Malicious DoH traffic. Data transfer, compared to other protocols like Web activity where attacks often take place. A malicious DNS attack can aim to exploit security vulnerabilities on the server that runs the DNS services and extract valuable data such as passwords, usernames, and other personal information. Since most of the Internet’s traffic is encrypted and served by large content delivery networks, in many cases, domain name systems are the only clear text sign about the specific service being accessed. DNS Tunneling is a method of cyber-attack that encodes the data of other programs or protocols in DNS queries and responses. DNS tunnels, established between the controlled host and master server disguised as the authoritative domain name server, can be used as a secret data commu- nication channel for malicious activities. Cybercriminals use multiple tunnelling techniques such as FTP-DNS tunnelling, HTTP-DNS tunnelling, HTTPS-DNS tunnelling, and POP3- DNS tunnelling to hide their identity[3]. Owing to the ready evasion of the DNS traffic to bypass the network security mechanism, DNS tunnelling can cause severe damage. DNS tunnelling often includes data payloads that can be added to an attacked DNS server and used to control a remote server and applications. Several earlier methods proposed to detect malicious DNS traffic include Network and DNS Traffic Analysis [4], [5], Domain name blacklisting, and Detailing of Web Page Content by the visual platform to protect top- level domain name servers against DDoS attacks [6]. Aiello et al.[4] combined principal component analysis (PCA) and mutual information (MI) to calculate a novel metric as the identification index, based on several statistical features. How- ever, they found that the different circumstances of DNS server size or the traffic encapsulated in DNS tunnelling would cause diverse manifestations of the value. Hence, the threshold could only be determined based on the condition of the non- overlapping of the mi value between legitimate and malicious traffic, which is affected by many environmental factors. In other words, it indicates the poor flexibility and generality of this method. A. DoH attack detection : Learning Techniques Other works have focused on predicting the validity of information coming from the DNS and do not take into account that DNS data for the malicious activity have statistical, temporal and payload related differences, so the results obtained were less effective [6], [7]. Some very recent research focused on analysing and de- tecting malicious and encrypted DNS traffic using various machine learning techniques. The research in [8] focused on the primary domain as a filter to classify the DNS traffic rather than the queries. The features have been extracted from sub-domains of multiple groups. The author used supervised machine learning for examining DNS traffic and filtering benign and malicious domains. However, this approach has a limitation of the inability to detect malicious queries in the main domain. In which the sub-domain is not enough for detecting the other types of attacks. Banadaki et al. [9] examined the dataset called CIRA-CIC-DoHBrw-2020[10] using several ML algorithms such as (Xgboost, Gradient Boosting, and Light Gradient Boosting algorithms). However, the preprocessing and optimization phase were unclear. In this research, we are proposing one of the very first explainable AI solutions to provide an accurate solution to detect and classify the DNS over HTTPS attacks. In the next subsection, we will discuss the introductory explainable AI methods used in this research for DoH attack detection. TRANSACTIONS ON INFORMATION FORENSICS AND SECURITY, VOL. 0, NO. 0, DECEMBER 2021 TABLE I TRAFFIC TYPES CAPTURED IN CIRA-CIC-DOHBRW-2020 DATASET [10] Traffic Type Instances captured Description of the Attack Non-DoH 889809.0 Traffic generated by accessing a website that uses HTTPS protocol is captured. In order to capture ample traffic to balance the dataset, thousands of websites from Alexa domain are browsed. Benign-DoH 19746.0 Benign DoH is non-malicious DoH traffic generated using Mozilla Firefox and Google Chrome web browsers. Malicious-DoH 249553.0 DNS tunneling tools such as dns2tcp, DNSCat2, and Iodine are used to generate malicious DoH traffic. These tools create tunnels of encrypted data. Therefore, DNS queries are sent using TLS-encrypted HTTPS requests to special DoH servers Fig. 2. Density plots displaying the difference in a number of features for the three types of traffic available in the dataset TABLE I TRAFFIC TYPES CAPTURED IN CIRA-CIC-DOHBRW-2020 DATASET [10] Traffic Type Instances captured Description of the Attack Non-DoH 889809.0 Traffic generated by accessing a website that uses HTTPS protocol is captured. In order to capture ample traffic to balance the dataset, thousands of websites from Alexa domain are browsed. Benign-DoH 19746.0 Benign DoH is non-malicious DoH traffic generated using Mozilla Firefox and Google Chrome web browsers. Malicious-DoH 249553.0 DNS tunneling tools such as dns2tcp, DNSCat2, and Iodine are used to generate malicious DoH traffic. These tools create tunnels of encrypted data. Therefore, DNS queries are sent using TLS-encrypted HTTPS requests to special DoH servers Fig. 2. Density plots displaying the difference in a number of features for the three types of traffic available in the dataset TABLE I Benign DoH is non-malicious DoH traffic generated using Mozilla Firefox and Google Chrome web browsers. DNS tunneling tools such as dns2tcp, DNSCat2, and Iodine are used to generate malicious DoH traffic. These tools create tunnels of encrypted data. Therefore, DNS queries are sent using TLS-encrypted HTTPS requests to special DoH servers Fig. 2. Density plots displaying the difference in a number of features for the three types of traffic available in the dataset 2. Density plots displaying the difference in a number of features for the three types of traffic available in the dataset along with non-DoH traffic. In the first layer, the Non-DoH activity is generated by accessing different web servers. The DoH traffic has been collected using Several DNS tunnelling tools have been used such as DNSCat2, Iodine, and dns2tcp [12], [13]. TRANSACTIONS ON INFORMATION FORENSICS AND SECURITY, VOL. 0, NO. 0, DECEMBER 2021 In layer 2 data collection, Malicious-DoH traffic is generated using the above-mentioned tunnelling tools, where these tools sent TLS-encrypted HTTPS data in DNS queries to DoH servers (Adguard, Cloudflare, Google, Quad9). Fig. 1 is shows the technical framework of DNS tunnels for generating Malicious DoH traffic. To capture Benign-DoH traffic, Several web browsers such as Chrome, Firefox, and safari have been used to generate Benign-DoH in the same mechanism as in scenario Non-DoH. by the sophisticated algorithm. Therefore, in this paper, we have addressed the XAI concept to enhance trust management by exploring the decision tree model in the area of IDS. Over the last few years, there has been significant progress on Explainable AI. The pursuit of converting these black- box models into transparent and interpretable algorithms has gained traction in both academia, industry and other users of AI and machine learning models. While many packages and methodologies have developed in recent years, one of the most popular methods today, SHAP (SHapley Additive exPlanations) is a game theory-based approach to explain the output of any ML model [11]. It connects optimal credit allocation with local explanations using the classic Shapley values from game theory and their related extensions. SHAP does a great job in decoding the strength of the influence of the input variables in the predictions with intuitive and engaging visualizations across various aspects of model explainability. SHAP values calculate the feature importance by comparing what a model predicts with and without the feature. However, since the order in which a model sees features can affect its predictions, this is done in every possible order, so that the features are fairly compared. A. Exploratory Feature Analysis The features of this dataset can be divided into multiple broad categories. Flow Statistics is one of the categories containing features such as the duration of the flow and the number of packets sent or received in that flow. The category Flow Bytes contains features describing the number of total bytes sent and/or received. Furthermore, there is a Packet Length category containing statistical features about the packet lengths such as mean value or standard deviation. There are similar statistical features calculated for the Packet Time and Inter-Packet Delay categories. At the time of writing this paper, there was not much explainable AI literature available for DoH attack detection and classification. Hence we will present a thorough discussion and the explanations from our proposed model using a model dashboard in Section VI in this paper. To learn the differences in characteristics of DoH and non- DoH, thorough feature analysis is performed in this section. The value distribution for the feature is plotted using a Kernel Density Estimation (KDE) plot. Fig. 2 shows the class- wise density distribution for Flow Bytes Received, packet length mean and variance features. A KDE plot is similar to a histogram, however, the KDE plot shows the estimation of the probability density function of a variable instead of B. Explainable AI for DoH attack detection Despite the growing popularity of machine learning models in cyber-security applications (e.g., an intrusion detection system (IDS)), most of these models are perceived as a black- box. The eXplainable Artificial Intelligence (XAI) has become increasingly important to interpret the machine learning mod- els to enhance trust management by allowing human experts to understand the underlying data and to understand the impact of the malicious data to detect any intrusion in the system. The previous studies focused more on the accuracy of the various classification algorithms for trust in IDS. They do not often provide insights into their behaviour and reasoning provided B. Pre-processing: Scaling and Normalization All numeric feature values are ranged between 0 and 1. We performed a min-max normalization on the feature vectors to prevent value related bias during training. D. Train-test Split The dataset has been split into training and testing sets of 90%, 10% respectively. As can be seen from the system overview diagram in Fig. 3, we have created three balanced splits from the 90% training data to feed three independent sub-models. We have used three different splits of Non-DoH data while sharing the same malicious samples over various divisions. We applied SMOTE up-sampling of the benign group to avoid any evident bias from the majority groups available in the dataset. With this sub-division, we had to use less amount of synthetic data from the minority group per sub-model. The training sets were further split into 10 folds to allow 10 fold cross-validation. To be noted, all of our experiments were performed on a Linux machine with an Intel Core i9 processor, 64 GB RAM and an NVIDIA RTX GPU. B. Balanced and Stacked Classifier for Higher Predictive Performance The simplest form of stacking can be described as an ensemble learning technique where the predictions of multiple classifiers are used as new features to train a meta-classifier [19]. The functional stages of the proposed algorithm is outlined in Fig. 3. The workflow is demonstrating the stacking scheme we used to train and implement our multi-class traffic detection model. The meta-classifier of our choice is a logistic regression model. All the sub-models in this diagram are Ran- dom forest models with numTrees=10, and has a maximum IV. MODEL IMPLEMENTATION discrete bins. The duration of a network flow is a feature clearly distinguishing malicious DoH from non-DoH, with DoH flows having comparatively longer duration. The non- DoH web traffic network flows have a short duration since the whole web page is fetched in only a few seconds. We noticed some differences in the Flow byte measurements as well, the number of bytes sent or received for malicious DoH is clearly higher compared to non-DoH and benign-DoH (shown in the KDE plot in Fig. 2(a). This is shown for the flow bytes received in As we looked into the variance of incoming packets (shown in the KDE plot in Fig. 2(c)), in general, DoH flows have more consistent packet lengths, resulting in a smaller variance compared to non-DoH. The outgoing packets showed similar properties. An interesting difference between benign and malicious DoH is that the variance for malicious DoH is always relatively high due to alternating small and larger packets. A full list of the 29 features in CIRA-CIC-DoHBrw- 2020 dataset can be found in [13] and also in Fig. 5. In this study, we attempted to identify DoH traffic generated by various malicious DNS tunnel tools. The payload of DoH traffic is encrypted; thus, its content cannot be accessed. There- fore, we have used the statistical features of the packets to analyze the traffic in detail. For the detection and classification task, we employed two main functional stages in our proposed model. A balanced-training layer with multiple sub-models and a stacked classifier for classification based on DNS over HTTPS intrusion features. We describe our intuition for using these components in the system development in the coming subsections. To be noted, we have also trained Decision Tree, Random Forest, and Xgboost Classifiers for the purpose of mode performance comparison before settling to our final Balanced and Stacked Random Forest Classifiers. A. Base-Classifiers: Random Forests For our model implementation, we are using a popular ensemble classifier called the Random Forest [15] which operates by constructing multiple decision tree models at the training time. It is one of the most accurate supervised learning methods in recent times. Each decision tree in a Random Forest represents one class of observations that are being considered. Decision trees are constructed during the learning process with the training data Random Forests mainly rely upon two parameters to control their growth: numTrees, the number of decision trees to be built and numFeatures, the number of random subset of features to assess at each tree node[16]. In our design, numTrees = 10 and numFeatures = 28. Each of the 10 decision trees is constructed in a top- down manner starting with a root node by selecting a set of N observations of size n at random with replacement from the training dataset and selecting the most significant features of these samples as the tree nodes. At each node a, the m number of features is selected at random from 28 features to grow the tree and the most significant feature that provides the best binary split on that node is selected among all according to an objective function. Feature significance is generally estimated using the Gini index[17]. To classify a new sample, the features values of the samples are tested with each of the decision trees present in the random forest. Each tree gives a classification score or “vote” and the class with the most votes is selected as the class to which the sample belongs. We have used the RandomForestClassifier from the sklearn.ensemble module in python for training the models [18]. C. Dataset resampling To deal with the class imbalance in the training data, we have used a one-sided selection with synthetic Minority Over- sampling Technique (SMOTE) technique [14] while preparing our training data. III. DATASET DESCRIPTION AND PRE-PROCESSING For experimenting, we have used the publicly available CIRA-CIC-DoHBrw-2020 dataset[10] from the Canadian In- stitute for Cybersecurity (CIC). In this dataset, a two-layered approach is used to capture benign and malicious DoH traffic TRANSACTIONS ON INFORMATION FORENSICS AND SECURITY, VOL. 0, NO. 0, DECEMBER 2021 5 TRANSACTIONS ON INFORMATION FORENSICS AND SECURITY, VOL. 0, NO. 0, DECEMBER 2021 6 Fig. 3. Functional stages of the Balanced Stacked Random Forest Classifier for Benign, Malicious DoH and NonDoH Classification nal stages of the Balanced Stacked Random Forest Classifier for Benign, Malicious DoH and NonDoH Classification Fig. 3. Functional stages of the Balanced Stacked Random Forest Classifier for Benign, Malicious DoH and NonDo Precision = TP TP + FP . (3) (3) branch depth of 5 in the individual decision trees to keep the computation fast enough during the prediction stage. For the implementation, we have used the StackingClassifier from the mlxtend.classifier module [20]. F1 Score = 2 ∗Precision ∗Recall Precision+Recall (4) (4) A. Model evaluation matrices If True Positive (TP ) is the number of attacks classified rightly as attack; True Negative (TN) is the number of normal events rightly classified normal; False Positive (FP ) is the number of normal events misclassified as attacks and False Negative (FN) is the number of attacks misclassified as normal, we can define accuracy, recall, precision and F1 values of a model using the following equations. • Accuracy: It is an indicator of the total number of correct predictions provided by the model and defined as follows: Accuracy = TP + TN TP + TN + FP + FN . (1) (1) B. Confusion Matrix Once the model is trained, we evaluated how well the model is performing on test data from various classes. For that, we have reported the scores such as accuracy, precision, recall, Area Under the ROC Curve (AUC), and F1-scores since these are directly comparable with other studies. Additionally, confusion matrices are reported to give insights into the strong and weak points of the classifiers, it shows which classes are often misclassified. A definition of the evaluation matrices is provided in the next sub-section. We presented the confusion matrix plot in Fig. 4, for our model when evaluated with the test data set. The columns correspond to the predicted class and the rows correspond to the true class (Actual Class). The diagonal cells in the confusion matrix correspond to observations that are correctly classified (TP and TN’s). The off-diagonal cells correspond to incorrectly classified observations (FP and FN’s). Both the number of observations and the percentage of the total number of observations are shown side by side. For the proposed balanced stacked random forest classifier, class-wise model performance for train and test set is shown in confusion matrices (a) and (b) respectively. As can be seen in Fig. 4 (b), the proposed stacked random forest was able to detect 24949 malicious out of 24955, with only six misclassified instances where the model predicted those as NonDoH. The major source of misclassification was observed in the model for benign instances classified as NonDoH, these errors are caused due to the test instance benign similar in nature to NonDoH in terms of models top predictive features such as duration, packet length etc, However, these errors are less damaging to the system because of their benign nature. E. Hyperparameter Tuning and Cross-Validation For finding the optimal hyperparameters, resulting in the best classification, we used the GridsearchCV function that is used for the exhaustive search. In the search, different models are trained covering all (manually) pre-configured parameter values. Each model is tested after training and the search was done with 10- fold cross-validation so that the selected parameters are less susceptible to outliers. TRANSACTIONS ON INFORMATION FORENSICS AND SECURITY, VOL. 0, NO. 0, DECEMBER 2021 A. Feature importance plots As DoH can be used for benign and malicious purposes, so if DoH is detected, the analysis of the features that are helping to detect DoH traffic would be highly beneficial. For our use case, Fig. 5 is highlighting a SHAP summary plot from the proposed model that is giving us a birds-eye view of global feature importance obtained from the training data. On the X- axis of the summary plot, we have the average impact ( mean absolute SHAP values) of a particular feature on the decision making of a particular sample. SHAP values show how much a given feature changed our prediction (compared to if we made that prediction at some baseline value of that feature). On Y- axis the features are presented according to their importance globally from the entire training set. From our visualization, we found out that the duration of a network flow and the packet length related features were the features that helped the model heavily to distinguish the malicious DoH from Non-DoH. The packet length related features were found to be most powerful in separating the benign DoH traffic from the dataset. Fig. 4. Class-wise model performance for train and test set is shown in confusion matrices (a) and (b) respectively. For each case, class-wise accuracy is shown as a percentage of predicted on the left and a count of instances was shown on the right. with SMOTE balancing. Compared to the other classifiers, the created ensemble framework misclassified a few samples from NonDoH and benign class but, there is only six wrong classi- fications in malicious class. And a very low misclassification for the malicious class would be desirable in this scenario. In the table, we have also compared the AUC score for the various classifiers we have developed for the task. B. Dependence plots and Interaction plots From the results showed in Fig. 4 and Table II, we noticed our system can identify malicious DNS traffic with more than 99% accuracy. The model can distinguish DoH traffic from normal HTTPS network traffic 99.9% of the time and the class-wise accuracy of Benign, Malicious and Non-DoH traffic on the test set was found to be 97.3%, 99.99% and 99.8% respectively. In Table II, we have also reported the AUC, F1-score, precision and recall value from the model. In comparison to state-of-the-art ensemble models such as gradient boosting and generic random forest, our proposed balanced and stacked random forest achieved slightly higher precision (99.91%), recall(99.92%) and F1 score (99.91%) which is desirable for the task at hand. For the purpose of comparison, the Xgboost model had a precision value of 99.56%), a recall value of 97.322% and an F1-score of 98.43% in this scenario. It is also possible to create local summary plots displaying positive SHAP-values indicative of a feature supporting the decision confidence. Negative SHAP-values are indicative of the feature negatively impacting the decision confidence. SHAP dependence plots provide useful insight if we want to delve into the impact of a single feature in terms of the samples the model has processed. In Fig. 6, we have plotted the Duration and FlowByteSent feature for all the samples in the test set, where each dot represents a row of the data. The horizontal location is the actual value from the dataset, and the vertical location shows the SHAP impact value for that prediction. Higher the SHAP value, the bigger the impact of the feature for one observation in its decision making. In these diagrams, the malicious traffic is indicated with positive SHAP values, benign and nonDoH traffic represented using negative shap values for classification. In Fig. 6(a) we plotted the duration of all the observations of the test set to monitor the impact of duration in the model’s classification. Most of the instances, that were classified as malicious DoH traffic by the model is having a duration above 40 seconds. In Fig. 6(b) We are showing an interaction plot of FlowBytesSent with FlowBytesReceived. The interesting grouping is revealed on the upper left cluster in this plot when bytes received sent in some instances is bigger than bytes C. Performance comparison • Recall, precision and F1 Score: Three of the most com- monly used performance measures with F1 score being the harmonic mean of recall and precision measures are defined as follows: • Recall, precision and F1 Score: Three of the most com- monly used performance measures with F1 score being the harmonic mean of recall and precision measures are defined as follows: For comparison purposes, we have also trained a Decision Tree, a Random Forest and an Xgboost (Gradient Boosting) Classifier along with our final Balanced Stacked Random Forest Classifier. As shown in Table II, the proposed classifier results are better than the generic ensemble learning frame- work such as the Gradient Boosting and The RF classifier Recall or True positive rate = TP TP + FN . (2) (2) TRANSACTIONS ON INFORMATION FORENSICS AND SECURITY, VOL. 0, NO. 0, DECEMBER 2021 7 TABLE II MODEL ACCURACY COMPARISON IN TERMS OF AREA UNDER THE CURVE (AUC), CLASSIFICATION ACCURACY (CA), PRECISION, RECALL AND F1-SCORE FOR NONDOH, BENIGN AND MALICIOUS DOH TRAFFIC CLASSIFICATION Three-class model AUC Accuracy F1 Precision Recall Decision Tree (Tree Depth=10) 0.8617 0.9770 0.8197 0.9658 0.7120 Gradient Boosting(Xgboost) 0.9986 0.9927 0.9843 0.9956 0.9732 Random Forest (number of Trees=10, SMOTE balanced) 0.9999 0.9998 0.9987 0.9989 0.9985 Proposed model (Balanced Stacked random forest) 0.9999 0.9998 0.9991 0.9991 0.9992 potential biases. It helped us characterize model accuracy, TABLE II A UNDER THE CURVE (AUC), CLASSIFICATION ACCURACY (CA), PRECISION, RECALL AND OH, BENIGN AND MALICIOUS DOH TRAFFIC CLASSIFICATION TABLE II potential biases. It helped us characterize model accuracy, transparency and outcomes to be validated by a human user. TRANSACTIONS ON INFORMATION FORENSICS AND SECURITY, VOL. 0, NO. 0, DECEMBER 2021 An interesting grouping is revealed on the upper left cluster in this plot when bytes received sent in some instances is bigger than bytes originally sent indicating the suspicious or probable malicious nature of these grouped instances. certain decision with the help of the dashboard functionalities we have put together for this. originally sent indicating the suspicious or probable malicious nature of the grouped instance. In Fig. 7, we are demonstrating a detailed explanation of a malicious test packet by the model. The deployed model dash- board provides a prediction probability (table and pie chart on the top left), which is 73.3% for this Malicious instance. The contribution plot below provides a further breakdown of which feature contributed positively or negatively in models decision making for this particular instance. For example, this test instance with a duration of 126.43 seconds, which is above the threshold for NonDoH and benign traffic contributed positively (+16.95%) to models decision for classifying this instance as TRANSACTIONS ON INFORMATION FORENSICS AND SECURITY, VOL. 0, NO. 0, DECEMBER 2021 8 Fig. 5. SHAP summary plot from the model that is giving us a birds-eye view of feature importance. From our analysis, we found the duration of a DoH traffic is the most important predictor of whether the traffic is malicious or not, followed by some features related to packet length and variance in packet time. On the right, the list of features is broadly categorised into flow statistics, flow bytes, packet length, packet time and Inter-packet delay features. Fig. 5. SHAP summary plot from the model that is giving us a birds-eye view of feature importance. From our analysis, we found the duration of a DoH traffic is the most important predictor of whether the traffic is malicious or not, followed by some features related to packet length and variance in packet time. On the right, the list of features is broadly categorised into flow statistics, flow bytes, packet length, packet time and Inter-packet delay features. Fig. 6. (a) SHAP dependence plot for Duration as a feature. On y-axis we have the SHAP Values for each observation. This plot holds the duration of all the observations of the test set to monitor the impact of duration in the model’s classification. Each dot represents a row of the data. We can infer from this diagram that the model is using a duration threshold above 40 seconds to determine malicious DoH traffic reflected by the positive SHAP Values above this range. (b) Interaction plot of FlowBytesSent and FlowBytesReceived. An interesting grouping is revealed on the upper left cluster in this plot when bytes received sent in some instances is bigger than bytes originally sent indicating the suspicious or probable malicious nature of these grouped instances. Fig. 6. (a) SHAP dependence plot for Duration as a feature. On y-axis we have the SHAP Values for each observation. This plot holds the duration of all the observations of the test set to monitor the impact of duration in the model’s classification. Each dot represents a row of the data. We can infer from this diagram that the model is using a duration threshold above 40 seconds to determine malicious DoH traffic reflected by the positive SHAP Values above this range. (b) Interaction plot of FlowBytesSent and FlowBytesReceived. VI. EXPLAINING THE DECISIONS USING XAI In this section, we highlighted our use of XAI methods to visualize the decision-making process of our proposed model. We used the methods available from the SHAP (SHapley Ad- ditive exPlanations) library to look into the model’s decision- making process, expected impact from various features and TRANSACTIONS ON INFORMATION FORENSICS AND SECURITY, VOL. 0, NO. 0, DECEMBER 2021 TRANSACTIONS ON INFORMATION FORENSICS AND SECURITY, VOL. 0, NO. 0, DECEMBER 2021 TRANSACTIONS ON INFORMATION FORENSICS AND SECURITY, VOL. 0, NO. 0, DECEMBER 2021 9 Fig. 7. Explaining a malicious test packet. On the top left, the dashboard provides a prediction probability and a pie chart shows the percentages. The label with the asterisk sign is the label the model outputs as the decision. On the right, we have a contribution table with the value of each feature of the data sample being processed by the model and their effect value or positive-negative contribution in models decision making. On the bottom left, we have the feature contribution plot as a waterfall plot, green bars displaying positive contributors and the red bars displaying negative contributors. Fig. 7. Explaining a malicious test packet. On the top left, the dashboard provides a prediction probability and a pie chart shows the percentages. The label with the asterisk sign is the label the model outputs as the decision. On the right, we have a contribution table with the value of each feature of the data sample being processed by the model and their effect value or positive-negative contribution in models decision making. On the bottom left, we have the feature contribution plot as a waterfall plot, green bars displaying positive contributors and the red bars displaying negative contributors. Fig. 8. Explaining a Non-DoH test packet. For this decision, we can see the model had an 86.1% confidence score for Non-DOH based on the feature averages and the impact the model has seen for various samples during the training phase. Fig. 8. Explaining a Non-DoH test packet. For this decision, we can see the model had an 86.1% confidence score averages and the impact the model has seen for various samples during the training phase. Fig. 8. Explaining a Non-DoH test packet. For this decision, we can see the model had an 86.1% confidence score for Non-DOH based on the feature averages and the impact the model has seen for various samples during the training phase. malicious. The next highest contribution was coming from a high packet time variance measurement for this case. However, the packet length skew from the mode for this transaction was very low, which affected the model confidence value negatively by 1.77%. that way. TRANSACTIONS ON INFORMATION FORENSICS AND SECURITY, VOL. 0, NO. 0, DECEMBER 2021 In this instance, the PacketTimeMedian value of this traffic was matching more to the average of the malicious group than to the Non-DOH group of traffic the model was trained with. C. Explaining a malicious and a non-DoH test packet We have deployed the model we have trained for this task on a live Explainer Dashboard. Fig. 7 and Fig. 8 is using a contribution table and a contribution plot to break down the contribution of various feature values for an exemplar malicious DOH traffic and a Non-DOH traffic sample from the dataset. We have used 29 features to classify DoH traffic in our model and it is possible to break down how much each feature contributed in generating the confidence value of a VII. CONCLUSIONS pp [9] Y. M. Banadaki, “Detecting malicious dns over https traffic in domain name system using machine learning classifiers,” Journal of Computer Sciences and Applications, vol. 8, no. 2, pp. 46–55, 2020. pp [10] Cira-cic-dohbrw-2020 dataset, 2020. [Online]. Available: https://www.unb.ca/cic/datasets/dohbrw-2020.html. [11] S. M. Lundberg and S.-I. Lee, “A unified approach to interpreting model predictions,” in Proceedings of the 31st international conference on neural information processing systems, 2017, pp. 4768–4777. [12] M. T. Jafar, M. Al-Fawa’reh, Z. Al-Hrahsheh, et al., “Anal- ysis and investigation of malicious dns queries using cira- cic-dohbrw-2020 dataset,” Manchester Journal of Artificial Intelligence and Applied Sciences, vol. 2, pp. 65–70, 2021. [13] M. Montazeri Shatoori, L. Davidson, G. Kaur, et al., “De- tection of doh tunnels using time-series classification of encrypted traffic,” in 2020 IEEE Intl Conf on Dependable, Autonomic and Secure Computing, 2020, pp. 63–70. [14] I. H. Witten, E. Frank, M. A. Hall, et al., Data Mining: Practical machine learning tools and techniques. Burlington: Morgan Kaufmann, 2016. g [15] L. Breiman, “Random forests,” Machine learning, vol. 45, no. 1, pp. 5–32, 2001. [16] D. Devetyarov and I. Nouretdinov, “Prediction with confi- dence based on a random forest classifier,” in IFIP Interna- tional Conference on Artificial Intelligence Applications and Innovations, Springer, 2010, pp. 37–44. Our future research will apply the explainable DoH detec- tion methods for deep neural network-based solutions [21]. Currently, DoH traffic is only distinguished from browser traffic. However, there might be HTTPS traffic created by more applications than browsers only, with characteristics more sim- ilar to DoH traffic. Other types of malicious use of DoH can also be an interesting topic for exploration. Botnets often use fast domain fluxing or Domain Generating Algorithms(DGA). DGAs in botnets might abuse DoH [7], [22]. Future extensions of the current work can aim to distinguish DGA related DoH traffic from other HTTPS traffic. p g pp [17] T. Ogwang, “A convenient method of decomposing the gini index by population subgroups,” Journal of Official Statistics, vol. 30, no. 1, p. 91, 2014. [18] M. Feurer, A. Klein, K. Eggensperger, et al., “Auto-sklearn: Efficient and robust automated machine learning,” in Auto- mated Machine Learning, Springer, Cham, 2019, pp. 113–134. [19] N. Hatami and R. Ebrahimpour, “Combining multiple clas- sifiers: Diversify with boosting and combining by stacking,” International Journal of Computer Science and Network Se- curity, vol. 7, no. 1, pp. 127–131, 2007. [20] S. VII. CONCLUSIONS In Fig. 8, we are showing a similar analysis of a Non-DOH test packet. Notice the value difference in Duration in both the decisions and their contribution differences. Additionally, for this decision, we can see the model had a 5.2% similarity of malicious traffic in some of the features. It is also possible to identify the features that impacted the model’s decision DoH technology has been developed to provide security and privacy for Internet users by encrypting the DNS traffic. However, over the past few years, DNS remained a prime target for hackers as it enables them to gain first entry into networks and gain access to data for exfiltration due to network TRANSACTIONS ON INFORMATION FORENSICS AND SECURITY, VOL. 0, NO. 0, DECEMBER 2021 10 [7] L. Vries, “Detection of DoH tunnelling: Comparing super- vised with unsupervised learning,” M.S. thesis, University of Twente, 2021. traffic generated by malware and malicious tools. Although many studies on encrypted network traffic classification and DNS tunnel detection have been reported before, as DoH is a new protocol we need new set of intrusion detection tools. In this paper, we reported an explainable AI model dashboard that can detect malicious DoH traffic accurately. To prove that our system can identify malicious DNS tunnel tools and evaluate the performance, we have used the publicly available CIRA-CIC-DoHBrw-2020 dataset. Our proposed model can distinguish DoH traffic from normal HTTPS network traffic 99.9% of the time and the class-wise accuracy of Benign, Malicious and Non-DoH traffic on the test set was found to be 97.3%, 99.9% and 99.8% respectively. We have also reported the AUC, F1-score, precision and recall value from the model. In comparison to state-of-the-art ensemble models such as gradient boosting and generic random forest, our proposed balanced and stacked random forest achieved slightly higher precision (99.91%), recall(99.92%) and F1 score (99.91%) which is desirable for the task at hand. Additionally, With the help of the SHAP values, we have also highlighted the feature contributions for the underlying classification decision by the model. We have also discussed the conditions under which high classification accuracy can be achieved by using these features. In summary, the proposed method provides an accurate solution to detect and classify the DNS over HTTPS attacks. [8] R. Preston, “Dns tunneling detection with supervised learn- ing,” in 2019 IEEE International Symposium on Technologies for Homeland Security (HST), IEEE, 2019, pp. 1–6. VII. CONCLUSIONS Raschka, “Mlxtend: Providing machine learning and data science utilities and extensions to python’s scientific comput- ing stack,” Journal of open source software, vol. 3, no. 24, p. 638, 2018. TRANSACTIONS ON INFORMATION FORENSICS AND SECURITY, VOL. 0, NO. 0, DECEMBER 2021 TRANSACTIONS ON INFORMATION FORENSICS AND SECURITY, VOL. 0, NO. 0, DECEMBER 2021 Dr. Tahmina Zebin is a Lecturer in the School of Computing Sciences at the University of East Anglia and is one of the academic leads at the On-device and Explainable AI Laboratory. Tahmina pursued her PhD studies at the University of Manchester and has been the recipient of the Presidents Doctoral Scholarship (2013-2016) in Electrical and Electronic Engineering. Her research expertise includes Ad- vanced Video and Signal Processing, Explainable and Inclusive AI, Human Activity Recognition, Risk Prediction modelling using various statistical ma- p learning techniques. Dr. Tahmina Zebin is a Lecturer in the School of Computing Sciences at the University of East Anglia and is one of the academic leads at the On-device and Explainable AI Laboratory. Tahmina pursued her PhD studies at the University of Manchester and has been the recipient of the Presidents Doctoral Scholarship (2013-2016) in Electrical and Electronic Engineering. Her research expertise includes Ad- vanced Video and Signal Processing, Explainable and Inclusive AI, Human Activity Recognition, Risk Prediction modelling using various statistical ma- chine learning and deep learning techniques. chine learning and deep l Shahadate Rezvy is a Lecturer in Computer nce at York St. John University, UK. He com- ed his PhD in Wireless Communications from dlesex University London, UK in 2016. He also s an M.Sc in Telecommunications. Shahadate ived his first degree in Computer Science and ineering. His current research interests include p Learning, AI, Telecommunications and Net- k Security, Wireless Communication, IoT based cular Communication Systems. Dr. Yuan Luo is a senior Lecturer in the of Science and Technology at Middlesex U UK. He completed his PhD in School puting, Staffordshire University, UK, in 2 is instructor of Cisco CCNA, CCNP, and His current research interests include netw tocols and operations, network programmab automation, network management, network cloud computing and visualization, machine and deep learning, AI, telecommunications communications. uter om- om lso ate and ude Net- sed Dr. Yuan Luo is a senior Lecturer in the Faculty of Science and Technology at Middlesex University, UK. He completed his PhD in School of Com- puting, Staffordshire University, UK, in 2002. He is instructor of Cisco CCNA, CCNP, and DevNet. His current research interests include network pro- tocols and operations, network programmability and automation, network management, network security, cloud computing and visualization, machine learning and deep learning, AI, telecommunications, wireless communications. Dr. REFERENCES [1] R. Fouchereau, 2021 Global DNS Threat Report, 2021. [On- line]. Available: https://www.efficientip.com/wp- content/ uploads/2021/06/2021-IDC-DNS-Threat-Report-Infobrief- final compressed.pdf. [21] S. Rezvy, Y. Luo, M. Petridis, et al., “An efficient deep learning model for intrusion classification and prediction in 5G and IoT networks,” in 2019 53rd Annual Conference on information sciences and systems (CISS), IEEE, 2019, pp. 1–6. [2] D. Vekshin, K. Hynek, and T. Cejka, “Doh insight: Detecting dns over https by machine learning,” in Proceedings of the 15th International Conference on Availability, Reliability and Security, 2020, pp. 1–8. [22] M. Behnke, N. Briner, D. Cullen, et al., “Feature engineering and machine learning model comparison for malicious activity detection in the dns-over-https protocol,” IEEE Access, vol. 9, pp. 129 902–129 916, 2021. [3] Y. Wang, A. Zhou, S. Liao, et al., “A comprehensive survey on dns tunnel detection,” Computer Networks, p. 108 322, 2021. [4] M. Aiello, M. Mongelli, and G. Papaleo, “Basic classi- fiers for dns tunneling detection,” in 2013 IEEE Symposium on Computers and Communications (ISCC), IEEE, 2013, pp. 000 880–000 885. [5] H. Zhao, Z. Chang, G. Bao, et al., “Malicious domain names detection algorithm based on n-gram,” Journal of Computer Networks and Communications, vol. 2019, 2019. [6] L. A. Trejo, V. Ferman, M. A. Medina-P´erez, et al., “Dns- advp: A machine learning anomaly detection and visual plat- form to protect top-level domain name servers against ddos attacks,” IEEE Access, vol. 7, pp. 116 358–116 369, 2019. 11 TRANSACTIONS ON INFORMATION FORENSICS AND SECURITY, VOL. 0, NO. 0, DECEMBER 2021 Yuan Luo is a senior Lecturer in the Faculty of Science and Technology at Middlesex University, UK. He completed his PhD in School of Com- puting, Staffordshire University, UK, in 2002. He is instructor of Cisco CCNA, CCNP, and DevNet. His current research interests include network pro- tocols and operations, network programmability and automation, network management, network security, cloud computing and visualization, machine learning and deep learning, AI, telecommunications, wireless communications. Dr. Shahadate Rezvy is a Lecturer in Computer Science at York St. John University, UK. He com- pleted his PhD in Wireless Communications from Middlesex University London, UK in 2016. He also holds an M.Sc in Telecommunications. Shahadate received his first degree in Computer Science and Engineering. His current research interests include Deep Learning, AI, Telecommunications and Net- work Security, Wireless Communication, IoT based Vehicular Communication Systems. communications.
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Efficacy and safety of erenumab in women with a history of menstrual migraine
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UC Riverside UC Riverside UC Riverside Previously Published Works UC Riverside UC Riverside Previously Published Works Title Efficacy and safety of erenumab in women with a history of menstrual migraine Permalink https://escholarship.org/uc/item/95x8t7hv Journal The Journal of Headache and Pain, 21(1) ISSN 1129-2369 Authors Pavlovic, Jelena M Paemeleire, Koen Göbel, Hartmut et al. Publication Date 2020-12-01 DOI 10.1186/s10194-020-01167-6 Peer reviewed UC Riverside UC Riverside Previously Published Works Title Efficacy and safety of erenumab in women with a history of menstrual migraine Permalink https://escholarship.org/uc/item/95x8t7hv Journal The Journal of Headache and Pain, 21(1) ISSN 1129-2369 Authors Pavlovic, Jelena M Paemeleire, Koen Göbel, Hartmut et al. Publication Date 2020-12-01 DOI 10.1186/s10194-020-01167-6 Peer reviewed UC Riverside UC Riverside Previously Published Works Title Efficacy and safety of erenumab in women with a history of menstrua Permalink https://escholarship.org/uc/item/95x8t7hv Journal The Journal of Headache and Pain, 21(1) ISSN 1129-2369 Authors Pavlovic, Jelena M Paemeleire, Koen Göbel, Hartmut et al. Publication Date 2020-12-01 DOI 10.1186/s10194-020-01167-6 Peer reviewed RESEARCH ARTICLE Open Access Powered by the California Digital Library University of California eScholarship.org Pavlovic et al. The Journal of Headache and Pain (2020) 21:95 https://doi.org/10.1186/s10194-020-01167-6 The Journal of Headache and Pain Efficacy and safety of erenumab in women with a history of menstrual migraine Jelena M. Pavlovic1,2*, Koen Paemeleire3, Hartmut Göbel4, Jo Bonner5, Alan Rapoport6, Risa Kagan7,8, Feng Zhang9, Hernan Picard9 and Daniel D. Mikol9 Abstract Background: We performed a post hoc, subgroup analysis of a phase 3, randomized, double-blind, placebo- controlled study of erenumab for prevention of episodic migraine (STRIVE) to determine the efficacy and safety of erenumab in women with self-reported menstrual migraine. Methods: Patients received placebo, erenumab 70 mg, or erenumab 140 mg subcutaneously once monthly during the 6-month double-blind treatment phase of STRIVE. Women who reported history of menstrual migraine and who were ≤50 years old were included in the analysis. Endpoints were change from baseline in monthly migraine days (MMD) and monthly acute migraine-specific medication days (MSMD; among patients who took acute migraine-specific medications at baseline), proportion of patients achieving ≥50% reduction from baseline in MMD, and incidence of adverse events. Results: Among 814 women enrolled in STRIVE, 232 (28.5%) reported a history of menstrual migraine and were ≤50 years old. Of the 232 patients, 214 (92%) had a baseline MMD > 5, suggesting a high proportion of women with attacks outside of the 5-day perimenstrual window (2 days before and 3 days after the start of menstruation). Information on “migraine days” includes (and does not discriminate between) perimenstrual and intermenstrual migraine attacks. Between-group differences from placebo over months 4–6 for erenumab 70 mg and 140 mg were −1.8 (P = 0.001) and −2.1 (P < 0.001) days for MMD and −1.6 (P = 0.002) and −2.4 (P < 0.001) days for acute MSMD, respectively. The odds of having a ≥50% reduction from baseline in MMD over months 4–6 were 2.2 (P = 0.024) and 2.8 (P = 0.002) times greater for erenumab 70 mg and 140 mg, respectively, than for placebo. Erenumab had an overall safety profile comparable to placebo. Conclusion: Data from this subgroup analysis of women with menstrual migraine are consistent with data from the overall STRIVE episodic migraine population, supporting the efficacy and safety of erenumab in women who experience menstrual migraine. p g Trial registration: ClinicalTrials.gov, NCT02456740. Registered 28 May 2015. Trial registration: ClinicalTrials.gov, NCT02456740. Registered 28 May 2015. Keywords: Erenumab, Headache, Episodic migraine, Migraine prevention, Pure menstrual migraine, Menstrually related migraine, Perimenstrual attacks Keywords: Erenumab, Headache, Episodic migraine, Migraine prevention, Pure menstrual migraine, Menstrually related migraine, Perimenstrual attacks Keywords: Erenumab, Headache, Episodic migraine, Migraine prevention, Pure menstrual migraine, Menstrually related migraine, Perimenstrual attacks * Correspondence: jpavlovi@montefiore.org 1Department of Neurology, Montefiore Headache Center, 1300 Morris Park Avenue, Van Etten 3C9, Bronx, NY 10461, USA 2Albert Einstein College of Medicine, Bronx, NY, USA Full list of author information is available at the end of the article Study design and patients STRIVE (ClinicalTrials.gov, NCT02456740) was a phase 3, randomized, double-blind, placebo-controlled study of erenumab in patients with episodic migraine [29]. In brief, the study consisted of a 7-week screening phase (including 4 weeks of baseline), a 6-month double-blind treatment phase, a 7-month dose-blinded active treat- ment phase, and a 3-month safety follow-up phase. Randomization was stratified by region (North America vs other) and prior preventive medication status (naïve vs prior use vs concomitant use). Placebo and erenumab 70 mg and 140 mg were administered subcutaneously once every month during the double-blind treatment phase; erenumab 70 mg or 140 mg were administered during the 28-week active treatment phase. There are no approved, specific preventive treatments for menstrual migraine. It has been proposed that women who do not respond to acute treatment options may be eligible to receive either short-term or long-term preventive treatments [14]. Several medications, including triptans, nonsteroidal anti-inflammatory drugs, cyclooxygenase-2 inhibitors, and estrogen supplementation, have been investigated for short- term prevention of menstrual migraine; however, these agents may delay rather than prevent attacks [14]. Long- term preventive treatment for menstrual migraine has been investigated with topiramate, which reduced the frequency but not the severity or duration of perimenstrual attacks [15]. Continuous use of hormonal contraceptives can reduce the severity and duration of migraine attacks [14, 16–18]. Al- though hormonal contraceptives containing estrogens are considered a viable treatment option for women with men- strual migraine [19], evidence of their effectiveness is limited [20], and they may be contraindicated because of their asso- ciation with increased risk of stroke [21–24]. According to the current guidelines, exogenous estrogens are contraindi- cated in all women with migraine with aura and in women with migraine without aura who are smokers and/or older than 35 years of age [25–27]; estrogen-containing options are therefore often not available to most women with mi- graine aged 35 or older [25, 26]. Eligible patients were 18–65 years old with a history of migraine with or without aura (based on medical records and/or self-reported) for at least 12 months before screening. Episodic migraine was defined as an average of 4–14 migraine days per month with fewer than 15 headache days per month (in accordance with ICHD- 3) during the 3 months before screening and during the 4-week baseline phase of the study. Background (CGRP) receptor [28]. In the 6-month double-blind treat- ment phase of the STRIVE trial of patients with episodic migraine, erenumab at 70 mg or 140 mg once monthly significantly reduced the number of monthly migraine days (MMD) and monthly acute migraine-specific medica- tion days (MSMD) and increased the odds of achieving ≥50% reduction from baseline in MMD [29]. Given the burden and challenges in the treatment of menstrual mi- graine, we performed a post hoc subgroup analysis of STRIVE to determine the efficacy and safety of erenumab in women with self-reported menstrual migraine. More than 50% of women self-report an association be- tween migraine and menses [1]. These perimenstrual at- tacks are commonly referred to as menstrual migraine if they occur within a 5-day window (2 days prior to men- struation and the first 3 days of menstruation). Menstrual migraine attacks are particularly burdensome, as they tend to be of longer duration [2–7] and are more severe and disabling [2, 3, 5, 7–9] than non-perimenstrual attacks. Furthermore, perimenstrual attacks are less responsive to acute therapy, making them difficult to treat [2, 4, 5]. Women who experience migraine attacks with the ma- jority of their menstrual periods (defined by The Inter- national Classification of Headache Disorders [ICHD] 3 beta [10] as at least two of three periods) are classified into either pure menstrual migraine (PMM; attacks that occur only during the 5-day perimenstrual window) or menstru- ally related migraine (MRM; attacks that occur during the 5-day perimenstrual window and at other times of the cycle). PMM is a rare condition that affects approximately 5%–8% of women with migraine [9, 11, 12], with most women self-reporting MRM [4]; however, the percentage varies widely depending on the study populations and diagnostic criteria used [2, 4, 11, 13]. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Pavlovic et al. The Journal of Headache and Pain (2020) 21:95 Page 2 of 9 Page 2 of 9 Study design and patients One concomi- tant migraine-preventive medication was allowed fol- lowing a protocol amendment that was introduced late during the enrollment period. Patients were excluded if they had no therapeutic response to > 2 migraine- preventive treatment categories, defined as no reduction in headache frequency, duration, or severity after adminis- tration of the medication for at least 6 weeks at the generally accepted therapeutic dose(s) based on the inves- tigator’s assessment. The study protocol was approved by the ethics com- mittee or institutional review board at each clinical site, and all patients provided signed informed consent before the start of any study-related procedures. The study was conducted in accordance with the International Council for Harmonisation Good Clinical Practice Guidelines and conforms to the provisions of the Declaration of Helsinki. The challenges associated with the treatment of men- strual migraine emphasize the need for novel, nonhor- monal, long-term preventive treatments. Erenumab is a fully human monoclonal antibody that selectively targets and blocks the canonical calcitonin gene-related peptide Page 3 of 9 Pavlovic et al. The Journal of Headache and Pain (2020) 21:95 Pavlovic et al. The Journal of Headache and Pain (2020) 21:95 Menstrual migraine subgroups Safety was monitored throughout the study, and ad- verse events were coded according to the Medical Dic- tionary for Regulatory Activities version 19.0. Women were asked if they had migraine attacks that oc- curred within a 5-day window (2 days prior to menstru- ation and the first 3 days of menstruation) in at least 2 out of the last 3 menstrual cycles prior to screening in accord- ance with the criteria for menstrual migraine diagnosis [10]. In industrialized countries, the average age for onset of perimenopause is 47.5 years and is influenced by several demographic, lifestyle, and biologic factors [30]. Based on this, for the current subgroup analysis, we included menstru- ating women aged ≤50 years with a self-reported history of menstrual migraine attacks. Since the data collected did not allow us to distinguish between women who had only men- strual attacks (PMM) and those who had both menstrual and non-menstrual attacks (MRM), both categories are in- cluded under the label “menstrual migraine” in our analyses. Statistical analysis Change from baseline in MMD and monthly acute MSMD was analyzed using a generalized linear mixed effects model, which included treatment, visit, treatment by visit interaction, stratification factors (North Amer- ica/other and naïve/prior use/concomitant use), and baseline value as covariates and assumed a first-order autoregressive covariance structure; missing data were not imputed. The proportion of responders was analyzed using a stratified Cochran-Mantel-Haenszel test after imputation of missing data as nonresponse. P values for the between-group differences (erenumab 70 mg and 140 mg vs placebo) are nominal P values without multi- plicity adjustment. Statistical significance was deter- mined based on the comparison of the nominal P values with a significance level of 0.05. Endpoints Efficacy endpoints were change from baseline in mean MMD, change from baseline in mean monthly acute MSMD among patients who took acute migraine-specific medica- tions at baseline, and the proportion of patients achieving a ≥50% reduction from baseline in MMD (proportion of re- sponders). Efficacy was assessed for each monthly interval from data collected daily using the patients’ electronic diaries; the primary time point of assessment in the study was the average monthly effect over months 4–6. Analysis of mi- graine frequency–related endpoints includes all (both peri- menstrual and intermenstrual) migraine days. Patient characteristics Among 814 women enrolled in STRIVE, 232 (28.5%) self-reported a history of menstrual migraine and were ≤50 years old. Baseline characteristics were fairly balanced among the treatment groups (Table 1). Of the 232 women with menstrual migraine, 65 (28%) were taking oral contraceptives/hormone therapy during Table 1 Baseline characteristics History of Menstrual Migraine Placebo N = 83 Erenumab 70 mg N = 68 Erenumab 140 mg N = 81 All Patients N = 232 Age, years, median (range) 37 (20–49) 38 (21–50) 37 (19–50) 37 (19–50) Race, n (%) White 71 (86) 61 (90) 77 (95) 209 (90) Black or African American 3 (4) 5 (7) 2 (3) 10 (4) Asian 4 (5) 0 (0) 2 (3) 6 (3) Other 5 (6) 2 (3) 0 (0) 7 (3) Migraine with aura,a n (%) 48 (58) 35 (52) 38 (47) 121 (52) Migraine without aura,a n (%) 71 (86) 61 (90) 72 (89) 204 (88) Received hormonal contraception, n (%) 20 (24) 18 (26) 27 (33) 65 (28) Treatment with migraine-preventive medication, n (%) Naïve 50 (60) 37 (54) 48 (59) 135 (58) Prior and/or concomitant use 33 (40) 31 (46) 33 (41) 97 (42) Baseline phase (4 weeks) Monthly migraine days, mean (SD) 8.6 (2.8) 8.3 (2.4) 8.4 (2.4) 8.4 (2.5) Monthly acute migraine-specific medication days, mean (SD) 3.2 (3.5) 3.1 (3.1) 3.7 (3.6) 3.3 (3.4) Acute migraine-specific medication use, n (%) 48 (58) 38 (56) 51 (63) 137 (59) aBased on self-report; categories are not mutually exclusive Abbreviations: SD standard deviation Page 4 of 9 Pavlovic et al. The Journal of Headache and Pain (2020) 21:95 the study: 18 (26%) in the erenumab 70 mg group, 27 (33%) in the erenumab 140 mg group, and 20 (24%) in the pla- cebo group. receiving exogenous hormones had similar efficacy re- sults compared to the total population with a history of menstrual migraine. ORa (95% CI) The analysis included randomized patients who received ≥1 dose of investigational product and had ≥1 postbaseline measurement during the double-blind treatment phase. Change from baseline in MMD and monthly acute MSMD was analyzed using a generalized linear mixed effects model, which included treatment, visit, treatment by visit interaction, stratification factors (North America/other and naïve/prior use/concomitant use), and baseline value as covariates and assumed a first-order autoregressive covariance structure; missing data were not imputed. The proportion of responders was analyzed using a stratified Cochran-Mantel-Haenszel test after imputation of missing data as nonresponse p g p Abbreviations: CI confidence interval, LSM least squares mean, MSMD migraine-specific medication days, OR odds ratio aThe common ORs and P values were obtained from a Cochran-Mantel-Haenszel test, stratified by prior/current treatment and region Abbreviations: CI confidence interval, LSM least squares mean, MSMD migraine-specific medication days, OR odds ratio aThe common ORs and P values were obtained from a Cochran-Mantel-Haenszel test, stratified by prior/current treatment with migraine-preventive medication and region Change from baseline in mean monthly migraine days During the study, both doses of erenumab resulted in statistically significantly greater reductions vs placebo in MMD as early as month 1 (Fig. 1). The mean MMD re- duction over months 4–6 was −1.4, −3.2, and −3.5 days in the placebo, erenumab 70 mg, and erenumab 140 mg groups, respectively (Table 2). Differences from placebo were statistically significant: –1.8 (P = 0.001) and −2.1 (P < 0.001) days for the erenumab 70 mg and 140 mg groups, respectively (Table 2). In the subgroup of patients who were taking acute migraine-specific medications at baseline, erenumab 70 mg and 140 mg vs placebo resulted in greater reductions in monthly acute MSMD starting at month 1; reductions were statistically significant at every month for the 140-mg dose group (Fig. 2). The mean reduction in monthly acute MSMD over months 4–6 was 0.4, 2.0, and 2.8 days in the placebo, erenumab 70 mg, and erenumab 140 mg groups, respectively (Table 2). Differences from placebo were statis- tically significant: –1.6 (P = 0.002) and −2.4 (P < 0.001) days for the erenumab 70 mg and 140 mg groups, respectively (Table 2). An analysis of MMD was performed for patients who were receiving exogenous hormones for contraception versus those who were not receiving exogeneous hor- mones (Table 3). Overall, the subgroup of patients Fig. 1 Change from baseline in MMD. Data are shown as LSM with 95% CIs. The gray shaded area represents months 4–6. Abbreviations: CI, confidence interval; LSM, least squares mean; MMD, monthly migraine days Fig. 1 Change from baseline in MMD. Data are shown as LSM with 95% CIs. The gray shaded area represents months 4–6. Abbreviations: CI, confidence interval; LSM, least squares mean; MMD, monthly migraine days Fig. 1 Change from baseline in MMD. Data are shown as LSM with 95% CIs. The gray shaded area represents months 4–6. Abbreviations: CI, confidence interval; LSM, least squares mean; MMD, monthly migraine days Pavlovic et al. The Journal of Headache and Pain (2020) 21:95 Page 5 of 9 Pavlovic et al. Proportion of patients achieving ≥50% reduction from baseline in monthly migraine days 4–6 were 2.2 (P = 0.024) and 2.8 (P = 0.002) times greater for the erenumab 70 and 140 mg groups, respectively, than for the placebo group (Table 2). Proportion of patients achieving ≥50% reduction from baseline in monthly migraine days Both doses of erenumab vs placebo resulted in a signifi- cantly higher proportion of patients achieving at least a 50% response at each time point except month 4 (Fig. 3). A ≥50% response over months 4–6 was achieved by 25.3%, 42.6%, and 49.4% of patients who received pla- cebo, erenumab 70 mg, and erenumab 140 mg, respect- ively. The odds of having a ≥50% response over months Both doses of erenumab vs placebo resulted in a signifi- cantly higher proportion of patients achieving at least a 50% response at each time point except month 4 (Fig. 3). Safety Erenumab had an overall safety profile comparable to placebo (Table 4). There were no cardiovascular adverse events in this subpopulation of patients. Table 3 Change From Baseline in Mean Monthly Migraine Days by Hormonal Contraception Status Received Hormonal Contraception Did Not Receive Hormonal Contraception Placebo N = 20 Erenumab 70 mg N = 18 Erenumab 140 mg N = 27 Placebo N = 63 Erenumab 70 mg N = 50 Erenumab 140 mg N = 54 Monthly migraine days at baseline, mean (SD) 9.5 (3.0) 8.5 (2.7) 8.9 (2.2) 8.3 (2.8) 8.2 (2.3) 8.2 (2.4) Change from baseline over months 4–6, LSM (95% CI) −1.6 (−3.4, 0.25) −2.9 (−4.8, −1.0) −3.9 (−5.5, −2.4) −1.4 (−2.2, −0.6) −3.3 (−4.2, −2.4) −3.4 (−4.2, −2.5) Difference from placebo −1.3 (−3.9, 1.2) P = 0.3 −2.4 (−4.7, −0.1) P = 0.045 −2.0 (−3.1, −0.8) P = 0.001 −2.0 (−3.1, −0.8) P < 0.001 Treatment by subgroup interaction P value over months 4–6 0.76 The analysis included randomized patients who received ≥1 dose of investigational product and had ≥1 postbaseline measurement during the double-blind treatment phase. Change from baseline in MMD and monthly acute MSMD was analyzed using a generalized linear mixed effects model, which included treatment, visit, treatment by visit interaction, stratification factors (North America/other and naïve/prior use/concomitant use), and baseline value as covariates and assumed a first-order autoregressive covariance structure; missing data were not imputed. P values for pairwise comparisons are nominal P values without multiplicity adjustment Abbreviations: CI confidence interval, LSM least squares mean Table 3 Change From Baseline in Mean Monthly Migraine Days by Hormonal Contraception Status Received Hormonal Contraception Did Not Receive Hormo The analysis included randomized patients who received ≥1 dose of investigational product and had ≥1 postbaseline measurement during the double-blind treatment phase. Change from baseline in MMD and monthly acute MSMD was analyzed using a generalized linear mixed effects model, which included treatment, visit, treatment by visit interaction, stratification factors (North America/other and naïve/prior use/concomitant use), and baseline value as covariates and assumed a first-order autoregressive covariance structure; missing data were not imputed. P values for pairwise comparisons are nominal P values without multiplicity adjustment The analysis included randomized patients who received ≥1 dose of investigational product and had ≥1 postbaseline measurement during the double-blind treatment phase. Change from baseline in mean monthly migraine days The Journal of Headache and Pain (2020) 21:95 Page 5 of 9 Table 2 Efficacy over months 4–6 of the double-blind treatment phase Table 2 Efficacy over months 4–6 of the double-blind treatment phase Placebo N = 83 Erenumab 70 mg N = 68 Erenumab 140 mg N = 81 Migraine days per month N 83 68 81 Change from baseline, LSM (95% CI) −1.4 (−2.2, −0.7) −3.2 (−4.0, −2.4) −3.5 (−4.3, −2.8) Difference from placebo, LSM (95% CI) −1.8 (−2.9, −0.7) P = 0.001 −2.1 (−3.1, −1.1) P < 0.001 Table 2 Efficacy over months 4–6 of the double-blind treatment phase Table 2 Efficacy over months 4–6 of the double-blind treatment phase Placebo N = 83 Erenumab 70 mg N = 68 Erenumab 140 mg N = 81 Migraine days per month N 83 68 81 Change from baseline, LSM (95% CI) −1.4 (−2.2, −0.7) −3.2 (−4.0, −2.4) −3.5 (−4.3, −2.8) Difference from placebo, LSM (95% CI) −1.8 (−2.9, −0.7) P = 0.001 −2.1 (−3.1, −1.1) P < 0.001 Acute MSMD per month among patients taking acute migraine-specific medications at baseline N 48 38 51 Change from baseline, LSM (95% CI) −0.4 (−1.1, 0.3) −2.0 (−2.8, −1.2) −2.8 (−3.5, −2.1) Difference from placebo, LSM (95% CI) −1.6 (−2.6, −0.6) P = 0.002 −2.4 (−3.4, −1.4) P < 0.001 Patients with ≥50% reduction from baseline in migraine days per month (≥50% response) N 83 68 81 n (%) 21 (25.3) 29 (42.6) 40 (49.4) ORa (95% CI) 2.2 (1.1, 4.4) P = 0.024 2.8 (1.5, 5.5) P = 0.002 Migraine days per month Acute MSMD per month among patients taking acute migraine-specific medications at baseline N 48 38 51 Change from baseline, LSM (95% CI) −0.4 (−1.1, 0.3) −2.0 (−2.8, −1.2) −2.8 (−3.5, −2.1) Difference from placebo, LSM (95% CI) −1.6 (−2.6, −0.6) P = 0.002 −2.4 (−3.4, −1.4) P < 0.001 Change from baseline, LSM (95% CI) −0.4 (−1.1, 0.3) −2.0 (−2.8, −1.2) Difference from placebo, LSM (95% CI) −1.6 (−2.6, −0.6) P Patients with ≥50% reduction from baseline in migraine days per month (≥50% response) N 83 68 81 n (%) 21 (25.3) 29 (42.6) 40 (49.4) ORa (95% CI) 2.2 (1.1, 4.4) P = 0.024 2.8 (1.5, 5.5) P = 0.002 The analysis included randomized patients who received ≥1 dose of investigational product and had ≥1 postbaseline measurement during the double-blind treatment phase. Change from baseline in MMD and monthly acute MSMD was analyzed using a generalized linear mixed effects model, which included treatment, visit, treatment by visit interaction, stratification factors (North America/other and naïve/prior use/concomitant use), and baseline value as covariates and assumed a first-order autoregressive covariance structure; missing data were not imputed. P values for pairwise comparisons are nominal P values without multiplicity adjustment Safety Change from baseline in MMD and monthly acute MSMD was analyzed using a generalized linear mixed effects model, which included treatment, visit, treatment by visit interaction, stratification factors (North America/other and naïve/prior use/concomitant use), and baseline value as covariates and assumed a first-order autoregressive covariance structure; missing data were not imputed. P values for pairwise comparisons are nominal P values without multiplicity adjustment Abb i i CI fid i l LSM l p y j Abbreviations: CI confidence interval, LSM least squares mean Pavlovic et al. The Journal of Headache and Pain (2020) 21:95 Page 6 of 9 Fig. 2 Change from baseline in monthly acute MSMD among patients with a self-reported history of menstrual migraine who took migraine- specific medications at baseline. Data are shown as LSM with 95% CIs. The gray shaded area represents months 4–6. Abbreviations: CI, confidence interval; LSM, least squares mean; MSMD, migraine-specific medication days Fig. 2 Change from baseline in monthly acute MSMD among patients with a self-reported history of menstrual migraine who took migraine- specific medications at baseline. Data are shown as LSM with 95% CIs. The gray shaded area represents months 4–6. Abbreviations: CI, confidence interval; LSM, least squares mean; MSMD, migraine-specific medication days achieving ≥50% reduction from baseline in MMD [29]. A subgroup analysis of MMD among women who re- ceived hormonal contraception suggests that exogenous hormones do not impact the efficacy of erenumab in this patient population; however, the sample sizes of these subgroups were too small to draw any definitive conclu- sions. Further investigation appears warranted, as several studies suggest that fluctuations of ovarian steroid hor- mone levels may modulate CGRP, with high estrogen states being related to an increase in CGRP levels in general, although the exact mechanistic interactions be- tween ovarian steroid hormones and CGRP are not fully understood [32]. Discussion Consistent with the overall STRIVE population, prevent- ive treatment with erenumab 70 mg and 140 mg vs pla- cebo resulted in statistically significant improvements in MMD and acute MSMD and achievement of ≥50% response in this subpopulation of patients with a self- reported history of menstrual migraine. The overall inci- dence of treatment-emergent adverse events was also consistent with the overall STRIVE population. Because of the frequency and burden of migraine in women with menstrual migraine, the majority qualify for preventive treatment [31]. However, although there are strategies for short-term prevention of menstrual mi- graine, limited options are available for long-term pre- vention [14]. It is, therefore, of interest that the efficacy and safety profiles of erenumab in this subgroup were similar to the overall episodic migraine population of STRIVE, in which erenumab significantly reduced the number of MMD and MSMD and increased the odds of The prevalence of menstrual migraine depends on how it is defined and recorded, and there may be sub- stantial differences in prevalence rates of menstrual mi- graine determined by self-report. For example, in population-based studies [11, 13], the reported preva- lence of menstrual migraine is about 20% of women with Pavlovic et al. The Journal of Headache and Pain (2020) 21:95 Page 7 of 9 Fig. 3 Proportion of patients achieving ≥50% reduction from baseline in MMD. Data are shown as percentages. The gray shaded area represents months 4–6. *Statistically significantly different from placebo. Abbreviations: MMD, monthly migraine days; OR, odds ratio Fig. 3 Proportion of patients achieving ≥50% reduction from baseline in MMD. Data are shown as percentages. The gray shaded area represents months 4–6. *Statistically significantly different from placebo. Abbreviations: MMD, monthly migraine days; OR, odds ratio ig. 3 Proportion of patients achieving ≥50% reduction from baseline in MMD. Data are shown as percentages. The gray sh months 4–6. *Statistically significantly different from placebo. In any of the treatment groups cBased on the following search criteria: ischemic central nervous system vascular conditions, ischemic heart disease, and peripheral arterial disease Adverse events were graded using CTCAE version 4.03. All serious adverse events were single-occurrence events Abbreviations: CTCAE Common Terminology Criteria for Adverse Events no grade 4 adverse events e treatment groups e following search criteria: ischemic central nervous system vascular conditions ischemic heart disease and peripheral arterial disease groups earch criteria: ischemic central nervous system vascular conditions, ischemic heart disease, and peripheral arterial disease sing CTCAE version 4.03. All serious adverse events were single occurrence events Terminology Criteria for Adverse Events se events Funding g This analysis was funded by Amgen Inc. and Novartis. Trial registration: ClinicalTrials.gov, NCT02456740. Registered 28 May 2015, https://clinicaltrials. gov/ct2/show/NCT02456740. g y Our exploratory analysis is limited by our inability to differentiate PMM and MRM, and our inability to exam- ine the effect of treatment on intermenstrual vs peri- menstrual migraine days. Given that the study required at least 4 MMDs and the fact that 214 (92%) patients had a baseline MMD > 5, it is reasonable to conclude that the majority of women likely belonged to the MRM group, ex- periencing both menstrual and non-menstrual migraine attacks, although this information was not collected. The patients categorized as having menstrual migraine in our study have similar characteristics to “real-world” patients with menstrual migraine, who are generally identified based on retrospective self-report of perimenstrual mi- graine attacks during clinical encounters with the treating clinician rather than with prospective headache diaries. Similar to the general population [34], approximately one- third of patients with menstrual migraine reported that they were taking oral contraceptives/hormone therapy during the study, which may be an important confounder in terms of efficacy in this subgroup of patients. In addition, women who experience menstrual migraine may be more likely to use continuous contraceptive/hormonal options and may have been misclassified if they were not currently experiencing perimenstrual attacks. Further- more, the relatively small sample size likely contributed to variability of effect and statistical significance at some time points. Variability was reduced, however, by analyzing the mean monthly efficacy over months 4–6, the primary pre- specified analytic approach. Authors’ contributions KP, HG, and JB collected data from patients enrolled in STRIVE. HP, DDM, and FZ were involved in the conception and design of the study. All authors were involved in the analysis and interpretation of the data, contributed to writing the manuscript, and approved the final version. Author details 1 1Department of Neurology, Montefiore Headache Center, 1300 Morris Park Avenue, Van Etten 3C9, Bronx, NY 10461, USA. 2Albert Einstein College of Medicine, Bronx, NY, USA. 3Ghent University Hospital, Ghent, Belgium. 4Kiel Migraine and Headache Center, Kiel, Germany. 5Mercy Clinic Neurology, St Louis, MO, USA. 6The David Geffen School of Medicine at UCLA, Los Angeles, CA, USA. 7Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, CA, USA. 8Sutter East Bay Medical Foundation, Berkeley, CA, USA. 9Amgen Inc., Thousand Oaks, CA, USA. Discussion Abbreviations: MMD, monthly migraine days; OR, odds ratio Table 4 Incidence of adverse events during the double-blind treatment phase Placebo N = 83 Erenumab 70 mg N = 68 Erenumab 140 mg N = 81 All treatment-emergent adverse events, n (%) 56 (67.5) 42 (61.8) 42 (51.9) Grade 3a 5 (6.0) 2 (2.9) 3 (3.7) Serious 2 (2.4) 2 (2.9) 2 (2.5) Leading to discontinuation of study drug 3 (3.6) 1 (1.5) 3 (3.7) Fatal 0 (0.0) 0 (0.0) 0 (0.0) Adverse events in ≥2% of patients, n (%)b Nasopharyngitis 10 (12.0) 8 (11.8) 8 (9.9) Upper respiratory tract infection 2 (2.4) 6 (8.8) 5 (6.2) Nausea 3 (3.6) 3 (4.4) 3 (3.7) Influenza 1 (1.2) 2 (2.9) 3 (3.7) Insomnia 0 (0.0) 0 (0.0) 3 (3.7) Fatigue 1 (1.2) 2 (2.9) 2 (2.5) Sinusitis 0 (0.0) 2 (2.9) 2 (2.5) Vomiting 3 (3.6) 1 (1.5) 2 (2.5) Injection site erythema 1 (1.2) 1 (1.5) 2 (2.5) Urinary tract infection 5 (6.0) 2 (2.9) 1 (1.2) Headache 0 (0.0) 2 (2.9) 1 (1.2) Cardiovascular events, n (%)c 0 (0.0) 0 (0.0) 0 (0.0) Adverse events were graded using CTCAE version 4.03. All serious adverse events were single-occurrence events Abbreviations: CTCAE Common Terminology Criteria for Adverse Events Table 4 Incidence of adverse events during the double-blind treatment phase Page 8 of 9 Pavlovic et al. The Journal of Headache and Pain (2020) 21:95 Page 8 of 9 Pavlovic et al. The Journal of Headache and Pain (2020) 21:95 Page 8 of 9 Page 8 of 9 migraine (approximately 7% of the general female popu- lation), compared with 11% of women with migraine when prospectively assessed in the context of clinic- based studies [33]. The prevalence of self-reported men- strual migraine determined in our analysis (28.5%) is higher than that observed in clinical trials that prospect- ively assessed menstrual migraine. Although our data on menstrual migraine were collected in the context of a prospective clinical trial, this finding may be due to self- reported data that were not confirmed with headache diaries during the study. Acknowledgments Kathryn Boorer, PhD, of KB Scientific Communications, LLC (funded by Amgen Inc.) provided medical writing support for the preparation of this manuscript. CGRP: Calcitonin gene-related peptide; ICHD: The International Classification of Headache Disorders; MMD: Monthly migraine days; MRM: Menstrually related migraine; MSMD: Migraine-specific medication days; PMM: Pure menstrual migraine Availability of data and materials Qualified researchers may request data from Amgen clinical studies. Complete details are available at http://www.amgen.com/datasharing. Competing interests JMP is a consultant/on advisory boards for Alder Biopharmaceuticals, Allergan, Amgen, Biohaven, Promius Pharma and receives research funding from NIH/NIA K23AG049466-05. KP has received personal compensation from Allergan, Amgen/Novartis, Eli Lilly, and Teva for consulting, serving on a sci- entific advisory board, and/or speaking and is a clinical trial investigator for Amgen/Novartis (erenumab), Eli Lilly (galcanezumab), and Autonomic Tech- nologies Inc. (sphenopalatine ganglion stimulation). HG has received research support from Allergan, Amgen, Bayer, Novartis, Menarini, and Teva and is a speaker for Allergan, Novartis, and Teva. JB has nothing to disclose. AR is an advisor for Allergan, Amgen, Amneal, Assertio, Autonomic Technologies Inc., Biohaven, Cala Health, Neurolief, Promius, Satsuma, Teva, Theranica, Xoc, and Zosano and is on a speakers’ bureau for Amgen and Teva. RK is a consultant for Amgen. FZ is an employee of Amgen Inc. HP and DDM are employees and stockholders of Amgen. Conclusions In summary, these exploratory data from a large phase 3 study of erenumab in patients with menstrual migraine attacks (including both PMM and MRM subgroups) are consistent with the overall STRIVE episodic migraine population and support the efficacy of erenumab in this specific subgroup of women. Received: 6 March 2020 Accepted: 27 July 2020 Received: 6 March 2020 Accepted: 27 July 2020 References 1. Macgregor EA (2009) Menstrual migraine: therapeutic approaches. Ther Adv Neurol Disord 2:327–336. https://doi.org/10.1177/1756285609335537 2. Couturier EG, Bomhof MA, Neven AK, van Duijn NP (2003) Menstrual migraine in a representative Dutch population sample: prevalence, disability and treatment. Cephalalgia 23:302–308. https://doi.org/10.1046/j.1468-2982. 2003.00516.x 3. MacGregor EA, Victor TW, Hu X, Xiang Q, Puenpatom RA, Chen W, Campbell JC (2010) Characteristics of menstrual vs nonmenstrual migraine: a post hoc, within-woman analysis of the usual-care phase of a nonrandomized Ethics approval and consent to participate The study protocol was approved by the ethics committee or institutional review board at each clinical site, and all patients provided signed informed consent before the start of any study-related procedures. The study was con- ducted in accordance with the International Council for Harmonisation Good Clinical Practice Guidelines and conforms to the provisions of the Declaration of Helsinki. Abbreviations CGRP C l i i 3. MacGregor EA, Victor TW, Hu X, Xiang Q, Puenpatom RA, Chen W, Campbell JC (2010) Characteristics of menstrual vs nonmenstrual migraine: a post hoc, within-woman analysis of the usual-care phase of a nonrandomized Page 9 of 9 Pavlovic et al. 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Vetvik KG, Macgregor EA, Lundqvist C, Russell MB (2014) Prevalence of menstrual migraine: a population-based study. Cephalalgia 34:280–288. https://doi.org/10.1177/0333102413507637 29. Goadsby PJ, Reuter U, Hallstrom Y, Broessner G, Bonner JH, Zhang F, Sapra S, Picard H, Mikol DD, Lenz RA (2017) A controlled trial of erenumab for episodic migraine. N Engl J Med 377:2123–2132 14. Allais G, Chiarle G, Sinigaglia S, Benedetto C (2018) Menstrual migraine: a review of current and developing pharmacotherapies for women. Expert Opin Pharmacother 19:123–136. https://doi.org/10.1080/14656566.2017. 1414182 g g 30. Gold EB (2011) The timing of the age at which natural menopause occurs. Obstet Gynecol Clin N Am 38:425–440. https://doi.org/10.1016/j.ogc.2011.05.002 31. Lipton RB, Bigal ME, Diamond M, Freitag F, Reed ML, Stewart WF, AMPP Advisory Group (2007) Migraine prevalence, disease burden, and the need for preventive therapy. 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Sacco S, Merki-Feld GS, Ægidius KL, Bitzer J, Canonico M, Gantenbein AR, Kurth T, Lampl C, Lidegaard O, MacGregor EA, MaassenVanDenBrink A, Mitsikostas DD, Nappi RE, Ntaios G, Paemeleire K, Sandset PM, Terwindt GM, Vetvik KG, Martelletti P, European Headache Federation (EHF), the European Society of Contraception and Reproductive Health (ESCRH) (2018) Effect of exogenous estrogens and progestogens on the course of migraine during reproductive age: a consensus statement by the European Headache Federation (EHF) and the European Society of Contraception and Reproductive Health (ESCRH). J Headache Pain 19:76. https://doi.org/10. 1186/s10194-018-0896-5 21. Xu Z, Li Y, Tang S, Huang X, Chen T (2015) Current use of oral contraceptives and the risk of first-ever ischemic stroke: a meta-analysis of observational studies. Thromb Res 136:52–60. https://doi.org/10.1016/j. thromres.2015.04.021
https://openalex.org/W2117665849
https://oaktrust.library.tamu.edu/bitstream/1969.1/153691/1/RYBKOWSKI_SHEPLEY_BALLARD_Target%20Value%20Design_Application%20to%20Newborn%20ICU.pdf
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Target Value Design: Applications to Newborn Intensive Care Units
HERD
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PA P E R S T H E O R Y PA P E R S T H E O R Y P TARGET VALUE DESIGN: APPLICATIONS TO NEWBORN INTENSIVE CARE UNITS Target Value Design: Applications to Newborn Intensive Care Units Zofia K. Rybkowski, PhD; Mardelle McCuskey Shepley, DArch; and H. Glenn Ballard, PhD Zofia K. Rybkowski, PhD; Mardelle McCuskey Shepley, DArch; and H. Glenn Ballard, PhD Author Affiliations: Dr. Rybkowski is Assistant Professor in the Depart- ment of Construction Science in the College of Architecture at Texas A&M University in College Station, TX. Dr. Shepley is a Professor in the Depart- ment of Architecture in the College of Architecture and Director, Center for Health Systems and Design, at Texas A&M University. Dr. Ballard is Adjunct Associate Professor and Research Director for Project Production Systems Laboratory (P2SL) at the University of California, Berkeley. Corresponding Author: Zofia K. Rybkowski, PhD, Assistant Professor, Department of Construction Science, 434 Langford A, College of Architec- ture, Texas A&M University, College Station, TX 77843-3137 (zrybkowski@ tamu.edu; 979-845-4354). Preferred Citation: Rybkowski, Z. K., Shepley, M. M., & Ballard, H. G. (2012). Target value design: Applications to newborn intensive care units. Health Environments Research & Design Journal, 5(4), 5–23. Preferred Citation: Rybkowski, Z. K., Shepley, M. M., & Ballard, H. G. (2012). Target value design: Applications to newborn intensive care units. Health Environments Research & Design Journal, 5(4), 5–23. One of the thrusts of EBD has been to examine the ROI of potentially expensive EBD measures. The recent convergence of two knowledge domains—EBD and Lean construction’s TVD— has highlighted the growing importance of devel- oping a more complete picture of the elements that constitute long-term value in NICU facility design. Early EBD indicators suggest that lower capital costs may not necessarily yield the most satisfactory healthcare outcomes long-term. Hav- ing a strong understanding of the true financial costs and benefits associated with specific NICU design elements is increasingly important as the implementation of TVD continues to expand. Part of the discussion regarding target value design (TVD) has centered on the impact of TVD on design approaches supported by evidence-based design (EBD). According to Hamilton (2006), EBD is “the conscientious and judicious use of current best evidence and its critical interpretation, to make significant design decisions for each unique project. These design decisions should be based on sound hypotheses related to measureable outcomes” (p. 31). One of the thrusts of EBD has been to examine the ROI of potentially expensive EBD measures. Although it is well known that construction costs represent a small portion of a hospital’s budget (Berry et al., 2004; Evans, Haryott, Haste, & Jones, 1998; Kirk & Dell’Isola, 1995), the impact of new and remodeled construction must be justified. TVD is a management practice that drives design to deliver customer value and design within project constraints (Ballard, 2011; Denerolle, 2011). TVD emerged from the Lean construc- tion community and its values. Therefore, to understand how and why TVD works requires some understanding of Lean principles. Lean construction, a project delivery process that seeks to minimize waste and add value, thus inducing flow, has been optimizing the three- legged stool of construction management—time, cost, quality and safety (as the seat) (Jackson, 2004). Value is defined as what customers need to accomplish their purposes. Historically, TVD de- veloped as an adaptation of target costing to con- struction within the context of Lean construction theory (Rybkowski, 2009); it is “a management practice that seeks to make customer constraints drivers of design for the sake of project delivery.” (Ballard, 2011, p. 79). Although the healthcare In the interest of addressing the issue of EBD and ROI in NICUs, this paper describes the TVD process and relevant case studies in healthcare facilities. Abstract In light of international and national efforts to provide both optimally designed and economical healthcare environments, a significant effort is un- derway to address the simultaneous accomplishment of these goals. A sophisticated analysis of the fiscal implications of a proposed healthcare environment is an essential tool for an informed design process. There is a need for greater understanding of the health impact of various design elements in neonatal intensive care units (NICUs) as well as cost-benefit information to make informed decisions about the long-term value of design decisions. This is particularly evident when design teams are considering the transition from open-bay NICUs to single-family-room (SFR) units. This paper introduces the guiding principles behind target value design (TVD)—a price-led design methodology that is gaining acceptance in healthcare facility design within the Lean construction methodology. The paper also discusses the role that set-based design plays in TVD and its application to NICUs. Although several healthcare projects have employed return on investment (ROI) to justify design deci- sions, neonatal intensive care units (NICUs) have not been thoroughly addressed. A description of tools for examining the financial impact of design decisions would be helpful to architects and organi- zations considering new construction. A significant number of new facilities have recently been com- pleted, and a host of others are in the programming and design process. Although there are currently more than 1,000 NICUs in the United States and Canada (American Academy of Pediatrics, 2011), the first NICU was established as recently as 1960 (Gluck, 1985). Key Words: NICU design, hospital design, healthcare facility design, private patient room, target value design, target cost- ing, Lean construction 5 HERD Vol. 5, No. 4, SUMMER 2012 • HEALTH ENVIRONMENTS RESEARCH & DESIGN JOURNAL 5 T H E O R Y HERD VOLUME 5, NUMBER 4, PP 5-22 COPYRIGHT ©2012 VENDOME GROUP, LLC T H E O R Y One of the thrusts of EBD has been to examine the ROI of potentially expensive EBD measures. Additionally, it provides a brief literature review of the construction costs associated with NICUs and recommendations for TVD tools in NICUs that can be used to compare the impact of open versus single- family rooms (SFRs). WWW.HERDJOURNAL.COM ISSN: 1937-5867 6 6 PA P E R S T H E O R Y facility owner, architecture, engineering, and construction (OAEC) community is still experi- menting with TVD, patterns of a shared meth- odology have emerged from organizations that have begun implementing it (Ballard, 2011). The following discussion attempts to outline TVD processes generally shared by these early pioneers. A working knowledge of the TVD framework is important for integrating long-term value into a NICU facility. waiting on workers and workers waiting on work. As simple as this task may sound superficially, reaching a state of perfect flow, especially when there are diverse individuals and complex sub- processes involved, is challenging. Although the Lean ideal may never be reached, it can be ever more approximated. Approaching the Lean ideal of minimum waste and maximum value customarily requires mul- tiple iterations; in Lean parlance, these iterations manifest as kaizen events (continuous improve- ment) where members of an organization collab- orate to plan and run experiments to see whether the recommended changes lead to reduced waste and/or enhanced value. Kaizen events require the participation of numerous individuals because in most organizations the repositories of organiza- tional knowledge are distributed. Lean Construction and the Emergence of Target Value Design Success principles that originated from the Toyota Production System, later termed “Lean” production (Liker, 2004; Womack, Jones, & Roos, 1990), were introduced not only to U.S. manufacturing (Black & Hunter, 2003; Car- reira, 2005; Davis, 2009; Standard & Davis, 1999), but also to healthcare processing (Arthur, 2011; Graban, 2009; Jimmerson, 2007, 2010; Kenney, 2011; Tapping, Kozlowski, Archbold, & Sperl, 2009; Zidel, 2006) and construction (Ballard, 2000; Koskela, 1992). Flow is a means to generate value and avoid waste. According to Womack & Jones (2003), Lean thinking can be condensed into five principles: “[P]recisely specify value by specific product, identify the value stream for each product, make value flow without interruptions, let the customer pull value from the producer, and pursue perfec- tion” (p. 10). This paper discusses the critical importance of deepening understanding of the impact of NICU facility design on patient outcomes, especially as the practice of TVD gains wider acceptance. The authors define TVD as it is practiced now—thus far by Sutter Health (based in California), Uni- fied Health Services (based in Tennessee), SSM Healthcare (based in Missouri), as well as other regions, and spreading to other parts of the United States (Christiansen, 2009; Rybkowski, 2010). They introduce the concept of set-based design and life-cycle cost analysis (two tools fundamen- tal to the TVD process). Finally, a summary of what is known about NICUs and suggestions for how TVD can be applied to improving their design is offered. The Lean ideal toward which processing is pulled might be defined as a state of perfect value-laden flow because it avoids both the waste of work 7 HERD Vol. 5, No. 4, SUMMER 2012 • HEALTH ENVIRONMENTS RESEARCH & DESIGN JOURNAL 7 T H E O R Y HERD VOLUME 5, NUMBER 4, PP 5-22 COPYRIGHT ©2012 VENDOME GROUP, LLC T H E O R Y Figure 1. Traditional costing begins with cost (black bar, left) and adds profit markup to determine price. Target costing, by contrast, begins with price (black bar, right) and subtracts profit markup to reveal the target cost at which the product must be produced to meet that price. Source: Adapted from Rybkowski, 2009, Fig. 46. Target Costing and Target Value Design Olaf Fieldhouse Completion date April 2000 August 2002 Gross square feet 85,414 114,000 Project duration 24 months 14 months Total cost (including A/E & CM * fees) $13,533,179 $11,716,836 Cost per square foot $158.44 $102.79 *A (Architecture); E (Engineering); CM (Construction Management) Table 1. A Comparison of Two Delivery Methods Note: OAEC stakeholders for the field house at St. Olaf engaged in target-costing exercises as a way to reduce cost and schedule—and enhance value. Source: Adapted from Ballard & Reiser, 2004. Critical to target costing is a cardinal rule: the tar- get cost cannot be exceeded (Clifton, Bird, Albano, & Townsend, 2004). In other words, if the target cost cannot be met, the project must be cancelled because it will not be economically feasible. OAEC stakeholders must be included in the process of identifying an appropriate target price because the “silo-ization” of OAEC professions has scattered the knowledge base of projects. Critical to target costing is a cardinal rule: the tar- get cost cannot be exceeded (Clifton, Bird, Albano, & Townsend, 2004). In other words, if the target cost cannot be met, the project must be cancelled because it will not be economically feasible. OAEC stakeholders must be included in the process of identifying an appropriate target price because the “silo-ization” of OAEC professions has scattered the knowledge base of projects. ences in construction times and total cost for the two projects are striking (Table 1). As it turned out, the St. Olaf director of facilities reported that Carleton College agreed that Tostrud was also better fit for its purpose. In 2005, the Project Production Systems Labo- ratory (P2SL) at the University of California, Berkeley, published a current best practice guide to target costing (Ballard, 2005). The recom- mended steps included a requirement that all key members of the team (designers, engineers, constructors, and client stakeholders) understand and be involved in the preparation of a feasibil- ity study and subsequent budget—and that the owner be an active and permanent member of the project delivery team, with real tasks to deliver and be accountable for. A cost-constrained building industry noted the benefits of target-costing, and in 2000 Nicolini and colleagues published one of the first research papers on target costing for the construction industry (Nicolini, Tompkins, Holti, Oldman, & Smalley, 2000). Target Costing and Target Value Design Target costing is “a structured approach to de- termine the life cycle cost at which a proposed product with specified functionality and qual- ity must be produced to generate the desired level of profitability over its life cycle when sold at its anticipated selling price” (Cooper & Slagmulder, 1997, p. 72). Unlike traditional costing that establishes product price by sum- ming component costs and a profit margin (and thus generates a price that may exceed a customer’s willingness or ability to pay), target costing begins with a price the customer is able and willing to pay and subtracts a desired profit margin to reveal the target cost. In other words, target costing is price-led costing, which can be expressed as: Figure 1. Traditional costing begins with cost (black bar, left) and adds profit markup to determine price. Target costing, by contrast, begins with price (black bar, right) and subtracts profit markup to reveal the target cost at which the product must be produced to meet that price. Source: Adapted from Rybkowski, 2009, Fig. 46. target cost = competitive market price - target profit (Ansari, Bell, & Group, 1997). Figure 2. To meet the target cost, multifunctional teams design collectively and continuously, re-estimat- ing the cost as the product’s design progresses. Source: Adapted from Rybkowski, 2009, Fig. 46. Adopting the target-costing process should not be interpreted as trying to produce a cheap product. In fact, if practiced properly, the pro- cess not only eliminates waste, it adds value—as will be discussed shortly in reference to the MacLeamy Curve. The target-costing concept is depicted in Figure 1. Once a viable target cost is established, a core team of OAEC stakehold- ers is engaged to iteratively redesign the project until the original design cost has been honed to its target cost, as shown in Figure 2. The pro- cess requires integrated cross-functional team engagement in value engineering where costs for successive stages of design are continually re-estimated (Clifton et al., 2004). Figure 2. To meet the target cost, multifunctional teams design collectively and continuously, re-estimat- ing the cost as the product’s design progresses. Source: Adapted from Rybkowski, 2009, Fig. 46. WWW.HERDJOURNAL.COM ISSN: 1937-5867 8 PA P E R S T H E O R Y Carleton College Recreation Center St. Target Costing and Target Value Design The authors indicated that target costing had failed, but Ballard and Reiser (2004) later reported suc- cess following target costing exercises at the Tostrud Fieldhouse of St. Olaf College in Min- nesota. They compared schedule and budget results from the St. Olaf field house with a similar project at Carleton College (Ballard & Reiser, 2004). The buildings were nonidentical and built in different years and cities, but their functions were similar enough that the differ- In 2005 and 2008, the term target value design formally entered the literature when Macomber and Barberio (2008) used it to refer to target costing in construction. The authors published foundational principles for the process of design, including: concurrently design the product and process in design sets; collaborate in small HERD Vol. 5, No. 4, SUMMER 2012 • HEALTH ENVIRONMENTS RESEARCH & DESIGN JOURNAL 9 9 T H E O R Y HERD VOLUME 5, NUMBER 4, PP 5-22 COPYRIGHT ©2012 VENDOME GROUP, LLC T H E O R Y The now well-publicized MacLeamy Curve demonstrates why early involvement of OAEC stakeholders is critical. Design decisions made early in the process have the greatest ability to affect cost and functional capabilities, as shown in Figure 3. Using a NICU as an example, sup- pose the architect selects low-emissivity glass for the windows in infant care areas associated with intense solar exposure. If OAEC stakeholders are involved early on, the mechanical engineer will be aware that he or she can downsize the chillers and minimize duct sizes, thus reducing project Design decisions made early in the process have the greatest ability to affect cost and functional capabilities. Design decisions made early in the process have the greatest ability to affect cost and functional capabilities. Design decisions made early in the process have the greatest ability to affect cost and functional capabilities. and diverse groups; and meet regularly in a big room. The purpose of working in a big room is to facilitate communication and develop creative synergies. A complete version of their 2008 list is included at the end of this paper. and diverse groups; and meet regularly in a big room. The purpose of working in a big room is to facilitate communication and develop creative synergies. A complete version of their 2008 list is included at the end of this paper. Figure 3. Source: Adapted from MSA, 2004, at http://www.msa-ipd.com/MacleamyCurve.pdf. Target Costing and Target Value Design The MacLeamy Curve demonstrates the logic of bringing OAEC members together early in the design process, where the ability to affect cost and function is greatest. Source: Adapted from MSA, 2004, at http://www.msa-ipd.com/MacleamyCurve.pdf. 1 Ability to impact cost and functional capabilities 2 Cost of design changes 3 Team during traditional (design-bid-build) design process 4 Team during IPD design process Traditional Delivery Construction Documents Agency Permit/ Bidding Schematic Design Predesign Design Development Construction Integrated Project Delivery Implementation Documents Agency Coord/ Final Buyout Criteria Design Conceptualization Detailed Design Construction 1 2 3 4 3 Traditional Delivery Figure 3. The MacLeamy Curve demonstrates the logic of bringing OAEC members together early in the design process, where the ability to affect cost and function is greatest. Source: Adapted from MSA, 2004, at http://www.msa-ipd.com/MacleamyCurve.pdf. Figure 3. The MacLeamy Curve demonstrates the logic of bringing OAEC members together early in the design process, where the ability to affect cost and function is greatest. Source: Adapted from MSA, 2004, at http://www.msa-ipd.com/MacleamyCurve.pdf. Figure 3. The MacLeamy Curve demonstrates the logic of bringing OAEC members together early in th design process, where the ability to affect cost and function is greatest. Source: Adapted from MSA, 2004, at http://www.msa-ipd.com/MacleamyCurve.pdf. WWW.HERDJOURNAL.COM ISSN: 1937-5867 10 PA P E R S T H E O R Y TARGET VALUE DESIGN: APPLICATIONS TO NEWBORN INTENSIVE CARE UNITS PA P E R S T H E O R Y cost. Similarly, once duct sizes are minimized, ad- ditional cost savings can be achieved by reducing floor-to-floor heights—if the structural engineer is in the room early on (Barnett, 2004). of agreement contract creates an environment where risk is shared among OAEC stakeholders and provides pain-sharing (above allowable cost) and gain-sharing (below allowable cost) incen- tives to motivate the team to collectively reach both allowable cost and target cost goals (Lichtig, 2005a, 2005b, 2006). By contrast, if a traditional design-bid-build delivery system had been used, many of these cost savings would have been discovered too late in the process to implement them. Target Costing and Target Value Design In addition to the financial benefits implied by the MacLeamy Curve, it is the authors’ experience that, unlike TVD, traditional design-bid-build systems pit architectural, engi- neering, and construction (AEC) service providers against the owner because owner aspirations have been both generated without an understanding of what the owner can actually afford, and financial risk has been contractually pushed downward from the owner through the AEC stakeholder team. The result is both an adversarial system of relationships and last-minute cost cutting severed from the project as a whole, resulting in compromise in design and construction quality. Thus far, experimentation with TVD on healthcare facility projects has yielded the preliminary results shown in Table 2. Tabulation of these results shows that the average cost savings during a TVD exercise reach nearly 13%. Because TVD experimentation is in its early days, some practitioners believe that much greater savings are possible. Set-Based Design Set-based design is integral to TVD and may be likened to setting a meeting appointment time Figure 4. The target-costing goal diagram (time versus cost) used by Sutter Health during its target-costing exercises for the CPMC Cathedral Hill Hospital in San Francisco, CA. Source: Adapted from Rybkowski, 2009. Time Estimated Cost ket Cost Allowable Cost Target Cost By contrast, with TVD providers and suppliers help the owner early on to establish an informed allowable cost (the make-or-break cost goal critical for a project to be economically fea- sible) and target cost (a “stretch” goal that would be desirable, though not critical, to reach), as shown in Figure 4. If there is a substantial gap between the market cost and the allowable cost, TVD spurs the creation of innova- tive solutions. An integrated form Ma Figure 4. The target-costing goal diagram (time versus cost) used by Sutter Health during its target-costing exercises for the CPMC Cathedral Hill Hospital in San Francisco, CA. Source: Adapted from Rybkowski, 2009. Figure 4. The target-costing goal diagram (time versus cost) used by Sutter Health during its target-costing exercises for the CPMC Cathedral Hill Hospital in San Francisco, CA. Source: Adapted from Rybkowski, 2009. HERD Vol. 5, No. 4, SUMMER 2012 • HEALTH ENVIRONMENTS RESEARCH & DESIGN JOURNAL 11 T H E O R Y HERD VOLUME 5, NUMBER 4, PP 5-22 COPYRIGHT ©2012 VENDOME GROUP, LLC T H E O R Y Project Size Year Market Cost Actual Cost Cost Reduction (sf) of Completion (%) 368,882 2009 $98,000,000 $89,200,000 9.0 230,000 2007 $22,000,000 $17,900,000 18.6 114,000 2002 $13,533,179 $11,717,000 13.4 75,362 2006 $13,600,000 $11,200,000 17.6 30,000 2010 $14,500,000 $13,700,000 5.5 12.8 Average % Cost reduction Table 2. Summary of Results From TVD Exercises Completed to Date Table 2. Summary of Results From TVD Exercises Completed to Date include a nurse, a doctor, a family member, and an administrator. Ward, Liker, Cristiano, and Sobek II (1995) contrast the set-based design approach with point-based design, where one alternative is modified through the contribution of various relevant stakeholders. Set-Based Design The problem with point-based design is that one alternative must be modified through multiple iterations, and designers of segments are often working both sequentially and in isolation (called “throw it over the wall” ap- proach); once the alternative reaches a “hard” constraint (i.e., a code violation or impassable owner requirement) upon evaluation by a later stakeholder, all earlier iterations performed on that alternative may be classified as waste. include a nurse, a doctor, a family member, and an administrator. Ward, Liker, Cristiano, and Sobek II (1995) contrast the set-based design approach with point-based design, where one alternative is modified through the contribution of various relevant stakeholders. The problem with point-based design is that one alternative must be modified through multiple iterations, and designers of segments are often working both sequentially and in isolation (called “throw it over the wall” ap- proach); once the alternative reaches a “hard” constraint (i.e., a code violation or impassable owner requirement) upon evaluation by a later stakeholder, all earlier iterations performed on that alternative may be classified as waste. with a group of parties via e-mail. If, for example, Tuesday at 10:00 a.m. is the only time the meet- ing organizer proposes, the e-mail must travel through multiple parties—back and forth—until a satisfactory time is reached; this is a potentially lengthy process. NICU staff members have in- tense and complex schedules and coordinating meeting times is challenging. In a set-based ap- proach, by contrast, a meeting organizer proposes a range of possible times using a meeting software program, allowing for the much faster identifica- tion of intersecting time slots available for all parties (Parrish, 2009; Parrish et al., 2007). Similarly, set-based design exercises challenge a group to simultaneously identify multiple alternatives to achieve the same functional- ity (Cooper & Slagmulder, 1997). As OAEC stakeholder groups successively evaluate each alternative according to whether the alterna- tive can accommodate specific constraints, the list of design options narrows. In the case of a NICU design process, the cluster might Whereas point-based design is a linear, throw-it-over-the-wall type of process, set-based design involves funneling. Whereas point-based design is a linear, throw-it-over-the-wall type of process, set-based design involves funneling. WWW.HERDJOURNAL.COM ISSN: 1937-5867 12 PA P E R S T H E O R Y P TARGET VALUE DESIGN: APPLICATIONS TO NEWBORN INTENSIVE CARE UNITS Figure 5. Set-Based Design During a traditional “throw-it-over-the-wall” point-based design approach (top), OAEC members consider each NICU configuration separately and sequentially in time. By contrast, during a “funneling” set-based design approach (bottom), a set of NICU configuration alternatives is considered, and alternatives are simultaneously assessed and revised or eliminated, greatly reducing the time it takes for the OAEC team to rework designs at points of constraint—and to reach a final consensus. Figure 5. During a traditional “throw-it-over-the-wall” point-based design approach (top), OAEC members consider each NICU configuration separately and sequentially in time. By contrast, during a “funneling” set-based design approach (bottom), a set of NICU configuration alternatives is considered, and alternatives are simultaneously assessed and revised or eliminated, greatly reducing the time it takes for the OAEC team to rework designs at points of constraint—and to reach a final consensus. A comparable situation in the design of a NICU would be to have the physicians and others meet in separate groups. Whereas point- based design is a linear, throw-it-over-the-wall type of process, set-based design involves fun- neling. The set of viable alternatives narrows increasingly as alternatives are simultaneously evaluated and successively eliminated by a core set of OAEC stakeholders. The example shown in Figure 5 represents one project team’s evalu- ation of four NICU configurations. Note that a different OAEC project team might evaluate the configurations differently from the one shown, subjecting them to its own code and owner constraints. paper (similar to the 11" × 17" standard cut) and posted publically on the wall of the design office for consideration by the full OAEC team. These ideas are evaluated concurrently. If life-cycle cost or ROI analysis as performed by engineers is used (ASTM, 2006; Bull, 1993; Kirk & Dell’Isola, 1995; Langston, 2005), practioners should take care to implement incremental analysis when comparing alternatives. The Cathedral Hill project instead implemented a process called “Choosing by Advantages” (Suhr, 1999) to evalu- ate and winnow alternatives—a decision-making methodology designed to develop consensus among stakeholders with different interests and criteria. Target Value Design and Set-Based Design Issues in NICUs When a project team is co-located, as was practiced during the design of Sutter Health’s CPMC Cathedral Hill Hospital, each design idea is documented individually on A3-sized A recent trend in employing ROI analyses is to study the impact of EBD features on long-term HERD Vol. 5, No. 4, SUMMER 2012 • HEALTH ENVIRONMENTS RESEARCH & DESIGN JOURNAL 13 T H E O R Y HERD VOLUME 5, NUMBER 4, PP 5-22 COPYRIGHT ©2012 VENDOME GROUP, LLC T H E O R Y staff and maintenance costs. A variation on this theme is to compare different approaches to particular design issues. In the case of neonatal intensive care, several recurring topics confront designers. These controversies include unit loca- tion in the context of the hospital, degree of homelike-ness, the number of babies per unit, supply location (centralized versus satellite versus bedside), access to nature, and the number of patients per room. Currently, the subject most frequently debated is the number of patients per room, specifically whether a unit should provide private family rooms, open bays, combined units, or shared rooms. The issue of shared versus private rooms and associated hybrids in NICUs has probably been the most frequently discussed issue in NICU design. roll, 2006). An open-bay NICU varies in the amount of space allocated to a child, although the minimum established by the Recommended Standards is 120 nsf per infant (White et al., 2007). Configurations for these spaces are shown in Figure 6. Private rooms, commonly called single-family rooms (SFRs), typically accommodate family space within the room and should be at least 160 net square feet (nsf), according to the New Recommended Standards for NICU Design (White, Smith, & Harrell, 2012). In a survey of 11 NICUs of varying room densities, the average private room was found to be 172 nsf (Harris, Shepley, White, Kolberg, & Har- The issue of shared versus private rooms and associated hybrids in NICUs has probably been the most frequently discussed issue in NICU de- sign. Most studies suggest that private rooms are beneficial for staff, infants, and families (Carter, Carter, & Bennett, 2008; Ortenstrand, Westrup, & Brostrom, 2010; Rosenblum, 2005; Shepley, Harris, & White, 2008; Shepley, Harris, White, Single-Family Room Double-Occupancy Open-Bay Combination Unit Figure 6. In set-based design, typical NICU configuration floor plans should be evaluated simultaneously as a set. Open-Bay Combination Unit Single-Family Room Figure 6. Target Value Design and Set-Based Design Issues in NICUs In set-based design, typical NICU configuration floor plans should be evaluated simultaneously as a set. WWW.HERDJOURNAL.COM ISSN: 1937-5867 14 PA P E R S T H E O R Y PA TARGET VALUE DESIGN: APPLICATIONS TO NEWBORN INTENSIVE CARE UNITS Previous Studies on the Cost Implications of Private Rooms & Steinberg, 2008; Smith, Schoenbeck, & Clay- ton, 2009; Stevens, Helseth, Khan, Munson, & Reid, 2011; Walsh,  McCullough, & White, 2006); however, some of these SFR studies do not support all outcomes (White, 2011). “Although families appreciate the increased privacy and ownership they feel in a private room, they can also feel somewhat isolated. Caregivers likewise can feel isolated from their colleagues, especially if a central gathering area is not provided” (White, 2011, p. 5). Most of the discussion around the pros and cons of private versus shared rooms has centered on the adult population. Chaudhury et al. provide a thorough summary of the literature on this topic, including a meta-analysis of cost information that suggests that operating costs drop, capital costs rise, occupancy rates increase, length of stay decreases, and medication errors and costs dimin- ish in private rooms (Chaudhury, Mahmood, & Valente, 2005). The Importance of Cost Research Regarding the Implications of SFRs Comparison of First (Capital) and Long-Term Costs for Semi-Private and Private Adult Patient Rooms—as well as Forecasted Simple and Discounted Payback Periods, Should Private Rooms Be Selected Over Semi-Private Rooms location are believed to recoup the additional construction cost within a few years (Calkins & Cassella, 2007). rooms for adult populations suggest that more expensive private rooms pay for themselves after 7.4 years, and 8.75 years, respectively, as shown in Table 3. Any savings garnered after the payback period is a long-term financial benefit for the owner. The Importance of Cost Research Regarding the Implications of SFRs According to Boardman and Forbes (2007), pri- vate rooms are more desirable to adult patients, they support recovery time, they require more space, and they are more expensive to construct and operate than shared rooms. In their study of the cost-benefit analysis of the two options, they found the increase in cost (land, construction, maintenance, and staffing) for each private room to be $228,000 (Canadian) based on the pres- ent value of a 50-year life span and a discount rate of 3.5%. Considering the health and social benefits (willingness to pay, patient transfers, reduced waiting time) of the private room, the difference is approximately $70,000 (Canadian) (Boardman & Forbes, 2007). The difference in construction cost alone is believed to be ap- proximately $61,000, assuming the shared room is 287 nsf per person and the private room is 436 nsf (Boardman & Forbes, 2007). g g p NICU operations play a significant role in the financial operation of a hospital. According to National Vital Statistics Reports, 12.18% of births in 2009 were premature (Martin et al., 2011). The annual hospitalization cost for these infants was $51,600 per infant (In- stitute of Medicine, 2006). Phillips notes that high-risk newborn care is the most profitable component of obstetric practice and cites an estimated 7% increase in NICU population between 2007 and 2015 (Phillips, 2010). These strong NICU volume trends offer a significant contribution profit oppor- tunity for hospitals offering neonatology services…. As VCM (variable contribution to margin) increases, hospitals can more easily underwrite the fixed cost associated with OB (obstetrics). Therefore NICUs of- fer hospitals and health care organizations tremendous profit opportunity. (Phillips, 2010, p. 4) A simple (undiscounted) payback and a discounted (3.5%) payback cost tally of Boardman and Forbes’ (2007) data for private 15 HERD Vol. 5, No. 4, SUMMER 2012 • HEALTH ENVIRONMENTS RESEARCH & DESIGN JOURNAL T H E O R Y HERD VOLUME 5, NUMBER 4, PP 5-22 COPYRIGHT ©2012 VENDOME GROUP, LLC T H E O R Y T H E O R Y HERD VOLUME 5, NUMBER 4, PP 5-22 COPYRIGHT ©2012 VENDOME GROUP, LLC Source: Adapted from Boardman & Forbes (2007). Table 3. Comparison of First (Capital) and Long-Term Costs for Semi-Private and Private Adult Patient Rooms—as well as Forecasted Simple and Discounted Payback Periods, Should Private Rooms Be Selected Over Semi-Private Rooms Table 3. Previous Studies on the Cost Implications of NICUs To the authors’ knowledge, there is only one study on the cost implications of SFRs, and only a handful of studies that address construc- tion costs in NICUs generally. Regarding the overall NICU, journal publications include: In general, authors have suggested that private room construction for adults is more expensive than shared rooms and open bays (Calkins & Cassella, 2007; Moon, 2005), although the operational costs due to flexibility of patient • Berens and Weigle examined the impact of *Adjusted to 2011 per U.S. Department of Labor http://www.bls.gov/data/inflation_calculator.htm Source: Walsh-Sukys et al., 2001) adjusted to 2011. Table 4. Costs for Renovation of a NICU Room Table 4. Costs for Renovation of a NICU Room WWW.HERDJOURNAL.COM ISSN: 1937-5867 16 PA P E R S T H E O R Y PA TARGET VALUE DESIGN: APPLICATIONS TO NEWBORN INTENSIVE CARE UNITS Figure 7. Cost per square foot of varying NICU configurations. Source: Harris et al., 2006. Figure 7. Cost per square foot of varying NICU configurations. Source: Harris et al., 2006. acoustical ceiling tile replacement in an open bay NICU (cost approximately $2,400), and found a decibel reduction level from 55 to 53 (p < 0.05) (Berens & Weigle, 1996). With specific regard to construction costs associ- ated with open-bay units versus SFRs, Harris et al. (2006) gathered data from 11 hospitals built between 1995 and 2005. The results are summa- rized in Figure 7; they suggest that combination units are the least expensive, followed by open- bay, SFR, and double-occupancy units (Harris et al., 2006). • Medical physician Robert White discusses the relative costs of flooring materials in the context of safety, economy, environmental impact, durability, comfort, sound control, and acoustics (White, 2007). • Walsh-Sukys and colleagues modified an existing NICU six-patient room to enhance the acoustical and lighting environment and were able to reduce noise and light levels and increase staff satisfaction relative to a control room at a 1997 cost of approximately $7,200 (Walsh-Sukys, Reitenbach, Hudson-Barr, & DePompei, 2001). These amounts are tabu- lated in Table 4. As part of the same study, the researchers found that the average area per infant station was low- est in a double-occupancy unit (340 nsf/infant), followed closely by the combination unit (370 nsf/infant). The open-bay units averaged 468 nsf/infant, and the average for the SFRs was 692 nsf/infant (Harris et al., 2006). Deciding whether to build an SFR versus open-bay NICU can be addressed using target value design set-based design analysis. dedicated to an infant in an open NICU was 107 nsf, whereas the average area in an SFR was 172 nsf. Previous Studies on the Cost Implications of NICUs According to Harris and colleagues (2006) the average area 17 HERD Vol. 5, No. 4, SUMMER 2012 • HEALTH ENVIRONMENTS RESEARCH & DESIGN JOURNAL 17 T H E O R Y HERD VOLUME 5, NUMBER 4, PP 5-22 COPYRIGHT ©2012 VENDOME GROUP, LLC T H E O R Y Deciding whether to build an SFR versus open-bay NICU can be addressed using TVD set-based design analysis. In a traditional lin- ear delivery system, the owner and designers might specify the type of NICU configuration they are most familiar with and then succes- sively iterate it until they reach a configuration they find most appropriate for their needs. In set-based design, by contrast, the OAEC will generate multiple design alternatives simulta- neously (e.g., open-bay, double rooms, SFRs, or combination units) and then evaluate the merits of each alternative simultaneously, mak- ing adjustments for owner priorities. A rough scoring model might be developed for a NICU, as shown in Table 5. (Because an explanation of the Choosing by Advantages system imple- mented by Sutter Health is beyond the scope Deciding whether to build an SFR versus open-bay NICU can be addressed using target value design set-based design analysis. Target Value Design and Single-Family- Room versus Open-Bay NICUs “A wise planner will balance innovation and ef- fectiveness.” (Harrell & Moon, 2008, p. 48) Note: Criteria are weighted by percentage (totaling 1) in alignment with a specific owner’s priorities and will vary by owner. Table 5. Example of Scoring Model Showing NICU Design Alternatives, Rated From 1– 5 (Least to Most Effective) for Each Desired Criteria Table 5. Example of Scoring Model Showing NICU Design Alternatives, Rated From 1– 5 (Least to Most Effective) for Each Desired Criteria Note: Criteria are weighted by percentage (totaling 1) in alignment with a specific owner’s priorities and will vary by owner. WWW.HERDJOURNAL.COM ISSN: 1937-5867 WWW.HERDJOURNAL.COM ISSN: 1937-5867 18 PA P E R S T H E O R Y P TARGET VALUE DESIGN: APPLICATIONS TO NEWBORN INTENSIVE CARE UNITS of this paper, the reader is advised to separately consult Suhr [1999] to understand the consensus- building decision-making process it employs—a methodology some consider superior to a scoring model.) Figure 8. The TVD process as developed by P2SL. Source: Adapted from Ballard (2008), as reported in Rybkowski (2009, Fig. 50). A NICU design team wishing to undertake TVD might follow the steps outlined in Figure 8. Note that each step in the process requires integrated collaboration among OAEC team members. There are also nodes to contemplate cancel- lation if it is discovered the project cannot be constructed at or below the allowable cost. In addition to the mechanics of TVD, it should be remembered that the TVD process is accomplished within the integrated project deliv- ery format of Lean. The Lean culture is one of collaboration, trust, shared decision making, reliable promises, and risk sharing (Lichtig, 2006). For designers of NICUs as well as other professionals, there are at least nine foundational practices key to a successful TVD process. Based on the general categories of Macomber and Barberio (2008) and customized for the NICU application, they are: Figure 8. T Source: Adapt Figure 8. The TVD process as developed by P2SL. Source: Adapted from Ballard (2008), as reported in Rybkowski (2009, Fig. 50 2. Lead the design effort for learning and innovation. 3. Design to a detailed estimate. 4. Collaboratively plan and replan the project. 5. Concurrently design the project and the process in design sets. 6. Design and detail in the sequence in which NICU staff, families, and patients will use the spaces. 1. Target Value Design and Single-Family- Room versus Open-Bay NICUs Engage deeply with the hospital-based NICU team members (e.g., medical staff, administrators, families) to establish the target value. 7. Work in small and diverse groups representing neonatologists and other medical staff, devel- opmental care teams, administrators, and cur- 19 HERD Vol. 5, No. 4, SUMMER 2012 • HEALTH ENVIRONMENTS RESEARCH & DESIGN JOURNAL 19 T H E O R Y HERD VOLUME 5, NUMBER 4, PP 5-22 COPYRIGHT ©2012 VENDOME GROUP, LLC T H E O R Y rent and previous NICU families—as well as representatives from the design, engineering, and construction stakeholder organizations. References American Academy of Pediatrics. (2011). Newborn intensive care units (NICUs) and neonatologists of the USA & Canada. Rock- ville, MD: Neonatology Today. 8. Work in a “big room” (ideally, a dedicated space near the existing NICU to facilitate user participation). Ansari, S., Bell, J., & CAM-I Target Cost Core Group. (1997). Target costing: The next frontier in strategic cost management. Austin, TX: Consortium for Advanced Manufacturing International. Arthur, J. (2011). Lean six sigma for hospitals: Simple steps to fast, affordable, flawless healthcare. New York, NY: McGraw-Hill. 9. Conduct retrospective presentations throughout the process to ensure learning and continuous improvement. ASTM. (2006). Standard practice for measuring life-cycle costs of buildings and building systems. Annual Book of ASTM Stan- dards. West Conshohocken, PA: Author. Ballard, H. G. (2000). The last planner system of production control. Doctorate, University of Birmington, United Kingdom. These practices are described in greater detail in the TVD literature (Ballard, 2005; Ballard, 2011; Ballard & Morris, 2010; Denerolle, 2011; Macomber & Barberio, 2008; Ryb- kowski, 2009). Any project team interested in undertaking a TVD exercise would be wise to study well the tools and culture that are inher- ent to success. Ballard, G. (2005).P2SL report: Current benchmark in target costing. Retrieved from http://p2sl.berkeley.edu/2009-05-26/P2SL%20 Report%20on%20the%20Current%20Benchmark%20in%20 Target%20Costing%202005-11-28.pdf Ballard, G. (2011). Target value design: Current benchmark (1.0). Lean Construction Journal, 79–84. Ballard, G., & Reiser, P. (2004). The St.Olaf College fieldhouse proj- ect: A case study in designing to target cost. Paper presented at the Proceedings of the 12th Annual Conference of the Interna- tional Group for Lean Construction (IGLC-12), August 3–5, 2004, Elsinore, Denmark. Ballard, H. G., & Morris, P. H. (2010). Maximizing owner value through target value design. AACE International Transactions, 1(1). Retrieved from http://connection.ebscohost.com/c/ar- ticles/52475948/maximizing-owner-value-through-target-value- design Conclusion One purpose of this paper was to give NICU own- ers and stakeholders a structured understanding of the NICU design process within the practice of TVD so they are better prepared to implement this new process as the practice of TVD spreads. Calculating capital cost per square foot is the first step necessary to evaluate set-based design alternatives for NICUs. However, it is critical to know the long-term costs and benefits associated with each NICU configuration to confidently select the option that delivers maximum long- term value for the customer. TVD is an effective means of allowing NICU design teams to under- stand the relative merits and cost implications of potential design alternatives. Barnett, D. L. (2004). A primer on sustainable building. Snowmass, CO: Rocky Mountain Institute. Berens, R., & Weigle, C. (1996). Cost analysis of ceiling tile re- placement for noise abatement. Journal of Perinatology, 16(3), 199–201. Berry, L. L., Parker, D., Coile, R. C., Hamilton, D. K., O’Neille, D. D., & Sadler, B. L. (2004). The business case for better buildings. Frontiers of Health Services Management, 21(1), 5–24. Black, J. T., & Hunter, S. L. (2003). Lean manufacturing systems and cell design. Dearborn, MI: SME. Boardman, A., & Forbes, D. (2007). A cost-benefit analysis of private versus semi-private inpatient rooms in a new hospital. Working paper. Phelps Centre for the Study of Government and Business, Sauder School of Business, Vancouver, BC. Bull, J. W. (1993). Life cycle costing for construction. London, Unit- ed Kingdom: Blackie Academic & Professional. Calkins, M., & Cassella, C. (2007). Exploring the cost and value of private versus shared bedrooms in nursing homes. Gerontolo- gist, 47(2), 169–183. Carreira, B. (2005). Lean manufacturing that works: Powerful tools for dramatically reducing waste and maximizing profits. New York, NY: AMACOM. WWW.HERDJOURNAL.COM ISSN: 1937-5867 20 PA P E R S T H E O R Y P TARGET VALUE DESIGN: APPLICATIONS TO NEWBORN INTENSIVE CARE UNITS Carter, B., Carter, A., & Bennett, S. (2008). Families’ views upon experiencing change in the neonatal intensive care unit envi- ronment from the “baby barn” to the private room. Journal of Perinatology, 28, 827–829. Koskela, L. (1992). Application of the new production philosophy to construction. In CIFE Technical Report #72 (pp. 1–81). Stanford, CA: Stanford University. Langston, C. A. (2005). Life-cycle cost approach to building evalu- ation. Amsterdam, The Netherlands: Elsevier. Chaudhury, H., Mahmood, A., & Valente, M. (2005). Conclusion Advantages and disadvantages of single versus multiple occupancy rooms in acute care environments: A review and analysis of the litera- ture. Environment & Behavior, 37(6), 760–786. Lichtig, W. A. (2005a). Sutter Health: Developing a contracting model to support Lean project delivery. Lean Construction Jour- nal, 2(1), 105–112. Christiansen, C. (2009). Embracing change. Health Environments Research & Design Journal, 3(1), 15–27. Lichtig, W. A. (2005b). Ten key decisions to a successful construc- tion project: Choosing something new—the Integrated Agree- ment for Lean Project Delivery. September 29–30, The Intercon- tinental Toronto Centre. Clifton, M. B., Bird, H. M. B., Albano, R. E., & Townsend, W. P. (2004). Target costing: Market-driven product design. New York, NY: Dekker. Lichtig, W. A. (2006). The integrated agreement for Lean project delivery. Construction Lawyer, 26(3), 1–8. Cooper, R., & Slagmulder, R. (1997). Target costing and value engi- neering. Portland, OR: Productivity Press. Liker, J. (2004). The Toyota way. New York, NY: McGraw-Hill. Davis, J. W. (2009). Lean manufacturing: Implementation strategies that work. New York, NY: Industrial Press. Macomber, H., & Barberio, J. (2008). Target value design: Nine foundational practices for delivering surprising client value. Retrieved from http://www.leanproject.com/wp-content/ uploads/2011/10/3-Target-Value-Design-LPC.pdf Denerolle, S. (2011). Technical report: The application of Target value design to 3 hospital projects. Project Production Systems Laboratory, University of California, Berkeley. Martin, J. A., Hamilton, B. E., Ventura, S. J., Osterman, M. J., Kir- meyer, S., Mathews, T. J., … Wilson, E. C. (2011). Births: Final data for 2009. In N. V. Statistics (Ed.), National Vital Statistics Reports (Vol. 60, No. 1). Hyattsville, MD: National Center for Health Statistics. Evans, R., Haryott, R., Haste, N., & Jones, A. (1998). The long term costs of owning and using buildings. London, UK: Royal Acad- emy of Engineering. Gluck, L. (1985). Conceptualization and initiation of a neonatal intensive care nursery in 1960. Paper presented at the Neo- natal Intensive Care: A History of Excellence, October 7, Bethesda, MD. Moon, S. (2005). Construction and costs—Going up. Modern Healthcare, 35(10), 30–42. Nicolini, D., Tompkins, C., Holti, R., Oldman, A., & Smalley, M. (2000). Can target costing and whole life costing be applied in the construction industry? Evidence from two case studies. Brit- ish Journal of Management, 11, 303–324. Graban, M. (2009). Lean hospitals: Improving quality, patient safety, and employee satisfaction. Boca Raton, FL: CRC Press. Hamilton, D. K. (2006). Evidence-based design supports evidence- based medicine in the ICU. Conclusion 4, SUMMER 2012 • HEALTH ENVIRONMENTS RESEARCH & DESIGN JOURNAL T H E O R Y HERD VOLUME 5, NUMBER 4, PP 5-22 COPYRIGHT ©2012 VENDOME GROUP, LLC T H E O R Y care unit: An evaluation of patient safety, staff satisfaction and costs. Journal of Perinatology, 21(4), 230–235. care unit: An evaluation of patient safety, staff satisfaction and costs. Journal of Perinatology, 21(4), 230–235. Shepley, M., Harris, D., White, R., & Steinberg, F. (2008). Impact of single-family rooms on family behavior. The AIA Report on University Research, 3, 174–185. Ward, A. C., Liker, J. K., Cristiano, J. J., & Sobek II, D. K. (1995). The second Toyota paradox: How delaying decisions can make better cars faster. Sloan Management Review, 36(3), 43–61. Smith, T. J., Schoenbeck, K., & Clayton, S. (2009). Staff percep- tions of work quality of a neonatal intensive care unit before and after transition from an open bay to a private room design. Work, 33, 211–227. White, R. (2007). Flooring choices for newborn ICUs. Journal of Perinatology, 27(S2), S29–S31. Standard, C., & Davis, D. K. (1999). Running today’s factory: A proven strategy for lean manufacturing. Cincinnati, OH: Hanser Gardner Publications. White, R. (2011). The newborn intensive care unit environment of care: How we got here, where we’re headed, and why. Semi- nars in Perinatology, 35(1), 2–7. Stevens, D. C., Helseth, C. C., Khan, M. A., Munson, D. P., & Reid, E. J. (2011). A comparison of parent satisfaction in an open-bay and single-family room neonatal intensive care unit. Health Envi- ronments Research & Design Journal, 4(3), 110–123. White, R., Browne, J., Cicco, R., Erickson, D., Graven, S., Greg- ory, S., . . . King, J. (2007). Recommended standards for newborn ICU design. Journal of Perinatology, 19(8), Part II, S2-S12. Suhr, J. (1999). The choosing by advantages decisionmaking sys- tem. Westport, CT: Quorum Books. White, R., Smith, J., & Harrell, J. (2012). New recommended stan- dards for NICU design. Paper presented at the The Gravens Conference: The Physical and Developmental Environment of the High Risk Infant, January 26–28, Clearwater Beach, FL. Tapping, D., Kozlowski, S., Archbold, L., & Sperl, T. (2009). Val- ue stream management for Lean healthcare: Four steps to planning, mapping, implementing and controlling improve- ments in all types of healthcare environments. Chelsea, MI: MCS Media Inc. Womack, J. P., & Jones, D. T. (2003). Lean thinking. New York, NY: Free Press. Walsh, W. Conclusion F., McCullough, K. L., & White, R. D. (2006). Room for improvement: Nurses’ perceptions of providing care in a single room newborn intensive care setting. Advances in Neo- natal Care, 6(5), 261–270. Womack, J. P., Jones, D. T., & Roos, D. (1990). The machine that changed the world. New York, NY: Free Press. Zidel, T. G. (2006). A Lean guide to transforming healthcare: How to implement Lean principles in hospitals, medical offices, clinics and other healthcare organizations. Milwaukee, WI: ASQ Quality Press. Walsh-Sukys, M., Reitenbach, A., Hudson-Barr, D., & DePompei, P. (2001). Reducing light and sound in the neonatal intensive Conclusion ICU Management Journal (Belgium), 6(3), 31. Ortenstrand, A., Westrup, B., & Brostrom, E. A. (2010). The Stock- holm neonatal family centered care study: Effects of length of stay and infant morbidity. Pediatrics, 125, e278–e285. Harrell, J., & Moon, R. (2008). Designs for the delicate. Health Fa- cilities Management, 21(12), 45–48. Parrish, K. (2009). Applying a set-based design approach to rein- forcing steel design. Doctoral dissertation, University of Califor- nia, Berkeley, Berkeley, CA. Harris, D., Shepley, M., White, R., Kolberg, J., & Harroll, K. (2006). The impact of single family room design on patients and care- givers: Executive summary. Journal of Perinatology, 26(Suppl 3), S38–S48. Parrish, K., Stransky, K. E., Rogers, C., Duncan, M. H., Smith, G. C., Neil, J., … Inder, T. (2007). Exploration of set-based design for re- inforced concrete structures. Paper presented at the International Group for Lean Construction (IGLC-15), MI, 213–222. Institute of Medicine. (2006). Preterm birth: Causes, conse- quences, and prevention. Washington, DC: National Acad- emy of Sciences. Phillips, C. R. (2010). Family-Centered Maternity Care Business Case. Santa Cruz, CA: Phillips + Fenwick. Jackson, B. (2004). Construction management jump start. India- napolis, IN: Wiley. Rosenblum, D. (2005). Single family room care: Before and after data. Paper presented at the Gravens Conference: The Physical and Developmental Environment of the High-Risk Infant, Janu- ary 26–28, Clearwater Beach, FL.. Jimmerson, C. (2007). A3 problem solving for healthcare: A practi- cal method for eliminating waste. Boca Raton, FL: CRC Press. Jimmerson, C. (2010). Value stream mapping for healthcare made easy. Boca Raton, FL: CRC Press. Rybkowski, Z. (2009). The application of root cause analysis and target value design to evidence-based design in the capital plan- ning of health care facilities. Doctoral dissertation, University of California, Berkeley, Berkeley, CA. Kenney, C. (2011). Value stream mapping for healthcare made easy. Boca Raton, FL: CRC Press. Rybkowski, Z. K. (2010). Letter to the editors. Health Environments Research & Design Journal, 3(2), 126–130. Kirk, S. J., & Dell’Isola, A. J. (1995). Life cycle costing for design professionals. New York, NY: McGraw-Hill. Shepley, M., Harris, D., & White, R. (2008). Open-bay and single family room neonatal intensive care units: Caregiver satisfaction and stress. Environment & Behavior, 40(2), 249–268. 21 HERD Vol. 5, No. Implications for Practice • The principals of Target Value Design are critical to responsible professional practice. By embracing the activities described in this article, designers will enhance the efficiency and effectiveness of healthcare environments. • The design process for NICUs will benefit particularly from integrated project teams because of the evolution that is occurring relative to open-bay versus private room unit configurations. • The collaboration of all design team members is necessary to make decisions regarding the implications of these two approaches. WWW.HERDJOURNAL.COM ISSN: 1937-5867 22 Copyright of Health Environments Research & Design Journal (HERD) is the property of Vendome Group LLC and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. Copyright of Health Environments Research & Design Journal (HERD) is the property of Vendome Group LLC and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. Copyright of Health Environments Research & Design Journal (HERD) is the property of Vendome Group LLC and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
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Effect of ethylene gas and Tryptophan amino acid in two oat varieties under soil stress conditions
Bionatura
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Introduction Avena sativa L oat crop. It is one of the annual herbs of the Poaceae family, and oat rank seventh in terms of econo- mic importance and production of grain crops; after wheat, rice, barley, corn, sorghum and millet, white oat is common- ly known in cold and humid areas are grown1. Studies have shown that oatmeal benefits heart health because it helps reduce Peterson's cholesterol levels2; although the oat crop is globally essential, its productivity in Iraq is still very li- mited. EL-Bassiouny3 explained that spraying wheat plants with 25, 50 and 100 mg-1 liters of tryptophan after 30, 45 and 75 days of cultivation increases grain product and that the concentration of 50 mg-1 recorded the highest average weight of 1,000 grains at 42,899 G, the highest average gra- in product was 2033 kg /acre (4.840 miga g.h-1), while the highest average straw product was 8,609 miga g.h-1. tryp- tophan gave the highest number of hooks at 3.92and 5.56 plant-1 enables, and the highest average dry weight of 4.39 and 6.42 g, The average weight of the grain was highest at 2.85 and 5.18 g, and the highest average grain snob was 55.48 and 69.44 snabel-1 grains. In the EL-Bassiouny3 study, the average protein content was found to have in- creased by 18.52% as a result of paper spraying of trypto- phan at a concentration of 50 mg-1 liters. Ethylene gas is a growth defect commonly used in grain crops, added to the plant in the form of spraying, and its effect depends on the spraying time and plant growth stage. Ethylene gas works by releasing ethylene into the plant by slow release mecha- nism4. DOI. 10.21931/RB/2023.08.01.78 Mohammed Radwan Mahmoud, Haider Rezaq Leiby and Husam S M Khalaf* 1 Department of Field Crops, Faculty of Agriculture, Muthanna University, Iraq. Corresponding author: husam.saadi@mu.edu.iq DOI. 10.21931/RB/2023.08.01.78 Mohammed Radwan Mahmoud, Haider Rezaq Leiby and Husam S M Khalaf* 1 Department of Field Crops, Faculty of Agriculture, Muthanna University, Iraq. Corresponding author: husam.saadi@mu.edu.iq Abstract: A field experiment was conducted at the Research Station, Agriculture Faculty, Al-Muthanna University. The investigation was applied using the split-plot design, according to RCBD, with three replicates, varieties occupied the main plots, amino acid tryptophan occupied the sub-plots, and ethylene levels occupied the sub-sub-plot. The results showed that Shifaa Variety was distinguished in the harvest index. Results showed its distinction in the characteristic of the Panicle number. The interaction between the varieties and the amino acids showed increased grain yield percentage and the Panicle numbers when the amino acid was added. The exchange of cultivars with ethylene showed a decrease in plant height at 250 ppm and an increase in the Panicle numbers, the tiller numbers and the number of grains Panicle-1. As for the interaction of amino acid with ethylene, the treatment of adding acid and concentration of 250 ppm was superior with increasing some traits, including the number of grains Panicle-1, grain yield tons ha-1 and biological yield. While in the triple interaction, the treatment of (oat 11 × amino acid × the addition of Ethylene 250 ppm) was superior on some traits, including the increase in, the Panicle numbers and 1000 grain weight. Key words: Ethylene gas, Tryptophan amino acid, oat, soil stress conditions. Volume 8 / Issue 1 / 78 / http://dx.doi.org/10.21931/RB/2023.08.01.78 Volume 8 / Issue 1 / 78 / http://dx.doi.org/10.21931/RB/2023.08.01.78 http://www.revistabionatura.com Citation: Mahmoud M R, Leiby H R and Khalaf H S M. Effect of ethylene gas and Tryptophan amino acid in two oat varieties under soil stress conditions. Revis Bionatura 2023;8 (1) 78. http://dx.doi.org/10.21931/RB/2023.08.01.78 Received: 15 January 2023 / Accepted: 25 February 2023 / Published: 15 March 2023 Effect of ethylene gas and Tryptophan amino acid in two oat varieties under soil stress conditions DOI. 10.21931/RB/2023.08.01.78 Publisher’s Note: Bionatura stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Introduction Therefore, by affecting crop components and other properties, it reduces leg elongation and resistance to re- flux, so this study aimed to know the optimal concentration of ethylene gas and Tryptophan amino acid in increasing the growth and production of the oat crop, Zarkani5 noted in his study of four varieties of oat (Shifaa, carlop, genza- nia, judo) found differences in the number of dalea, grain yield, bio-product, and harvest index between varieties, as the variety carlop outperformed better than the rest of the varieties in the first season, in giving it the highest average number of Panicles amounted to 940.70 Panicle m-2 and in a study conducted by the pilgrims6 in a field experiment on oat varieties in Tikrit, he found moral differences between the varieties in the category of the number of grains in dalal, and gave the item Hamel The highest average was 56.44 Panicles.m-2, while the Icarda tall gave the lowest average of 34.92 Panicles.m-2 grains. In a field study conducted by Mut et al.7 in Turkey, on 25 genetic compositions of oat and found that there were moral differences in the characteristic of grain quotient between these genetic compositions; the field study conducted by Mut et al.7 included the cultivation of 25 genetic compositions of oat, the results showed that the designs differed in the proportion of protein in grains, where the genetic composition was recorded G23 was the highest average of 13.3%, while the average values of G2 and G25 were the lowest at 12.0% for both in the protein ratio in cereals. Materials and methods Citation: Mahmoud M R, Leiby H R and Khalaf H S M. Effect of ethylene gas and Tryptophan amino acid in two oat varieties under soil stress conditions. Revis Bionatura 2023;8 (1) 78. http://dx.doi.org/10.21931/RB/2023.08.01.78 Received: 15 January 2023 / Accepted: 25 February 2023 / Published: 15 March 2023 Publisher’s Note: Bionatura stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Experience sitei A field experiment was carried out at the Research Sta- tion of the College of Agriculture - University of Al-Muthan- na, located on the Euphrates River (9 km northeast of the Publisher’s Note: Bionatura stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Mohammed Radwan Mahmoud, Haider Rezaq Leiby and Husam S M Khalaf Volume 8 / Issue 1 / 78 • http://www.revistabionatura.com Mohammed Radwan Mahmoud, Haider Rezaq Leiby and Husam S M Khalaf Volume 8 / Issue 1 / 78 • http://www.revistabionatura.com city of Samawa). During the 2021-2020 agricultural season, random soil samples were taken at a depth of 30 cm and then analyzed the physical and chemical qualities described in table (1). equal impulses first after emergence and the, second at the branching stage and third at the elongation phase, and the fourth lining stage and in quantity (180 kg). N ha-1 (Hasa- ni,2014), phosphate fertilizer was added in the form of su- perphosphate triphosphate (P2O5 46%), at a rate of 100kg H-1 (1995) at once before cultivation, and potassium fertili- zer was added in the form of potassium sulfate (K (42% in the branching phase by 60 kg ). K-1 (Taher,2005), irrigation and weeding were carried out whenever needed, and plants were harvested when they reached full maturity. equal impulses first after emergence and the, second at the branching stage and third at the elongation phase, and the fourth lining stage and in quantity (180 kg). N ha-1 (Hasa- ni,2014), phosphate fertilizer was added in the form of su- perphosphate triphosphate (P2O5 46%), at a rate of 100kg H-1 (1995) at once before cultivation, and potassium fertili- zer was added in the form of potassium sulfate (K (42% in the branching phase by 60 kg ). K-1 (Taher,2005), irrigation and weeding were carried out whenever needed, and plants were harvested when they reached full maturity. Number of Panicles m-2 Table 2 shows that oat 11, with the highest average number of Panicles m-2 recorded at 418.06 Panicles m-2, while the variety recorded the lowest average recovery of the number of Panicles m-2 and reached 390.7 Panicles m-2, may be due to the different genetic structure to which the ability to form fertile traits is associated. The same table shows that there is effect of amino acid in increasing the number of Panicles m-2 where the treatment of the addi- tion of amino acid recorded the highest average of 429.57 Panicles m-2, while the control treatment (not adding amino acid) recorded the lowest average of 378,81 Panicles m-2, the reason for this is the increase in the number of Panicles m-2. Table (2) shows the effect of ethylene spraying in the Study factor The experiment included the study of three factors, na- mely: i First: the first factor is two varieties of oat 1-V1 Shifaa Variety 2-V2 Oat 11 The following growth qualities have been studied: The following growth qualities have been studied: • Number of Panicles m-2 • Number of grains in the Panicle m-1 • Weight of 1000 grains (g) • Grain Product (Mega Gram. h-1) • biological yield (Mega Gram h-1) • Harvest index % Percentage of protein in cereals% The following growth qualities have been studied: Second: Tryptophan (contains 7.56% organic nitrogen • Number of Panicles m-2 N, C organic carbon 22.8% and total amino acid 46.8%) N, C organic carbon 22.8% and total amino acid 46.8%) , g ) 1-B0 without addition 2-B1 Add 15 ml per liter of water Third: the third factor is the addition of ethylene gas : 1-E0 without addition • Number of grains in the Panicle m-1 • Weight of 1000 grains (g) 2-B1 Add 15 ml per liter of water Third: the third factor is the addition of ethylene gas : 1-E0 without addition Third: the third factor is the addition of ethylene gas : Third: the third factor is the addition of ethylene gas : 1-E0 without addition • Grain Product (Mega Gram. h-1) • biological yield (Mega Gram h-1) 2-E1 add 250 ppm active substance h-1 • Harvest index % • Percentage of protein in cereals% 3-E2 add 500 ppm active substance h-1 2.5. Statistical analysis: The data were statistically analysed using the Genstat statistical programme in a way that analysed all the variations and traits studied, and the calculation averages were compared using the lowest moral difference8 (LSD) at 0.05. Design experience The experiment was carried out using the order of the renegade panels - plots design Split - Split - According to the design of the entire randomized sectors (RCBD) and three bis, the main-plot panels and sub-plot were occupied by main-plot, while sub-sub-plot levels were occupied, and the convocation was distributed randomly within each sec- tor, bringing the total units to Experimental (2×2×3×3=36 experimental units). Field operations As we note from table (2) that the combination (the ethylene gas × ethylene gas spraying) recorded the highest ave- rage when treated as oat 11 × adding ethylene gas at a concentration of 250 ppm at 438.11 Panicles m-2 While the treatment recorded Shifaa ×the addition of ethylene gas is the lowest average at 380.11 Panicles m-2, which may be due to the genetic difference between varieties or the di- fference in varieties by the extent to which they respond to the spraying× of ethylene gas) The overlap between them was moral and recorded the highest average when treating the addition of amino acid ×the addition of ethylene gas at the concentration of 250 ppm, where it was 479,82 Panicles m-2, While the transaction recorded the non-addition of ami- no acid× the addition of ethylene gas at the concentration of 250 ppm the lowest average at 348.32 Panicles m-2. As Table 2 shows, the triple interaction of the study factors was significant of oat 11 ×in addition of amino acid ×the addition of ethylene gas recorded at the concentration of 250 ppm the highest average number of Panicles m-2 reached 525.44 Panicles m-2 and recorded a Shifaa treatment ×in addition of amino acid ×the addition of ethylene gas at the concentra- tion of 250 ppm the lowest average number of Panicles m-2 and reached 350.77 Panicles m-2. Field operations Tillage, softening and leveling operations were carried out. The land was divided according to the design used into panels of an area of (2m× 2m = 4 m2) and the experimental units were separated by a distance (50 cm) to prevent inter- ference between transactions. Agriculture was carried out on - 20202021 and at an seed rate of 120 kg H-1 (Moham- med, 2017) Tryptophan was added in the elongation and counterfeiting phases to the plant and even complete wet- ness. The addition of ethylene gas in the elongation phase sprayed the plant to complete wetness and urea fertilizer (N 46%) was used as a nitrogen source. It was added in Table 1 Some chemical and physical qualities of field soil before agriculture Table 1. Some chemical and physical qualities of field soil before agriculture. Effect of ethylene gas and Tryptophan amino acid in two oat varieties under soil stress conditions Table 2. The effect of varieties, amino acid and ethylene gas and the overlap between them in the number of Pani- cles m-2 number of Panicles m-2 and recorded the highest average concentration at 250 ppm, at 413.77 Panicles m-2, recorded the lowest average concentration at 500 ppm, which rea- ched 396.1 Panicles m-2, this is in line with Hashim14 and Al-Dahari15, who explained that the addition of ethylene gas leads to an increase in the number of Panicles m-2 in the wheat, barley and oat yield. As we note from table (2) that the combination (varieties×amine acid) increased the num- ber of Panicles m-2 and morally difference when spraying the amino acid of the oat 11 and this characteristic recorded the highest average number of Panicles m-2 of treatment oat 11 × amino acid addition and reached 458.38 Panicles m-2, The lowest average treatment of oat was 11×in addition of amino acid, which amounted to 376.09 Panicles m-2 and was not morally different from the Shifaa combination of × no acid addition, which amounted to 382. Panicles m-2. Number of grains. Panicles.m-2 The results of table 3 showed the variety oat 11 where the highest average number of grains was recorded. Those who pointed out that the difference in varieties in this cha- racteristic is due to the fact that the character of the number of grains in Panicle is a quantitative trait positively associa- ted with genetic and environmental factors. In table 3 we note that the number of grains in Panicle increased as a result of the spraying of amino acid, where the treatment of adding acid (adding amino acid) recorded the highest ave- rage number of grains in Panicle and reached 24.43 grains. Panicles.m-2, while the control spray treatment recorded the lowest average at 22. 63 grains. Panicles.m-2 may be due to the role of amino acid, which has increased the rate of fertility and reduced the rate of egg abortion in addition to the role of amino acid in increasing the amount of carbo- hydrates transferred from manufacturing areas to effective growth sites.f In table 3 we note the effect of ethylene gas on the number of grains in Panicle, where the proportion of grains in Panicle increased with the addition of ethylene gas, whe- re the highest average concentration was recorded at 250 Mohammed Radwan Mahmoud, Haider Rezaq Leiby and Husam S M Khalaf Volume 8 / Issue 1 / 78 • http://www.revistabionatura.com Mohammed Radwan Mahmoud, Haider Rezaq Leiby and Husam S M Khalaf Volume 8 / Issue 1 / 78 • http://www.revistabionatura.com Mohammed Radwan Mahmoud, Haider Rezaq Leiby and Husam S M Khalaf Volume 8 / Issue 1 / 78 • http://www.revistabionatura.com Table 3. The effect of varieties, amino acid and ethylene gas and the overlap between them in the number of grains Panicles m-2 ppm and reached 26 grains. varieties or different varieties by the extent to which they respond to a spray withathylene gas. In table 3 we note that bilateral interference (varieties ×ethylene gas) was moral, with oatmeal treatment recor- ded 11 × addition ethylene gas at a concentration of 250 ppm, the highest average number of grains in Panicle at 29.78 grains. Panicles.m-2, while the lowest average Shifaa treatment was recorded ×addestion ethylene gas at a con- centration of 500 ppm. It reached 19.00 grains. The weight of 1000 grains Table 4 of the results show a decrease in the weight of 1000 grains with the addition of ethylene gas, where the highest average was recorded when the control was treated 45.00 g, while the lowest average was recorded at 250 ppm where it reached 43. 99 g, this may be due to an increase in the number of ethylene gas sprays, which has increased the competition for photosynthesis products and thus reduced the amount of manufactured materials that reach the grain during the full period. We note in table 4 that the interfe- rence of (varieties ×ethylene gas) as a result of spraying withethylene gas was recorded at the highest average when Shifaa × not adding ethylene gas at 45.06 g and was not morally different from the treatment of oat 11× the addition of ethylene gas when concentrating 250 ppm, 46 g, while the treatment recorded Shifaa × the addition of ethylene gas at the concentration of 250 ppm the lowest average at 42 g, which may be due to the genetic difference between Number of grains. Panicles.m-2 As we note from table 4, the interference (acid × ethyle- ne gas) was the Significant statistical differences and re- corded the highest average when treating the addition of amino acid ×the addition of ethylene gas (compared) to 45.95 g, it did not differ Significant from the treatment of not adding amino acid × the addition of ethylene gas at a concentration of 500 ppm, 46.5 g, while the transaction re- corded the addition of amino acid × adding ethylene gas at a concentration of 500 ppm lowest average at 43.06 g. As we note from table 4, the triangular overlap of the study fac- tors was moral, with the treatment of oat recorded 11 × the addition of amino acid × the addition of ethylene gas at the concentration of 250 ppm the highest average of 49 g and was not Significant different from the treatment of oat 11 × the addition of amino acid × add athylene gas and the treat- ment of oat 11 × addition of amino acid × adding ethylene gas at a concentration of 500 ppm was recorded the lowest average was 41.04 g. In table 3 we note that the bilateral interference (amino acid × ethylene gas) where the transaction recorded the addition of amino acid × the addition of ethylene gas at the concentration of 250 ppm, the highest average of 28.38 grains. Panicles.m-2, while the transaction recorded the ad- dition of amino acid ×the addition of ethylene gas at the concentration of 500 ppm the lowest average 20.23 gra- ins.Panicles.m-2. Harvest index% As we note from table 5, the tripartite interaction of the study factors was significant, with the treatment recorded Shifaa × in addition of amino acid × the addition of ethyle- ne gas at a concentration of 250 ppm higher. The average grain product was 6.55 miga g.h-1, while the lowest average oatmeal treatment was 11 × in addition of amino acid × the addition of ethylene gas was 3.07 mega g.h-1. The varieties showed Significant statistical differences between them in the harvest index, where the variety Shifaa recorded the highest average of the harvest index and rea- ched 45.07%, while the oat 11 recorded the lowest average for this characteristic at 35.71%, and may be due to the di- fference in genetic composition that controls the ability to convert photosynthesis products into an economic product. We note in table 7 that interference (varieties ×ethylene gas) . Shifaa × not adding ethylene gas gave 47.60%, while oat 11 treatment × no addition of ethylene gas recorded the lowest as. %32.91. Yield miga g.h-1 Table 5 shows the Significant statistical differences when spraying ethylene gas where the highest average was recorded at a concentration of 250 ppm and reached 5.24 miga g.h-1, while the control transaction recorded the lowest average of 4.17 miga g.h-1, due to an increase in the num- ber of Panicles and the number of grains. We note in table 5 that interference (varieties of ×amine acid) increased the grain product when spraying amino acid for a Shifaa varie- ty, while oat 11 decreased the grain product when spraying amino acid. This status recorded the highest average grain for treatment Shifaa ×in addition of amino acid was 5.42 miga g.h-1, while the lowest average treatment for oat 11 × in addition of amino acid, which was 4.15 mg.h-1. As table Effect of ethylene gas and Tryptophan amino acid in two oat varieties under soil stress conditions Table 4. The effect of varieties, amino acid and ethylene gas and the overlap between them in the weight of 1000 grains. Table 5. The effect of varieties, amino acid and ethylene gas and the overlap between them in the characteristic of the grain quotient megagram.ha-1. ct of varieties, amino acid and ethylene gas and the overlap between them in the weight of 1000 grains. Table 5. The effect of varieties, amino acid and ethylene gas and the overlap between them in the characteristic of the grain quotient megagram.ha-1. Table 5. The effect of varieties, amino acid and ethylene gas and the overlap between them in the characteristic of the grain quotient megagram.ha-1. Mohammed Radwan Mahmoud, Haider Rezaq Leiby and Husam S M Khalaf Volume 8 / Issue 1 / 78 • http://www.revistabionatura.com of amino acid ×the addition of ethylene gas at the concen- tration of 250 ppm the highest average of 12. 96 mega g.h- 1, while the transaction recorded no addition of amino acid ×the addition of ethylene gas recorded the lowest average of 10.65mega g.h-1. 5 shows, the interference (amino acid× ethylene gas) whe- re the treatment recorded the addition of amino acid ×the addition of ethylene gas at the concentration of 250 ppm the highest average of 5.86 miga g.h-1, while the addition of amino acid was recorded × the addition of athylene gas the lowest average of 3.97 miga g.h-1. The biological yield (mega g.h-1)i Table 6 shows the Significant statistical differences in biological yield when using ethylene gas and recorded the highest average at a concentration of 250 ppm gave 11.90 mega g.h-1, while the control transaction recorded the lowest average of 10.47 mega g.h-1, which is consistent with Hashim,2006, where the addition of athylene gas positively affected the increase in The biological yield. Discussion 6. Al Hajog, Y.A.M. The effect of some environmental factors in genetic compositions of oat in the conditions of Salaheddine province. Ph.D. Thesis - Faculty of Agriculture - Tikrit Univer- sity. 2019. The results were consistent with the findings of (9,10) in the difference in oat varieties in the number of Panicles m-2. The availability of conditions helped to increase the number of Panicles m-2 when adding amino acid, which is consis- tent with (11-13), who pointed to the effect of amino acid in increasing the number of Panicles m-2 for wheat crops. In Table 3 we note the effect of ethylene gas on the number of grains in Panicle, this is consistent with (14,16) of the moral effect of different ethylene gas compositions in the number of grains in spike for wheat. (4-17), they explained that there is an increase in grain yield for barley, wheat and oat crop when the concentration of medium ethylene gas but the grain product decreases at high concentration. Mo- reover, (15) pointed out that the harvest index values in the oat crop differed by varieties and genetic composition. Fina- lly when spraying amino acid, the treatment of amino acid spray recorded the highest average grain protein synth, this is may be due to the fact that free amino acid when added is a source of essential nitrogen in the construction and for- mation of protein and enzymes and energy processing for vital processes (18,19). 7. Mut, Z, H. Akay and Ö. D. E. Köse. Grain yield, quality traits and grain yield stability of local oat cultivars. Journal of Soil Science and Plant Nutrition, 2018. 18 (1): 269-281. 8. Al-Rawi, K.M. and Khalafallah, A. Design and analysis of agri- cultural experiments. 1980. 9. Al-Hajjuj, Y.A. and A.H.A. Anis. The role of the number of irri- gations in the performance of several genetic models of avena sativa oat . L for its quantitative qualities. 1st International And Scientific Conference, Faculty of Science, Tikrit University (December 17-18, 2018). 10. Abdulateef, S.M. ,O.K. Atalla, MQ. Al-Ani, TH. T. Mohammed, F.M. Abdulateef, O.M. Abdualmajeed , K. Mahmod. The effect of the electric shock on embryonic development and neuro- physiological traits in the chick's embryo . IOP Conference Se- ries: Earth and Environmental Science.2021, 761(1), 012090. 11. Baqir, H.A. Physiological behavior of three varieties of wheat bread under the influence of amino acid and yeast powder. Discussion Doctoral thesis .University of Baghdad. Faculty of Agricultural Engineering Sciences. Field Crops Department. 2018.f 12. Mohammedi, M.H.M. The effect of the spraying date with nu- tr-green amino acid compound concentrations on the qualities of growth and product and its components and quality recipes for varieties of chlamyli wheat tritikal master's thesis - Universi- ty of Tikrit - Faculty of Agriculture - Department of Field Crops. 2018. Protein in cereals% Table 8 shows when spraying amino acid where the treatment of amino acid spray recorded the highest ave- rage grain protein synth of 18.23% and a moral difference from the control treatment where recorded the lowest ave- rage of 14.24%, and may be due to the fact that the free amino acid when added is a source of essential nitrogen in the construction and formation of protein and enzymes and energy processing for vital processes. Table (8) shows when spraying ethylene gas where the highest average grain protein was recorded at a concentration of 500 ppm We note in table 6 that interference (ethylene gas × va- rieties) shows the Significant statistical differences in biolo- gical yield ethylene gas x Shifaa treatment added ethylene gas at concentration of 250 ppm at 12.57mega g.h-1, while Shifaa treatment ×no addition Ethylene gas was recorded the lowest average at 9.69 mega g.h-1. Table 6 also shows that the interference (amino acid × ethylene gas) where the treatment recorded the addition Table 6. The effect of varieties, amino acid and ethylene gas and the overlap between them in the mega g.h-1. Effect of ethylene gas and Tryptophan amino acid in two oat varieties under soil stress conditions Table 7. The effect of varieties, amino acid and ethylene gas and the overlap between them in harvest index %. The effect of varieties, amino acid and ethylene gas and the overlap between them in harvest index %. Table 8. The effect of varieties, amino acid and ethylene gas and the overlap between them in grain protein. Mohammed Radwan Mahmoud, Haider Rezaq Leiby and Husam S M Khalaf Volume 8 / Issue 1 / 78 • http://www.revistabionatura.com 4. Abdalbakee, H. N. & Mohammed, Th. T. Effect of using differ- ent levels of azolla as a substitute for soybean meal in the pro- duction performance of fish carp. Plant Archives. 2019, 19(1): 573-577. Doi: 10.13140/RG.2.2.23568.15367. 18.60%, and there was no Significant statistical differences from the treatment of 250 ppm to 16. 51% protein in cereals, while the control treatment (no ethylene gas addition) recor- ded the lowest average of 14.53%. 5. Zarkani, M.S.M. The effect of soaking seeds with peridoxin and boron sprays in the grain product and its ingredients for four varieties of Avena sativa oat. L Doctoral Thesis, Faculty of Agriculture, University of Baghdad. 2017.f Conclusions The interaction of cultivars with ethylene showed a de- crease in plant height at 250 ppm and increase the Panicle numbers, the tiller numbers and the number of grains Pa- nicle -1. As for the interaction of amino acid with Eth-ylene, the treatment of adding acid and concentration of 250 pp- mwas superior with increasing some traits, in-cluding the number of grains Panicle -1, grain yield tons ha-1 and bio- logical yield. While in the triple interaction, the treatment of (oat 11 × amino acid × the addition of Ethylene 250 ppm) were superior on some traits, including the increase in, the Panicle numbers and 1000 grain weight. 13. Abdul Karim, S.J.A. The effect of paper spraying with amino acid and copper in the product and quality of varieties of soft wheat Tritivuma estivum. L. Master's Thesis. 2020.f 14. Hashim, R.T.J. The effect of planting dates on the growth and product of different genetic compositions of the Avena sativa oat crop. L Master's Thesis, Faculty of Agriculture, Muthanna University. 2014.f 15. Al-Dahir, A.M.S. The effect of the paper feeding of athyphon and boron in the growth and pollen of oat (Avena sativa). L) . Doctoral Thesis, Faculty of Agriculture, Anbar University. 2020. 16. Al-Obaidi, M.S. Use of clatar and athyphon to improve growth, outcome and dry endurance of two varieties of wheat (Triticum aestivum ). L) Doctoral Thesis, Department of Field Crops, Faculty of Agriculture, University of Mosul. 2001.f 3. EL-Bassiouny, H.M.S. physiological responses of wheat to sa- linity alleviation by nicotinamide and tryptophan. Int. J. Agric. Biol., 2005. 7(4): 653-659 . Bibliographic references 17. Abdul Mughni, A.M.T. The effect of celtar and athion in the growth and product of some varieties of wheat (Triticum aes- tivum ). L) Doctoral thesis, Department of Field Crops, Faculty of Agriculture, University of Baghdad. 2001. 1. Soratto , R.P. ; C. A.C. Crusciol; G.S. A. Castro ; C. H.M. D. Costa and J. F. Neto. Leaf application of silicic acid to white oat and wheat .R. Bras. Sci . Solo, 2012. 36:14538-1644.f 2. Taher, F.M. The effect of paper feeding with iron, zinc and po- tassium in the growth and product of triticum aestivum wheat . L Doctoral Thesis. Faculty of Agriculture - University of Bagh- dad. 2004. 18. Khudair, M.Y Alyassein, R.N, Jasim, F.M. Improving the Quali- ty of Ground Water in Some Areas of Al-Anbar Governorate by Recharging with Rainwater. IOP Conference Series: Earth and Environmental Science.2021, 761(1), 012009f 3. EL-Bassiouny, H.M.S. physiological responses of wheat to sa- linity alleviation by nicotinamide and tryptophan. Int. J. Agric. Biol., 2005. 7(4): 653-659 . 19. Al Salman, N.T.Sh. and J.K.M. Al-Gharawi. Effect of Eucalyp- tus leaves water extract on some productive traits of broilers. Plant Archives Vol. 19, Supplement 1, 2019 pp. 920-923.
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Kang Yuwei on the origins of Western Civilisation
Rossiâ i mir: naučnyj dialog
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ТЕОРИЯ И МЕТОДОЛОГИЯ УДК: 94(510)+316.722 DOI: 10.53658/RW2022-2-1(3)-36-52 DOI: 10.53658/RW2022-2-1(3)-36-52 РОССИЯ И МИР: НАУЧНЫЙ ДИАЛОГ № 1(3) | март 2022 РОССИЯ И МИР: НАУЧНЫЙ ДИАЛОГ № 1(3) | март 2022 РОССИЯ И МИР: НАУЧНЫЙ ДИАЛОГ № 1(3) | март 2022 Казанский (Приволжский) федеральный университет (Каз у Аннотация. Статья посвящена вопросу о формировании концепции цивилизационного развития Кан Ювэя (1858–1927). На материале «Путешествия в  Италию» (1904) рассмотрено видение западной цивилизации с  точки зрения конфуцианского мыслителя. Кан Ювэй утверждал, что единение является основным законом истории. С его точки зрения, Римская империя была примером государства, создание которого отвечало законам эволюционного развития природы и  общества. Современная ему Италия рассматривалась как молодое государство, которое еще не преодолело последствий «родовой травмы». Формируясь в  синоцентрической парадигме, Кан Ювэй рассматривал мировую цивилизацию как единый эволюционный процесс восхождения от варварства к вершинам культуры. Соответственно, позитивный опыт Запада можно использовать для исправления недостатков Китая и возвращения стране высокого политического и геостратегического статуса. Ключевые слова: Кан Ювэй, цивилизация, глобализм, синоцентризм, конфуцианство, Великое единение (Да тун) Об авторе: МАРТЫНОВ Дмитрий Евгеньевич. Доктор исторических наук. Профессор кафедры алтаистики и китаеведения Института международных отношений Казанского (Приволжского) федерального университета. ORCID 0000-0001-5385-1915. Адрес: 420111, Российская Федерация, Казань, ул. Пушкина, 1/55. dmitrymartynov80@mail.ru. Мартынов Д.Е. Казанский (Приволжский) федеральный университет (Казань, Россия) 1 Кан Ювэй (康有为). Идали юцзи (Путешествие в Италию, 意大利游记) // Цюаньцзи (Полное со­ брание соч., 康有為全集); Гл. ред. Цзян Ихуа, Чжан Жунхуа (姜义华, 张荣华编校). Т. 7. Пекин: Чжунго жэнь­ минь дасюэ чубаньшэ, 2007. С. 347–406. [Kang Youwei. Yidali youju. Quanji, Jiang Yihua, Zhang Ronghua bianjiao. Di 7 ji. Beijing: Zhongguo renmin daxue chubanshe]. Далее цитируется как: «Кан Ювэй, 2007». Введение Наше исследование посвящено проблеме осмысления сущности западной ци­ вилизации выдающимся китайским мыслителем и  реформатором Кан Ювэем (康有 为, 1858–1927). Важнейшей особенностью его социокультурного и интеллектуального опыта является длительное пребывание за пределами Китая (в 1898–1914 годах), воз­ можность непосредственного ознакомления с жизнью более тридцати государств тог­ дашнего мира. Интерпретация его впечатлений от образа жизни Запада только начи­ нается и вызывает в национальных синологиях Европы и США, России и восточных государств, включая Китайскую Народную Республику, существенные разногласия. В статье мы излагаем наши воззрения на интеллектуальные достижения Кан Ювэя, ко­ торый воспринимал себя пророком – основателем новой глобальной религии. Соответ­ ственно, в его глобализаторской исторической доктрине опыт западной цивилизации занимал немалое место. 36 RUSSIA & WORLD: SCIENTIFIC DIALOGUE No. 1(3) | March 2022 RUSSIA & WORLD: SCIENTIFIC DIALOGUE ( ) | h CIENTIFIC DIALOGUE No. 1(3) | March 2022 Основной задачей данного исследования является разрешение вопроса о  трансформации конфуцианского сознания, носителем которого является выдаю­ щийся мыслитель, получивший традиционное китайское воспитание. Обретенный внешний опыт выражается в теоретической саморефлексии философа относительно данного опыта. Кан Ювэй был одним из первых конфуцианцев Китая, которые имели возможность длительного общения с носителями западной культуры, не будучи ско­ ванными служебными ограничениями или необходимостью выражения ангажиро­ ванной государством точки зрения. Материалы и методы Методология исследования интеллектуальных перемен в Китае на рубеже XIX– XX веков была отрефлексирована выдающимся китайским философом Лян Цичао (1873–1929). Он предложил так называемый метод аналогии культурных ценностей, который и сделался основной базой сопоставления западной и китайской интеллек­ туальной истории с функционалистской точки зрения. С позиции метода аналогии Лян Цичао обозначал своего учителя Кан Ювэя как «Мартина Лютера конфуциан­ ства» (Мартынов 2010, с. 35). Метод аналогии был неотделим и от аксиологической компаративистики, которая широко применялась множеством китайских мысли­ телей, включая Лю Жэньхана (1884–1938), и  позднейшими исследователями. При множестве ограничений данный метод остается базовым в современной синологии, хотя при его некритическом использовании превращает интеллектуальную историю в «ухудшенную версию истории философии». Базовый набор персоналий при этом восходит к тезаурусу все того же Лян Цичао (Elman 2010, p. 376). Основными источниками являются сборник путевых впечатлений Кан Ювэя «Путешествие в Италию» (1904)1, а также базовый философский трактат мыслителя «Книга о Великом единении» (впервые издан в 1935 году)2. j j j g gg у 2 Кан Ювэй (康有为). Датун шу (Книга о Великом единении, 大同书) / Ред. и предисловие Тан Чжицзюня (汤志钧导读). Шанхай: Шанхай гуцзи чубаньшэ, 2005. 18, 292 c. [Kang Youwei. Datong shu / Tang Zhijun daodu. Shanghai: Shanghai guji chubanshe, 2005]. Далее цитируется как: «Кан Ювэй, 2005». Интеллектуальный фундамент учения Кан Ювэя Вся философская система Кан Ювэя основана на эволюционном учении. Одним из наиболее сложных для его биографов вопросов является время усвоения мыслите­ 1 Кан Ювэй (康有为). Идали юцзи (Путешествие в Италию, 意大利游记) // Цюаньцзи (Полное со­ брание соч., 康有為全集); Гл. ред. Цзян Ихуа, Чжан Жунхуа (姜义华, 张荣华编校). Т. 7. Пекин: Чжунго жэнь­ минь дасюэ чубаньшэ, 2007. С. 347–406. [Kang Youwei. Yidali youju. Quanji, Jiang Yihua, Zhang Ronghua bianjiao. Di 7 ji. Beijing: Zhongguo renmin daxue chubanshe]. Далее цитируется как: «Кан Ювэй, 2007». 2 Кан Ювэй (康有为). Датун шу (Книга о Великом единении, 大同书) / Ред. и предисловие Тан Чжицзюня (汤志钧导读). Шанхай: Шанхай гуцзи чубаньшэ, 2005. 18, 292 c. [Kang Youwei. Datong shu / Tang Zhijun daodu. Shanghai: Shanghai guji chubanshe, 2005]. Далее цитируется как: «Кан Ювэй, 2005». Вся философская система Кан Ювэя основана на эволюционном учении. Одним из наиболее сложных для его биографов вопросов является время усвоения мыслите­ 37 37 РОССИЯ И МИР: НАУЧНЫЙ ДИАЛОГ № 1(3) | март 2022 лем эволюционистских взглядов. Еще младшие современники мыслителя обращали внимание на то, что Кан Ювэй датировал свои новейшие работы задним числом, что­ бы оправдать миф о том, что по вдохновлению Неба создал все свои работы до дости­ жения тридцатилетнего возраста (Мартынов 2010, с. 27–28) (Alitto, Liang Shuming 2010, p. 156). Это стало основой теории Тан Чжицзюня о позднем формировании философии истории Кан Ювэя. В предисловии к изданию «Датун шу» Тан утверждал, что это про­ изошло не ранее отъезда Кана в эмиграцию, то есть не ранее 1899 года, где он познако­ мился с трактатом Томаса Гексли «Эволюция и этика», переведенным выдающимся философом Янь Фу (1854–1921) под названием «Теория природного развития»3. лем эволюционистских взглядов. Еще младшие современники мыслителя обращали внимание на то, что Кан Ювэй датировал свои новейшие работы задним числом, что­ бы оправдать миф о том, что по вдохновлению Неба создал все свои работы до дости­ жения тридцатилетнего возраста (Мартынов 2010, с. 27–28) (Alitto, Liang Shuming 2010, p. 156). Это стало основой теории Тан Чжицзюня о позднем формировании философии истории Кан Ювэя. В предисловии к изданию «Датун шу» Тан утверждал, что это про­ изошло не ранее отъезда Кана в эмиграцию, то есть не ранее 1899 года, где он познако­ мился с трактатом Томаса Гексли «Эволюция и этика», переведенным выдающимся философом Янь Фу (1854–1921) под названием «Теория природного развития»3. Принципиальной инновацией Кан Ювэя – философа был синтез конфуциан­ ской доктрины с понятием изменений (как в природе, так и в человеческих уста­ новлениях), что выразилось в учении о «Трех эрах» (сань ши). Концепция «Трех эр» в различных модификациях существовала в китайской мысли от времени Конфу­ ция и была существенно развита в эпоху Хань (III век до н.э. – III век н.э.), но после­ довательное ее изложение предложил только Кан Ювэй. Традиционное китайское мировоззрение рассматривает мир как пространственно-временной континуум; учение о Трех эрах предполагало корреляцию стадиальных изменений как во вре­ мени, так и в пространстве. Иными словами, концепция трех эпох / эр имела для Кан Ювэя общеметодологический смысл. Если до Кана концепция была инволю­ ционной  – от идеального состояния Поднебесной в  глубокой древности, которое сменяется «затмением Дао-пути», то новаторством Кан Ювэя стала трансформация временной последовательности в эволюционную. Первая эпоха, в которую живет и сам Кан Ювэй, это Эра хаоса (цзюй луань ши). 3 Кан Ювэй 2005, с. 9, 17. Вся философская система Кан Ювэя основана на эволюционном учении. Одним из наиболее сложных для его биографов вопросов является время усвоения мыслите­ Далее последует эпоха Рождающе­ гося мира (или Подъема к равновесию, шэн пин ши), к которой, по мысли Кана, уже близки некоторые страны современной ему Европы и Америки. Далее, в неопреде­ ленно далеком будущем, настанет эпоха Великого равновесия, или Великого мира (тай пин ши), в которую во всем мире будет реализовано Великое единение (да тун). С каждой эпохой соотносится эволюция общественного и социального строя: само­ державие и патриархальная семья в Эру хаоса; конституционная монархия и ну­ клеарная семья с равноправием полов в Эру рождающегося мира; демократическая республика и коммунизм (с полным устранением всех границ, включая государ­ ственные, социальные, половые и даже межвидовые) Великого единения. В каждой из эпох выделялась троичная структура, что должно было в философско-методоло­ гическом смысле показать неограниченность развития даже после наступления Великого единения (Куан Болинь 1980, с. 91; Кобзев 2002, с. 467–468). Концепция Кан Ювэя являлась конфуцианской по своим глубинным философ­ ским основаниям. Это связано как с понятием жэнь-гуманности, которую Кан Ювэй провозгласил вселенской константой, так и с тем, что под «развитием» мыслитель понимал нравственный прогресс и лишь затем – материальный. Каждая последую­ щая эра является более прогрессивной и цивилизованной по сравнению с предше­ 38 RUSSIA & WORLD: SCIENTIFIC DIALOGUE No. 1(3) | March 2022 RUSSIA & WORLD: SCIENTIFIC DIALOGUE | No. 1(3) | March 2022 ственницей; Великое единение – это истинный рай на земле, в котором каждое живое существо освободится от страданий и достигнет состояния Будды4. Человеческое об­ щество (не суть важно, западное или восточное) обязательно проходит через Три эры, поскольку они имеют значение всеобщего закона исторического развития. Развитие при этом происходит постепенно, точные временные границы этого процесса не мо­ гут быть установлены, однако очередность смены эр не может быть нарушена (Куан Болинь 1980, с. 91). ственницей; Великое единение – это истинный рай на земле, в котором каждое живое существо освободится от страданий и достигнет состояния Будды4. Человеческое об­ щество (не суть важно, западное или восточное) обязательно проходит через Три эры, поскольку они имеют значение всеобщего закона исторического развития. Развитие при этом происходит постепенно, точные временные границы этого процесса не мо­ гут быть установлены, однако очередность смены эр не может быть нарушена (Куан Болинь 1980, с. 91). Современный итальянский синолог Федерико Брусаделли, рассматривая ин­ теллектуальную эволюцию Кан Ювэя, впервые попытался поставить китайского мыслителя в контекст интеллектуальной истории, цитируя Р. Козеллека и М. Фуко (Brusadelli 2020, p. 2, 4). Собственно, и А.И. Вся философская система Кан Ювэя основана на эволюционном учении. Одним из наиболее сложных для его биографов вопросов является время усвоения мыслите­ Кобзев рассматривал учение о Великом единении (которое единогласно объявляется главным теоретическим достиже­ нием Кан Ювэя) в терминологии «унификаторского глобализма» и «экуменизма» (Кобзев 2002, с. 469). Ф. Брусаделли также рассматривал деятельность мыслителя в религиозном ключе, обозначая учение, изложенное в «Датун шу», не как филосо­ фию истории, а как ее телеологию. Собственно, именно в этом контексте он напо­ минает о М. Фуко, так как Кан Ювэй не признавал «разрыва времени», и его теория является не аллегорией о несбыточных мирах, а вполне позитивистским описани­ ем действия и  исполнения объективных законов общества и  истории (Brusadelli 2020, p. 5). Ф. Брусаделли истолковывает Датун шу как «глобальную историю», на­ писанную с позиции ее завершения в неопределенном, но не слишком отдаленном будущем. 4 Кан Ювэй 2005, с. 290-292. 5 Кан Ювэй 2007, с. 406. Отдельно обозначено, что это наборное, а не ксилографическое издание. 6 То есть 22 марта 1904 года. То есть 22 марта 1904 года. 7 В этом разделе помещено описание помпейских руин. 8 В заголовках 5 и 6 использован термин яньгэ, то есть «эволюция, процесс последова­ тельных изменений», а не лиши (собственно, «прошлое, ход событий»). 9 Так Кан Ювэй аттестовал себя в «Автобиографии» (второй год Гуансюй, 1876). 10 Специфический жанр китайской авторской прозы, по преимуществу создаваемый государственными чиновниками. Единственным конструирующим законом жанра была ав­ торская воля, однако произведения в жанре бицзи включали «рассказы об удивительном», бес­ сюжетные заметки и зарисовки, стихотворные вставки и рассуждения на самые разные темы. Хватает и авторов, которые занимались изысканиями в разных областях знания – от лингвисти­ ки и антиквариата до музыки и астрономии (Алимов, Кравцова, 2014, с. 1244-1256). Трактат «Путешествие в Италию» «Путешествие в Италию» было опубликовано в Шанхае в 1905 году в составе сборника «Путешествий по одиннадцати странам Европы»5. По форме это эклекти­ ческое сочинение в традиционном жанре, которое сочетает дневниковые записи (из­ ложение начинается с полуслова: от начала путешествия в Европу, датированного 6 днем второй луны 30-го года эры Гуансюй)6, разнообразные суждения, поэтические вставки и мини-эссе, которые в совокупности образуют философский комментарий. Перечислим структурные части, выделяемые автором (в оригинале заголовки не ну­ меруются): 1. «Путь по Южно-Китайскому морю и Индийскому океану». 2. «От Адена по Красному морю». 3. «От Суэцкого канала до Порт-Саида». 4. «Средиземное море». 4. «Средиземное море». 5. «Песнь о Средиземном море» (поэма). 39 РОССИЯ И МИР: НАУЧНЫЙ ДИАЛОГ № 1(3) | март 2022 6. «Путешествие в Италию». 6. «Путешествие в Италию». 6. «Путешествие в Италию». 7. «Мои ощущения, когда я высадился в Европе после морского путешествия» (от­ крывается стихотворением, далее текст прозаический)7. 7. «Мои ощущения, когда я высадился в Европе после морского путешествия» (от­ крывается стихотворением, далее текст прозаический)7. 8. «Созерцаю море и весь Неаполь из кабачка у подножья Везувия» (открывается стихотворением, далее текст прозаический). 9. «Восемь отрывочных стансов о Рафаэле» (прозаический текст с многочислен­ ными поэтическими вставками). Внутри разделов 8–9 содержатся обширные вставные рассуждения, представ­ ляющие собой самостоятельные эссе. Китайский социолог Ван Минмин выделил 11 внутритекстовых комментариев (Ван Минмин 2019, с. 3): 1. «О том, что римские дворцы не столь велики и изысканны, как китайские ди­ настий Цинь и Хань». 1. «О том, что римские дворцы не столь велики и изысканны, как китайские ди­ настий Цинь и Хань». 2. «Китайцам следует сохранять свои культурные реликвии, чтобы не отстать от европейцев». 3. «Четыре сотни храмов Рима уступают Собору Св. Петра». 3. «Четыре сотни храмов Рима уступают Собору Св. Петра». 4. «О том, почему парламентаризм как система возник на Западе, а не на Востоке». 5. «Удачи и неудачи в ходе исторического развития Рима». 6. «История Италии»8. 7. «Гражданская политика Италии». 8 Р ( 3. Четыре сотни храмов Рима уступают Собору Св. Петра . 4. «О том, почему парламентаризм как система возник на Западе, а не на Востоке». 5. «Удачи и неудачи в ходе исторического развития Рима». 6. «История Италии»8. 4. «О том, почему парламентаризм как система возник на Западе, а не на Востоке». 5. «Удачи и неудачи в ходе исторического развития Рима». 8. «Римско-католическая доктрина (с приложенным рассуждением, что учение Иисуса произошло от буддизма)». 9. «О древнеримской диалектике». 9. «О древнеримской диалектике». 10. р ф ( р у ) 10 Специфический жанр китайской авторской прозы, по преимуществу создаваемый государственными чиновниками. Единственным конструирующим законом жанра была ав­ торская воля, однако произведения в жанре бицзи включали «рассказы об удивительном», бес­ сюжетные заметки и зарисовки, стихотворные вставки и рассуждения на самые разные темы. Хватает и авторов, которые занимались изысканиями в разных областях знания – от лингвисти­ ки и антиквариата до музыки и астрономии (Алимов, Кравцова, 2014, с. 1244-1256). , ( , р , д ) Так Кан Ювэй аттестовал себя в «Автобиографии» (второй год Гуансюй, 1876). 7 В этом разделе помещено описание помпейских руин. 8 В заголовках 5 и 6 использован термин яньгэ, то есть «эволюци тельных изменений», а не лиши (собственно, «прошлое, ход событий»). р , ц изменений», а не лиши (собственно, «прошлое, ход событий»). 7 В этом разделе помещено описание помпейских руин. 7 В этом разделе помещено описание помпейских руин. 8 В заголовках 5 и 6 использован термин яньгэ, то есть «эв тельных изменений», а не лиши (собственно, «прошлое, ход событ Трактат «Путешествие в Италию» «Сравнение Древнего Рима и китайской империи Хань; пять причин, по кото­ рым Рим не уступал Китаю». 11. «Об истоках цивилизации пяти морей и трех континентов». Ван Минмин говорит о двойственности подходов к рассуждениям Кан Ювэя. С одной стороны, Кан Ювэй представал в роли этнографа, оказавшегося в ситуации первого контакта с Другим (Ван Минмин 2019, с. 4). Разумеется, он не имел ни ма­ лейшего понятия об этнографии, однако, поставив целью «стать мудрецом, пере­ читав все книги еще до того, как мне исполнится тридцать лет»9 (Мартынов 2010, с. 103), прекрасно знал о сборниках бицзи, в которых было множество записей о раз­ нообразных заморских диковинах10. Иными словами, традиционная китайская 40 RUSSIA & WORLD: SCIENTIFIC DIALOGUE No. 1(3) | March 2022 RUSSIA & WORLD: SCIENTIFIC DIALOGUE ( ) | h No. 1(3) | March 2022 ученость располагала необходимым тезаурусом и формами выражения впечатле­ ний от разнообразия не-китайского Иного; поэтому Кан Ювэй не был в этом отно­ шении оригинален и не сделался образцом для подражания (Ван Минмин 2019, с. 4)11; «Путешествие в Италию» не было первым описанием западной цивилизации со стороны образованного китайца (Findeisen 2000, p. 91). ученость располагала необходимым тезаурусом и формами выражения впечатле­ ний от разнообразия не-китайского Иного; поэтому Кан Ювэй не был в этом отно­ шении оригинален и не сделался образцом для подражания (Ван Минмин 2019, с. 4)11; «Путешествие в Италию» не было первым описанием западной цивилизации со стороны образованного китайца (Findeisen 2000, p. 91). Если рассматривать «Путешествие в  Италию» как документ, который свиде­ тельствует не об описываемой реальности, а о личности самого Кан Ювэя, тогда его значение существенно возрастает. Для иллюстрации приведем перевод двух заклю­ чительных абзацев трактата Кан Ювэя (собственно, из рассуждения «Об истоках ци­ вилизации пяти морей и трех континентов»): Полагаю, что носители цивилизации Европы  – сыновья Рима и  внуки Греции. Прародители земель пяти морей и  трех континентов произошли оттуда; прародительницы их  – из Аравии. Новая же цивилизация Америки  – суть отпрыск, который совсем недавно захлестнул Индию и  Японию и  сотрясает нашу страну. Европа сравнительно молода, ее культура произросла из древних Египта и Вавилона, которые являются ее семенем; неужели это не удивительно?! Император Данте12 основал столицу столь блистательную и  величественную, что она простояла тысячу лет, великолепием и  прочностью превосходя все, известное на земле, хотя вызов ей бросали турки и мусульмане13. Магомет основал новое государство в Мекке, объединив владения множества правителей от Испании на западе до Индии на востоке, севера Африки на юге и Каспийского моря на севере. 11 Напротив, Чжу Цзыцин (1898–1948) свидетельствовал, что в молодости был заворожен литературой о путешествиях и в особенности «Путешествием в Италию» Кан Ювэя. Во время по­ сещения Италии в 1932 г. он пользовался сочинением Кан Ювэя как своего рода путеводителем и «камертоном наблюдателя» (Findeisen 2000, p. 90). р д ( , p ) 12 Так в оригинале. 13 Вероятно, Кан Ювэй имел в виду Константинополь-Стамбул и Багдад. В Стамбуле он побывал в дни Младотурецкой революции 1908 г. 14 Китайская верста (ли) во времена Кан Ювэя примерно равнялась 571,5 м., в современ­ ном Китае приравнена к 500 м. 15 Так Кан Ювэй истолковал разделение последователей ислама на суннитов и шиитов. 16 Кан Ювэй использует двусмысленное сочетание гуайи (怪异), которое означает как «странный, диковинный, чудесный; фантастический; чудовищный, невероятный», так и «чрез­ вычайно изменчивый». 17 Тубо – название Тибета в эпоху Тан (VII–IX вв ) 11 Напротив, Чжу Цзыцин (1898–1948) свидетельствовал, что в молодости был заворожен литературой о путешествиях и в особенности «Путешествием в Италию» Кан Ювэя. Во время по сещения Италии в 1932 г. он пользовался сочинением Кан Ювэя как своего рода путеводителем и «камертоном наблюдателя» (Findeisen 2000, p. 90). 12 Так в оригинале. 13 Вероятно, Кан Ювэй имел в виду Константинополь-Стамбул и Багдад. В Стамбуле он побывал в дни Младотурецкой революции 1908 г. 14 Китайская верста (ли) во времена Кан Ювэя примерно равнялась 571,5 м., в современ ном Китае приравнена к 500 м. 15 Так Кан Ювэй истолковал разделение последователей ислама на суннитов и шиитов. 16 Кан Ювэй использует двусмысленное сочетание гуайи (怪异), которое означает как «странный, диковинный, чудесный; фантастический; чудовищный, невероятный», так и «чрез вычайно изменчивый». 17 Тубо – название Тибета в эпоху Тан (VII–IX вв.). 17 Тубо – название Тибета в эпоху Тан (VII–IX вв.). Трактат «Путешествие в Италию» Арабская цивилизация раскинулась на протяжении 20 000 вёрст-ли14, пересекая моря и континенты; и так появилась великая держава, которая торговала с Римом и Китаем, и была устроена совершенно по-новому. Халифат охватил все земли Индии, Персии, Рима и  Китая, создав новое правление и новую религию. Однако турки и персы разделились на два царства, имеющих каждый свою религию15, и в свою очередь покатились волной завоеваний. Европа была потеряна для арабов со времен крестовых походов, и с тех пор европейская цивилизация стала распространяться по всей земле. Поистине, страны пяти морей и  трех континентов чрезвычайно сильны и культурны, и поразительно быстро развиваются16. Ныне весь мир переживает цикл великих перемен. Среди стран пяти морей и  трех континентов владения ислама самые бедные и застойные, государства Ту и Бо17 самые Ныне весь мир переживает цикл великих перемен. Среди стран пяти морей и  трех континентов владения ислама самые бедные и застойные, государства Ту и Бо17 самые 11 Напротив, Чжу Цзыцин (1898–1948) свидетельствовал, что в молодости был заворожен литературой о путешествиях и в особенности «Путешествием в Италию» Кан Ювэя. Во время по­ сещения Италии в 1932 г. он пользовался сочинением Кан Ювэя как своего рода путеводителем и «камертоном наблюдателя» (Findeisen 2000, p. 90). 2 р 13 Вероятно, Кан Ювэй имел в виду Константинополь-Стамбул и Багдад. В Стамбуле он побывал в дни Младотурецкой революции 1908 г. 4 й ( ) Ю кая верста (ли) во времена Кан Ювэя примерно равнялась 571,5 м., в современ­ внена к 500 м. Ювэй истолковал разделение последователей ислама на суннитов и шиитов 15 Так Кан Ювэй истолковал разделение последователей ислама на суннитов и шиитов. 16 Кан Ювэй использует двусмысленное сочетание гуайи (怪异), которое означает как «странный, диковинный, чудесный; фантастический; чудовищный, невероятный», так и «чрез­ вычайно изменчивый». б б ( ) 41 РОССИЯ И МИР: НАУЧНЫЙ ДИАЛОГ № 1(3) | март 2022 РОССИЯ И МИР: НАУЧНЫЙ ДИАЛОГ № 1(3) | март 2022 закрытые и ретроградные. Но разве бывает, чтобы дух земли был истощён без остатка?! Бывает и так, что первопроходец отстанет, и круговорот Дао начнется заново. Поистине, через далекое время все без исключения страны изменятся, тогда как турки и персы, оставшись неизменными, едва ли смогут уцелеть. Европа – географический центр стран пяти морей и трех континентов, и непременно должна сделаться образцом и центром притяжения всемирной цивилизации. Не исчезнет ли она в  противном случае? <…> Кавказская раса своим упорством известна по всему свету, подданные императора Данте очень белые и  отличаются огромными носами. 18 Далее ошибочно перечислено и Красное море, описанное в этом же трактате. 19 «Четырнадцатилетнее» изгнание следует понимать как метафору, сознательную под­ гонку жизненных обстоятельств Кан Ювэя к биографии Первоучителя. В действительности эми­ грация Кан Ювэя продлилась 16 лет (Ван Минмин 2019, с. 12), как и будет показано в следующей секции. 18 Далее ошибочно перечислено и Красное море, описанное в этом же тр ф Трактат «Путешествие в Италию» Я  не добрался до пределов всех пяти морей и трех континентов, и лишь мысленно побывал на Чёрном и Каспийском море и в Персидском заливе18. На эти земли мои соотечественники должны обратить внимание (Кан Ювэй 2007, с. 405–406). Эмигрантский период жизни Кан Ювэя между 1899 и 1913 годами может рас­ сматриваться как «изгнание», хотя Сяо Гунцюань утверждал, что сам мыслитель воспринимал свою жизнь после провала реформаторского движения «как радост­ ный отдых после тяжких трудов, время, когда можно без ограничений странство­ вать по свету и удовлетворять давно осознанную тягу к перемене мест» (Hsiao 1975, p. 28). Данный подход справедлив, но должен ныне рассматриваться как поверх­ ностный. Мы сами в свое время посвятили отдельную работу самоуподоблению Кан Ювэя Конфуцию. В рамках искренней веры Кан Ювэя в то, что он является современ­ ным Конфуцием, его путевые записки приобретают совершенно иное звучание. Па­ раллели изгнанничества были таковы: и Конфуций, и Кан Ювэй были вынуждены оставить родину в результате провала всеобъемлющего политического плана; оба формулировали основные положения своего учения в изгнании; осознали в этой обстановке несбыточность собственной миссии; продолжительность эмиграции в обоих случаях составила 14 лет19 (Мартынов 2014, с. 297, табл. на с. 305-306). Европейский опыт Кан Ювэя имел свои особенности, проанализированные тайваньским исследователем Ван Юнцзу. Согласно его мнению, именно путеше­ ствие в  Европу (1904) окончательно убедило Кан Ювэя в  том, что материальная цивилизация в основе своей космополитична, не может быть ни китайской, ни за­ падной, что стало причиной осознания универсальности общественной и цивили­ зационной эволюции, этапы которой одинаковы для всех (Мартынов 2014, с. 296). Впрочем, не следует забывать, что вызревание доктрины Кан Ювэя было чрезвы­ чайно длительным, поскольку еще в  юности, после первого посещения Гонкон­ га в 1879 году, он убедился, что англичан нельзя считать «варварами» (Мартынов 2010, с. 109). В силу конфуцианских мировоззренческих установок Кан Ювэй больше был обращен к древности, а не к современности. Вдобавок, не владея европейскими языками, он не осознавал глубины межнациональных противоречий, рассматри­ 42 RUSSIA & WORLD: SCIENTIFIC DIALOGUE No. 1(3) | March 2022 RUSSIA & WORLD: SCIENTIFIC DIALOGUE No. 1(3) | March 2022 вая многообразие Европы по аналогии с  разными провинциями Китая, которым были свойственны этнонациональные различия и  разные диалекты. Поскольку Запад для Кан Ювэя был «ящиком Пандоры», таившим как вероятную гибель Цин­ ской империи, так и  секрет ее спасения, мыслитель в  первую очередь обращал внимание на детали обстановки, которые были доступны на поверхностном уров­ не. Отсюда подмеченные им «хутуны Неаполя». Трактат «Путешествие в Италию» В этом плане Ван Юнцзу был прав, когда утверждал, что путешествие Кан Ювэя по Италии только лишь укрепило уже созданную в его сознании умозрительную конструкцию, а увиденное служило толь­ ко для обоснования априори выстроенных конструкций (Мартынов 2014, с. 299). Но Ван Юнцзу, как нам кажется, преувеличивал простоту построений Кан Ювэя; на­ против, Ван Минмин подчеркивал, что «Путешествие в Италию» является одним из наиболее теоретичных и систематизированных текстов, созданных носителями традиционного китайского сознания, оказавшихся в  инокультурном окружении (Ван Минмин 2019, с. 5). вая многообразие Европы по аналогии с  разными провинциями Китая, которым были свойственны этнонациональные различия и  разные диалекты. Поскольку Запад для Кан Ювэя был «ящиком Пандоры», таившим как вероятную гибель Цин­ ской империи, так и  секрет ее спасения, мыслитель в  первую очередь обращал внимание на детали обстановки, которые были доступны на поверхностном уров­ не. Отсюда подмеченные им «хутуны Неаполя». В этом плане Ван Юнцзу был прав, когда утверждал, что путешествие Кан Ювэя по Италии только лишь укрепило уже созданную в его сознании умозрительную конструкцию, а увиденное служило толь­ ко для обоснования априори выстроенных конструкций (Мартынов 2014, с. 299). Но Ван Юнцзу, как нам кажется, преувеличивал простоту построений Кан Ювэя; на­ против, Ван Минмин подчеркивал, что «Путешествие в Италию» является одним из наиболее теоретичных и систематизированных текстов, созданных носителями традиционного китайского сознания, оказавшихся в  инокультурном окружении (Ван Минмин 2019, с. 5). 20 В этой секции все подробности приведены по сводке внука Кан Ювэя – профессора Ло Жунбана (сына дочери Ювэя – Кан Тунби) в нашем переводе (Мартынов 2010, с. 231-287). Контекст: Кан Ювэй – путешественник20 После бегства из Китая в сентябре 1898 года семейство Кан Ювэя длительное время не имело постоянного пристанища. 25 октября 1898 года по приглашению япон­ ского правительства мыслитель отправился в Токио; все расходы оплачивались из го­ сударственного бюджета. После смены правительства Кан Ювэй решил отправиться за океан в поисках поддержки императору Гуансюй (1875–1908), оказавшемуся под до­ машним арестом. 7 апреля 1899 года Кан Ювэй прибыл в Канаду, и 10 мая в Оттаве был принят генерал-губернатором британского доминиона – сэром У. Лорье. Заручившись поддержкой официальных лиц, 31 мая 1899 года мыслитель прибыл в Великобрита­ нию. В Лондоне он квартировал у бывшего лорда Адмиралтейства адмирала Бересфор­ да, с которым был знаком еще до государственного переворота. Однако рассмотрение вопроса о давлении на императрицу Цыси с целью установления конституционного строя в Палате общин (9 июня 1899 года) полностью провалилось. Во второй полови­ не года Кан Ювэй пребывал в Канаде, основав Общество защиты императора, которое стало первой китайской политической партией глобального масштаба. В самом конце года мыслитель вернулся в Гонконг, при этом цинский двор объявил Кан Ювэя в меж­ дународный розыск и организовал на него «охоту» со стороны спецслужб. После попытки покушения Кан Ювэй отбыл в  Сингапур, куда высадился 1 февраля 1900 года. Уже в день высадки на мыслителя было совершено нападение; в дальнейшем произошло еще несколько покушений, и с 23 февраля Кан Ювэй жил под охраной индийских солдат, назначенных губернатором колонии. После начала Ихэтуаньского восстания Кан Ювэй и Лян Цичао начали подготовку вооруженного выступления, намеченного на август; Кан руководил из Сингапура пропагандист­ ской кампанией. Поскольку на остров были посланы наемные убийцы, с 26 июля по 43 РОССИЯ И МИР: НАУЧНЫЙ ДИАЛОГ № 1(3) | март 2022 РОССИЯ И МИР: НАУЧНЫЙ ДИАЛОГ № 1(3) | март 2022 № 1(3) | март 2022 9 августа 1900 года Кан Ювэй жил под охраной в резиденции губернатора, а затем ему было предложено переселиться в Пинанг, в колонии Малайя. Проведя в Пинан­ ге почти полтора года, в декабре 1901 года Кан Ювэй выехал в Северную Индию, где был мягкий горный климат. В январе 1902 года мыслитель обосновался в Дарджи­ линге, в котором прожил до весны 1903 года, занимаясь писательским трудом. Период с четвертого по девятый лунный месяц 1903 года Кан Ювэй провел в  путешествии по Бирме, Таиланду, Вьетнаму и  Индонезии, откуда перебрался в Гонконг. В 1904 году в британской колонии прошел всемирный съезд отделений Общества спасения императора. После оформления глобальной политической и экономической организации Кан Ювэй отправился в европейское турне. Контекст: Кан Ювэй – путешественник20 1(3) | March 2022 C C D OGU No. 1(3) | March 2022 сику. Собирался он поехать и в Россию, однако царское правительство выпустило постановление о немедленном аресте и депортации в Пекин, и планы пришлось отменить. сику. Собирался он поехать и в Россию, однако царское правительство выпустило постановление о немедленном аресте и депортации в Пекин, и планы пришлось отменить. После банкротства американских и мексиканских предприятий Общества за­ щиты императора Кан Ювэй в апреле 1908 года отправился в Германию, 5 мая он присутствовал на праздновании 60-летия правления императора Франца-Иосифа в Вене. В мае – июне 1908 года Кан Ювэй жил в Швеции и Норвегии, а далее решил вернуться в Гонконг. Путь был кружным: 21 июля мыслитель прибыл в Белград, да­ лее побывал в Софии, а 27 июля в Стамбуле оказался свидетелем победы Младоту­ рецкой революции и установления конституции. Побывав в Афинах, Кан Ювэй от­ правился в Швейцарию и из Италии отплыл на Цейлон. После прибытия в Пинанг мыслитель получил известия о кончине императора Гуансюй (14 ноября 1908 года). Оправившись, весной 1909 года он отправился в Египет, обозрел Каир, Луксор и Асу­ ан. Далее он проехал в Палестину, посетил Иерусалим и побывал на Мертвом море. Через Швейцарию и Францию он проехал в Британию, посетив Бат и Хэмптон-Корт. Посетив Канаду, летом 1909 года Кан Ювэй прожил месяц в Потсдаме и вернулся 16 августа в Пинанг. Далее неугомонный путешественник отправился обозревать свя­ тыни Южной Индии, посетил Мадрас и Бомбей, а далее – вновь задержался в буддий­ ских храмах Цейлона. Весной 1910 года Кан Ювэй обосновался в Сингапуре, проведя четыре месяца в Гонконге у матери. Повторив поездку в Гонконг в мае 1911 года, в июне мыслитель отправился в Японию, где приобрел дом по соседству со своим учеником Лян Цичао, обитавшим в стране постоянно. Здесь он получил известия, что Цинское правитель­ ство сняло с мыслителя все обвинения и отменило указы об аресте и казни. Начало Синьхайской революции и установление республиканского строя не позволило Кану вернуться в Китай. Он оставался в Японии, общался с бывшим министром Инукаи Цуёси, подолгу жил в Осаке и Никко, побывал в Хаконэ, Киото и Хиросиме. В начале 1913 года в Токио ему была прооперирована язва желудка, 9 августа того же года скон­ чалась его мать. По нездоровью Кан Ювэй не смог побывать на ее похоронах в Гон­ конге и погрузился в положенный конфуцианским ритуалом годичный траур. 21 Кан Ювэй 2007, с. 346–348. Контекст: Кан Ювэй – путешественник20 Лишь в 1914 году, получив три приглашения от президента Юань Шикая, Кан Ювэй вернул­ ся в Китай после шестнадцатилетнего перерыва. Все его многочисленное семейство воссоединилось на китайский Новый год – 14 февраля 1915 года. Контекст: Кан Ювэй – путешественник20 Он от­ плыл из Гонконга 22 марта 1904 года. Посетив Бангкок, Малакку и Пинанг, китай­ ский мыслитель продолжил путь через Цейлон, Красное море и Суэцкий канал. 16 июня он прибыл в Бриндизи. Обозрев за три дня Неаполь, Помпеи и Геркуланум, Кан Ювэй 19 июня прибыл в Рим; в Вечном городе он пробыл неделю. 26 июня через Флоренцию и Милан Кан Ювэй отбыл в Париж, где также пробыл три дня. Далее его маршрут включал Швейцарию, Австрию и Венгрию. Полмесяца в июле – августе заняло пребывание в Великобритании, затем последовал вояж в Данию. В Копенга­ гене Кан Ювэй был принят министром иностранных дел, который одновременно занимал должность премьер-министра. Пребывание в Осло (Норвегия) заняло один день, надолго Кан Ювэй задержался в Швеции, где 24 августа был принят мини­ стром иностранных дел. 23 сентября китайский мыслитель отправился в Бельгию и Голландию, обозрел поле Ватерлоо и домик царя Петра I. С 29 сентября до 3 но­ ября Кан Ювэй изучал промышленные предприятия Великобритании, после чего отбыл в Монреаль. В Канаде он оформил текст «Путешествия по одиннадцати стра­ нам Европы». После тяжелой болезни 12 февраля 1906 года Кан Ювэй выехал в США. Из-за нездоровья он останавливался на месяц в Сиэтле и на два месяца в Лос-Анджелесе. Приехав в Вашингтон, 16 июня 1905 года он побывал на заседании Палаты пред­ ставителей и  посетил с  экскурсией мемориал Дж. Вашингтона в  Маунт-Верноне. 24 июня 1905 года мыслитель был приглашен в Белый дом на неофициальный обед с президентом Т. Рузвельтом, с которым обсуждал отмену законов, дискриминиру­ ющих китайцев в США. Далее, много времени заняли поездки по штатам Новой Ан­ глии и Среднего Запада. 2 декабря 1905 года Кан Ювэй пересек границу Мексики. Посетив Мехико, штаты Идальго, Оахака и Юкатан, в августе 1906 года китайский философ отбыл в Милан. Через Флоренцию он отправился в Германию, а на вось­ мом лунном месяце обосновался в Швеции, где приобрел недвижимость. 4 января 1907 года Кан Ювэй вернулся в Берлин, далее обозрел Рейнскую область и через Брюссель и Париж прибыл в Монте-Карло. 29 января 1907 года Кан уже находился в Мадриде, в феврале добрался до Кордовы и Гранады. Далее он пересек Гибрал­ тар, побывав в Танжере и Фесе, где даже удостоился аудиенции короля Марокко. В феврале – марте 1907 года Кан Ювэй побывал в Португалии, Франции и Велико­ британии, откуда двинулся в США. В пятый лунный месяц он вновь посетил Мек­ 44 RUSSIA & WORLD: SCIENTIFIC DIALOGUE No. Итальянские впечатления Кан Ювэя По замечанию Ван Минмина, «Путешествие в  Италию» было выдержано в  традиционном стиле «рассказов об удивительном» (Ван Минмин 2019, с. 13). Дневниковые записи перечисляют многочисленные военные объекты Британ­ ской империи21. Это сразу задавало общий тон повествования, но, по мнению Ван 45 РОССИЯ И МИР: НАУЧНЫЙ ДИАЛОГ № 1(3) | март 2022 № 1(3) | март 2022 Минмина, проистекало из ожиданий потенциальной читательской аудитории Кан Ювэя. Великобритания для Китая начала ХХ века была ведущей империали­ стической державой и одновременно образцом для подражания; соответственно, Италия и Франция рассматривались в одном ряду с ней, что сразу ориентировало читателей. Те, кто не ездил в Европу, полагают, что эти места напоминают обитель небожителей, сплошь населенную мудрецами и  бессмертными; откуда же им знать о  непролазной грязи и неустранимом беспорядке, и о том, что повсюду воры и мошенники! Поэтому, как говорится, “сто раз услышанное не превосходит единожды виденного”. Когда я прежде странствовал по Европе и Америке, и самой Англии, увиденное было очень далеко от того, что я привык представлять, когда учился, и это порождало разочарование. Потому- то, сходя на берег, достигнув цели, испытываешь великую растерянность (К Ю й 2007 351) (Кан Ювэй 2007, с. 351). По мнению Ван Минмина, эта сентенция обращена к  молодому поколению китайцев, которые привыкли представлять страны Запада как своего рода будущее Китая. Поэтому в  путевых записках постоянно идет сопоставление (Ван Минмин 2019, с. 14). По пути из Бриндизи в  Неаполь Кан Ювэй сообщает противоречащие друг другу сведения. Так, он описывает, как его обманули при расчете стоимости провоза багажа, и заявляет, что «итальянцы самые бедные и самые лживые, воров много»; описывая окрестности Везувия, он упоминал толпы нищих и заключал, что картина «ничем не отличается от Индии». Здесь же он пытается объяснить эту си­ туацию, дабы не отпугнуть своих читателей. Объяснение таково: существующее королевство Италия было создано «на одиннадцатом году правления под девизом Сяньфэн22, четвертом году моей жизни». То есть Италия – молодое государство, по­ рядки в котором еще не успели устояться. Далее, описывая сельские пейзажи, Кан Ювэй проводил параллели с Китаем, уточняя, что крестьянские хижины здесь на­ поминают египетские или индийские («стены их грубы и не обработаны, весьма не­ красивы»), а «склоны гор так же возделаны, как на острове Ява». Ландшафт и почва напоминают таковые в Цзянсу и Чжэцзяне, а деревенские проулки подобны трущо­ бам Гуанчжоу. Из этого делается вывод, что образ жизни всех древних цивилизаций единообразен, поэтому, созерцая дома и поля Италии и Китая, «можно узнать след­ ствия эволюции». 22 Сяньфэн – девиз правления цинского императора Ичжу (в 1850–1861 гг.). Одиннадца­ тый год Сяньфэн соответствует 1860 г. по западному исчислению. 23 Кан Ювэй 2007 с 351 353 ф у 23 Кан Ювэй 2007, с. 351–353. фэн – девиз правления цинского императора Ичжу (в 1850–1861 гг.). Одиннадца­ фэн соответствует 1860 г. по западному исчислению. Кан Ювэй 2007, с. 351–353. Итальянские впечатления Кан Ювэя Уровень развития их существенно уступает странам Северной Европы. Однако бедные кварталы всего мира одинаковы: заваленные конским на­ возом и гниющими отбросами переулки Неаполя, в которых телеги выбили в улич­ ном покрытии колеи, «подобны пекинским хутунам». Это происходит оттого, что «исправление порядков в древнем государстве подобно сносу старого дома, то есть чрезвычайно затруднительно». Тем не менее китайцы способны извлечь уроки из образа жизни Запада23. 46 RUSSIA & WORLD: SCIENTIFIC DIALOGUE No. 1(3) | March 2022 RUSSIA & WORLD: SCIENTIFIC DIALOGUE No 1(3) | March 2022 C C OGU No. 1(3) | March 2022 27 Афан – главная резиденция Цинь Шихуана (238–209 годы до н.э.), объедин , 25 Кан Ювэй 2007, с. 353. Чжугэ Лян (181–234) – государственный и военный деятель эпо­ хи Восточной Хань и Троецарствия. Сделался персонажем фольклора и классической китайской литературы, как олицетворение стратегических талантов, великой мудрости и изобретатель­ ности, личного бескорыстия и служения сюзерену. й р 26 Кан Ювэй 2007, с. 354. 24 Кан Ювэй 2007, с. 393-396. 25 Кан Ювэй 2007, с. 353. Чжугэ Лян (181–234) – государственный и военный деятель эпо­ хи Восточной Хань и Троецарствия. Сделался персонажем фольклора и классической китайской литературы, как олицетворение стратегических талантов, великой мудрости и изобретатель­ ности, личного бескорыстия и служения сюзерену. 26 Кан Ювэй 2007, с. 354. 27 Афан – главная резиденция Цинь Шихуана (238–209 годы до н.э.), объединителя Китая. 24 Кан Ювэй 2007, с. 393-396. 26 Кан Ювэй 2007, с. 354. ф ( ) б Цивилизация Древнего Рима и уроки для Китая Если обобщать концепцию, выраженную в философских эссе Кан Ювэя, выяс­ нится, что он именовал первопричиной отсталости Италии («самой слаборазвитой из больших европейских стран») распад Римской империи и феодализацию средневеко­ вой Европы. Воссоздав в 1860 году единое государство, Италия была вынуждена тра­ тить значительные средства для содержания армии и флота, что и создавало разрыв с более процветающими странами к северу от Альп. Все это расписано в заключитель­ ной секции «Путешествия по Италии», где Кан Ювэй рассматривает устройство мест­ ного самоуправления в Италии, ее транспорта, экономики, налоговой системы и так далее, вплоть до устройства национального банка и системы коммерческих банков24. Описание Неаполя открывается поэмой о Камилло Кавуре, вдохновленной его бронзовым памятником, в которой герой Италии сравнивается с Чжугэ Ляном25. Это ключ к его методу универсальных аналогий, поскольку мыслитель объявил, что изу­ чающий эволюцию мировой цивилизации должен сначала рассмотреть эволюцию Европы, «а кто захочет познать эволюцию Западной Европы, должны начать с руин Древнего Рима»26. Собственно, главной целью итальянского турне Кан Ювэя и была древнеримская история. Именно на руинах Помпей он изрек сентенцию, которая была процитирована Сяо Гунцюанем (Hsiao 1975, p. 28): Некогда произошло катастрофическое извержение вулкана, ныне мы, живущие, способны исследовать старину непосредственно. Если бы не вулкан, как бы мы соприкоснулись с людьми Древнего Рима? И как бы мы судили об империи Цинь, если бы не было Великой стены? Под Небесами сменяют друг друга потери и  прибытки, в природе всё взаимно дополняет друг друга. Поэтому тот, кто рассуждает о Дао, не может не понимать смены Инь и  Ян. Поэтому вулкан, причинивший великое зло [людям древности], в какой-то степени облагодетельствовал последующие поколения; как же можно не восхищаться таким порядком вещей?! Мыслители невеликого ума, видящие лишь одну сторону явлений, как могут они рассуждать о Дао?! (Кан Ювэй 2007, с. 354). Посещение исторических памятников Помпей и Римского форума, а также Ва­ тиканских музеев подвигло Кан Ювэя на рассуждение о сохранении исторических реликвий в  Китае. В  рассуждении «Китайцам следует сохранять свои культурные реликвии, чтобы не отстать от европейцев» Кан Ювэй перечисляет императорские дворцы разных династий, уничтоженные в гражданских конфликтах, начиная от ве­ ликого дворца Афан, сожженного Сян Юем27. Эти примеры служат доказательством 47 РОССИЯ И МИР: НАУЧНЫЙ ДИАЛОГ № 1(3) | март 2022 того, что китайцы намного уступают итальянцам в плане сохранения культурных ре­ ликвий. Кан Ювэй выделял две причины: императоры древности «не любили старое и воздвигали новое», а последующие династии уничтожали наследие предшествен­ ников. у 28 Кан Ювэй 2007, с. 370-371. Кан Ювэй писал это до эпохальных археологических откры­ тий первой трети ХХ века, когда были найдены материальные следы китайской цивилизации вплоть до эпохи неолита. Историко-архитектурные экспедиции Лян Сычэна (сына ученика Кана Лян Цичао) и его жены Линь Хуэйинь в 1932–1937 годы позволили открыть полностью со­ хранившиеся деревянные постройки IX–X веков. Сян Юй (232–202 годы до н.э.) – мятежный полководец царства Чу, уничтоживший династию Цинь и ее столицу. Проиграл в борьбе с основателем династии Хань Лю Баном и покончил жизнь самоубийством. д р 9 Кан Ювэй 2005, с. 4. р д р р 29 Кан Ювэй 2005, с. 4. Цивилизация Древнего Рима и уроки для Китая В обычном для него стиле Кан Ювэй приводит в пример земляков-гуандунцев, у которых принято разбивать прекрасные сады, погибающие после того, как семей­ ство основателей разоряется, да так, что и следов не остается. Он сурово заключает: «Если народ не умеет чтить героев и сохранять древности – то это варвары». Китайцы сохранили память о былых достижениях в литературе, но не имеют материальных памятников старины. Впрочем, одной из причин недолговечности материальной стороны китайской культуры является то, что с древнейших времен постройки были деревянными, за исключением немногих пагод. Поэтому китайцы должны овладеть искусством каменного строительства и научиться сохранять материальные культур­ ные ценности28. В тексте «Путешествия в  Италию» Кан Ювэй часто использовал понятие «цивилизация» (вэньмин, 文明). Мыслитель раскрывал его через бинарные про­ тивопоставления: примеры высокого или низкого цивилизационного развития. Неслучайно использование им термина «диалектика» (не существующей в  тра­ диционной китайской философии): Кан Ювэй, прекрасно осознавая наличие ма­ териальной и  нематериальной цивилизации, подчинял эти стороны политике. Несмотря на то, что Кан Ювэй рассматривал Европу как целостное цивилизаци­ онное поле, он отлично видел различия стран. При этом он полагал, что с точки зрения материальной цивилизации Италия не уступала ни Англии, ни Германии, ни даже Китаю. С точки зрения духовной культуры точек соприкосновения между Китаем и Западом намного больше, чем в сфере материального, поскольку циви­ лизация – это процесс, а не состояние (Ван Минмин 2019, с. 17-18). Цивилизация – это универсальное выражение прогресса от «варварства» к  «культурности». При этом Кан – конфуцианец, в Датун шу он утверждал, что человек единотелесен всем живым существам, в том числе лишенным разума животным и низшим из насе­ комых, и должен всемерно помогать им освободиться от страданий и смерти29, но это не относилось к  цивилизованному и  нецивилизованному состоянию челове­ чества. Иными словами, некультурность типологически может выражать как низ­ шее положение по эволюционной лестнице (и такие общества надлежит спасать, то есть «подвергать эволюции»), так и регресс, остановку в развитии, вырождение. Эволюционный взгляд на борьбу с хаосом позволял Кан Ювэю пропагандировать единство человеческой цивилизации и призывать своих соотечественников рас­ д р Кан Ювэй 2005, с. 4. 48 RUSSIA & WORLD: SCIENTIFIC DIALOGUE No. 1(3) | March 2022 RUSSIA & WORLD: SCIENTIFIC DIALOGUE ( ) | h No. 1(3) | March 2022 сматривать людей Запада как венец эволюции, которые во всем превосходят ки­ тайцев. Китайский народ, будучи порождением и наследником китайской циви­ лизации, несет ответственность за достижения прошлого во имя будущего (Ван Минмин 2019, с. 18-19). Цивилизация Древнего Рима и уроки для Китая сматривать людей Запада как венец эволюции, которые во всем превосходят ки­ тайцев. Китайский народ, будучи порождением и наследником китайской циви­ лизации, несет ответственность за достижения прошлого во имя будущего (Ван Минмин 2019, с. 18-19). В «Книге о Великом единении» мыслитель максимально широко трактовал «цивилизационную эмпатию», призывая даже изучать цивилизации, зародивши­ еся на других небесных телах, «разделяя их радости и печали»30. Цивилизация при этом является составной частью и  диагностическим признаком эволюционной эры. Основным законом эволюции является стремление разумных существ к еди­ нению и  ликвидации конкуренции. Множество мелких вождеств образовывали государства, соперничающие между собой, как уделы древнего Китая, Египет, Элладу, Вавилон, Персию. С  течением веков государства увеличиваются и  явно демонстрируют тенденцию к объединению в глобальном масштабе31. Однако Кан Ювэй был далек от признания гомогенности исторического времени и  утверж­ дал, что дикость и  цивилизованность вполне могут сосуществовать. Более того, он пришел к  выводу, что жестокость проявлений войны прямо пропорциональ­ на степени развития материальной цивилизации32, но это не ведет к осуждению цивилизации, ибо ценностные ориентации носят относительный характер. Кан Ювэй не использовал понятие цивилизации для описания исторических фактов, которые существуют для него сами по себе и моральных уроков сами по себе не несут. Не связывал он цивилизацию и с любой формой организованной религии. Подобного рода теоретические выкладки прекрасно прослеживаются по тексту «Путешествия в Италию», например, в описании Колизея и деяний Юлия Цезаря и  Октавиана Августа33. При этом Римская империя представала в  описании Кан Ювэя как государство, возникшее естественным образом в соответствии с законом эволюции34. 30 Там же. 31 Там же, с. 56. 32 Там же, с. 68. 33 Кан Ювэй 2007, с. 365–366. 34 Там же, с. 367. Выводы Изучение интеллектуального наследия Кан Ювэя чрезвычайно важно для на­ шей современности, переживающей процесс цивилизационного изменения. Мыс­ литель воспитывался в русле традиционной синоцентрической парадигмы, в соот­ ветствии с которой Китай занимал центральное место в Поднебесной, тогда как все остальные государства и культуры занимали периферийное, подчиненное положе­ ние. Подобная «архитектура мироздания» пронизывала все мышление Кан Ювэя, определяя даже сакральную географию Датун шу, согласно которой все связующие материки Земли горные хребты – Гималаи, Кордильеры, Анды – являются лишь от­ 49 РОССИЯ И МИР: НАУЧНЫЙ ДИАЛОГ № 1(3) | март 2022 РОССИЯ И МИР: НАУЧНЫЙ ДИАЛОГ № 1(3) | март 2022 № 1(3) | март 2022 ветвлениями срединного Кунь-Луня35 (Мартынов 2017, с. 273). Это свидетельствова­ ло о том, что, пережив множество жизненных и политических катастроф, Кан Ювэй сохранил ощущение неразрывного единства мира: то, что пригодно для развития Китая, пригодно и для остального человечества (там же). Ван Минмин описывал из­ менение его мировоззрения в категориях смены «модели данничества» на «модель паломничества», что в  известной степени определяло риторический стиль. Кан Ювэй был любознательным изгнанником, а  не носителем империалистического «бремени» (Ван Минмин 2019, с. 29). Впрочем, учитывая его религиозное мировоззре­ ние, можно истолковать деятельность Кан Ювэя как миссию по спасению императо­ ра Гуансюй. Масштабы мира неимоверно расширяются, но при этом Кан совершенно сознательно соотносил все увиденные и познанные сущности с традиционным лето­ счислением и событийной канвой истории Китая (Рим был основан в семнадцатом году правления чжоуского Пин-вана), используя григорианские даты лишь тогда, когда не мог подобрать им китайского аналога. В известном смысле это превращен­ ная модель «данничества». ветвлениями срединного Кунь-Луня35 (Мартынов 2017, с. 273). Это свидетельствова­ ло о том, что, пережив множество жизненных и политических катастроф, Кан Ювэй сохранил ощущение неразрывного единства мира: то, что пригодно для развития Китая, пригодно и для остального человечества (там же). Ван Минмин описывал из­ менение его мировоззрения в категориях смены «модели данничества» на «модель паломничества», что в  известной степени определяло риторический стиль. Кан Ювэй был любознательным изгнанником, а  не носителем империалистического «бремени» (Ван Минмин 2019, с. 29). Впрочем, учитывая его религиозное мировоззре­ ние, можно истолковать деятельность Кан Ювэя как миссию по спасению императо­ ра Гуансюй. Масштабы мира неимоверно расширяются, но при этом Кан совершенно сознательно соотносил все увиденные и познанные сущности с традиционным лето­ счислением и событийной канвой истории Китая (Рим был основан в семнадцатом году правления чжоуского Пин-вана), используя григорианские даты лишь тогда, когда не мог подобрать им китайского аналога. В известном смысле это превращен­ ная модель «данничества». 35 Кан Ювэй имел в виду Кунь-Лунь даосско-буддийской китайской мифологии: священ­ ная гора, скрывающая Западный рай, место, где соединяются Небо и Земля. Выводы и Кан Ю-вэй [The Idea of Great Unity (Datong) in the History of Chinese Social Thought at the Turn of the 19th – 20th Cent. and Kang Youwei]. Казань: Изд-во Академии наук РТ. g g ] д д у Alitto, G.S., Liang Shuming (2010), Has Man a  Future?: Dialogues with the Last Confucian. Beijing: Foreign Languages Teaching and Research Press. g g g g Brusadelli, F. (2020), Confucian concord: reform, utopia and global teleology in Kang Youwei’s Datong Shu. Leiden – Boston: Brill. Elman, B.A. (2010), “The Failures of Contemporary Chinese Intellectual History,” Eig Studies, Vol. 43, No. 3, pp. 371-391. 010), “The Failures of Contemporary Chinese Intellectual History,” Eighteenth-Century 43, No. 3, pp. 371-391. , , , pp Findeisen, R.D. (2000), “In the hutong of Naples. From Chinese Travelogues of the Republican Era,” Cina, No. 28, pp. 89-106.̈ , , pp Hsiao Kung-ch’üan (1975), A modern China and a New World: K’ang Yu-wei, Reformer and Utopian, 1858–1927. Seattle, L.: University of Washington Press. DOI: 10.53658/RW2022-2-1(3)-36-52 УДК: 94(510)+316.722 DOI: 10.53658/RW2022-2-1(3)-36-52 DOI: 10.53658/RW2022-2-1(3)-36-52 Выводы Модель цивилизации, построенная Кан Ювэем, в тексте «Путешествия в Ита­ лию» еще не успела оформиться до конца, но при этом хорошо соотносится с логикой изложения в  итоговом философском тексте «Книги о  Великом единении». Задачей Кана в «Путешествии» было развенчание представления о Западе как буддийском рае «Чистой Земли». Для этого он использовал не только прямые сопоставления из опыта настоящего и прошлого Запада и Китая, но и увязывал различия со стадия­ ми цивилизационного развития. В «Путешествии в Италию» еще нет утверждения из «Датун шу», что Швейцария, США и скандинавские страны почти подошли к по­ рогу перехода в следующую эру цивилизационного развития. В свою очередь, длин­ ные описания римского прошлого призваны проиллюстрировать разные варианты действия основного закона истории – закона единства: объединения в исторически краткие сроки с последующим распадом (Рим), долгого объединения с замедлением темпов развития (Китай). Исторически для Кан Ювэя эти государства были равно­ значны и равноценны, поскольку становление Римской империи происходило одно­ временно с утверждением империй Цинь и Западная Хань. 50 RUSSIA & WORLD: SCIENTIFIC DIALOGUE No. 1(3) | March 2022 RUSSIA & WORLD: SCIENTIFIC DIALOGUE Источники Алимов, И.А., Кравцова, М.Е. (2014), История классической китайской литературы с древности и до XIII в.: поэзия, проза [The history of Classical Chinese literature from the antiquity to 13th c.: Poetry. Prose]. СПб.: Петербургское востоковедение. Ван Минмин (王铭铭) (2019), “Шэнпин чжи цзин: цун «Идали юцзи» кань Кан Ювэй Оуя вэньмин лунь” (“Предел Рождающегося мира: взгляд Кан Ювэя на европейскую цивилизацию” в [трактате] “Путешествие в Италию” [The Ambit of Flourishing Peace: Kang Youwei’s Perspective of Eurasian Civilizations as Seen in “The Travelogue of Italy”], 升平之境:从《意大利游记》看康有为欧 亚文明论), Шэхуй цзачжи, Т. 39, № 3, с. 1–56. ) у Кобзев, А.И. (2002), Философия китайского неоконфуцианства [Philosophy of Chinese Neo- Confucianism]. Москва: Восточ. лит. ] Куан Болинь (邝柏林) (1980), Кан Ювэй дэ чжэсюэ сысян (Философские идеи Кан Ю-вэя, 康有为的哲 学思想). Пекин: Чжунго шэхуй кэсюэ чубаньшэ. у у у Мартынов, Д.Е. (2010), Кан Ю-вэй: Жизнеописание [Kang Youwei: A  Biography]. Казань: Ин-т истории АН РТ им. Ш. Марджани. Мартынов, Д.Е. (2014) “Путешествия Кан Ю-вэя, западная цивилизация и модернизация Китая” [Travels of Kang Youwei, Western civilization and modernization of China], В пути за Китайскую стену. К 60-летию А.И. Кобзева. Ученые записки Отдела Китая, Вып. 12. Москва, с. 296-306. у д Мартынов, Д.Е. (2017), Идея Великого единения (Да тун) в истории китайской общественной мысли рубежа XIX–XX вв. References Alimov, I.A., Kravtsova, M.E. (2014) The history of Classical Chinese literature from the antiquity to 13th century: Poetry. Prose. [Istoriya klassicheskoii kitaiiskoii literatury s drevnosti i do XIII c.: poesiya, proza,] Saint-Petersburg: St. Petersburg Center for Oriental Studies Publ. (In Russian) p ] g g ( ) Alitto, G.S., Liang Shuming (2010). Has Man a Future?: Dialogues with the Last Confucian. Beijing: Foreign Languages Teaching and Research Press. Brusadelli, F. (2020) Confucian concord: reform, utopia and global teleology in Kang Youwei’s Datong Shu. Leiden – Boston: Brill. Elman, B.A. (2010), “The Failures of Contemporary Chinese Intellectual History,” Eighteenth-Century Studies. Vol. 43, No. 3, pp. 371-391. Elman, B.A. (2010), The Failures of Contemporary Chinese Intellectual History, Eighteenth-Century Studies. Vol. 43, No. 3, pp. 371-391. Findeisen R D (2000) “In the hutong of Naples From Chinese Travelogues of the Republican Era ” Cina pp (2000) “In the hutong of Naples. From Chinese Travelogues of the Republican Era,” Cina -106.̈ pp Findeisen, R.D. (2000) “In the hutong of Naples. From Chinese Travelogues of the Republi No. 28, p. 89-106.̈ , p Hsiao Kung-ch’üan. (1975) A modern China and a New World: K’ang Yu-wei, Reformer and Utopian, 1858–1927. Seattle, London: University of Washington Press.i y g Kobzev,A.I. (2002), Philosophy of Chinese Neo-Confucianism[Filosofiya kitaiiskogo neokonfutsianstva].. Moscow: Vostochnaya literatura. (In Russian) y Kobzev,A.I. (2002), Philosophy of Chinese Neo-Con Moscow: Vostochnaya literatura. (In Russian) y ( ) Kuang Bolin (1980), Kang Youwei de zhexue sixiang [Kang Youwei’s Philosophical Thought]. Beijing: Zhongguo shehui kexue chubanshe. (In Chinese) gg ( ) Martynov, D.E. (2010), Kang Youwei: A Biography[Kan Youwei: Zhizneopisanie . Kang Youwei: Biografiya]. Kazan: Institut Istorii Akademii Nauk Respubliki Tatarstan imeni Sh. Mardzhani. (In Russian) ) Martynov, D.E. (2014), “Travels of Kang Youwei, Western civilization and modernizati Martynov, D.E. (2014), Travels of Kang Youwei, Western civilization and modernization of China [“Puteshestvie Kan Youweiya, zapadnaya tsivilizatsiya I modernizatsiya Kitaiya” V puti za kitaiskuyu stenu. K 60-letiyu A.I.Kobzeva. Uchenyi zapiski otdela Kitaiya],” On the way to the Chinese wall. To the 60th anniversary of A.I. Kobzev. Moscow: Institut vostokovedeniya Rossiyskoy Akademii nauk, pp. 296-306. (In Russian) Martynov, D.E. (2017), The Idea of Great Unity (Datong) in the History of Chinese Social Thought at the Turn of the 19th – 20th Cent. and Kang Youwei[Ideya velikogo edineniya. (Da tun) v istorii kitaiskoi obshestvennoi mysli rubezha XIX – XX vv. I Kan You Wei]. Kazan: Izdatel’stvo Akademii Nauk Respubliki Tatarstan. Kang Youwei on the origins of western civilization Dmitry E. Martynov Kazan (Volga Region) Federal University (Kazan, Russia) Dmitry E. Martynov Kazan (Volga Region) Federal University (Kazan, Russia) Abstract. The article is devoted to the research in the shaping of concept of civilizational development made by the prominent Chinese philosopher Kang Youwei (1858-1927). The vision of Western civilization from the point of view of a Confucian thinker is considered on the basis of the material “Travels to Italy” (1904). Kang Youwei argued that Unity is the basic law of history. From Kang’s point of view, the Roman Empire was an example of a state, which was founded according to the laws of the evolutionary development of nature and society. 19th century Italy was seen as a young state that which had not yet overcome the consequences of the “birth trauma”. Shaping his views within the Sino centric paradigm, Kang Youwei considered the world civilization as a single evolutionary process of ascending from barbarism to the heights of culture, respectively, the positive experience of the West can be used to correct China’s shortcomings and return a high political and geostrategic status to the country. 51 РОССИЯ И МИР: НАУЧНЫЙ ДИАЛОГ № 1(3) | март 2022 РОССИЯ И МИР: НАУЧНЫЙ ДИАЛОГ № 1(3) | март 2022 Keywords: Kang Youwei, Civilization, Globalism, Sinocentrism, Confucianism, Great Unity (Da tong) Keywords: Kang Youwei, Civilization, Globalism, Sinocentrism, Confucianism, Great Unity (Da tong) About the author: DMITRY E. MARTYNOV – Dr.Sci. (History), Professor of the Department of Altai and Chinese Studies, Kazan (Volga Region) Federal University. ORCID 0000-0001- 5385-1915. Address: 420111, Russia, Kazan, Pushkin st., 1/55. dmitrymartynov80@mail.ru. About the author: DMITRY E. MARTYNOV – Dr.Sci. (History), Professor of the Department of Altai and Chinese Studies, Kazan (Volga Region) Federal University. ORCID 0000-0001- 5385-1915. Address: 420111, Russia, Kazan, Pushkin st., 1/55. dmitrymartynov80@mail.ru. Wang Mingming (2019), “The Ambit of Flourishing Peace: Kang Youwei’s Perspective of Eurasian Civilizations as Seen in “The Travelogue of Italy,” Chinese Journal of Sociology, Vol. 39, No. 3, pp. 1-56. (In Chinese) References (In Russian) Wang Mingming (2019), “The Ambit of Flourishing Peace: Kang Youwei’s Perspective of Eurasian Civilizations as Seen in “The Travelogue of Italy,” Chinese Journal of Sociology, Vol. 39, No. 3, pp. 1-56. (In Chinese) 52
https://openalex.org/W2741874029
https://boris.unibe.ch/107200/1/Kaiser%20MJ%202017.pdf
English
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Generation of transgenic rodent malaria parasites by transfection of cell culture-derived merozoites
Malaria journal
2,017
cc-by
10,182
© The Author(s) 2017. 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. METHODOLOGY ARTICLE METHODOLOGY ARTICLE Abstract Background:  Malaria research is greatly dependent on and has drastically advanced with the possibility of geneti‑ cally modifying Plasmodium parasites. The commonly used transfection protocol by Janse and colleagues utilizes blood stage-derived Plasmodium berghei schizonts that have been purified from a blood culture by density gradient centrifugation. Naturally, this transfection protocol depends on the availability of suitably infected mice, constituting a time-based variable. In this study, the potential of transfecting liver stage-derived merozoites was explored. In cell culture, upon merozoite development, infected cells detach from the neighbouring cells and can be easily harvested from the cell culture supernatant. This protocol offers robust experimental timing and temporal flexibility. Methods:  HeLa cells are infected with P. berghei sporozoites to obtain liver stage-derived merozoites, which are harvested from the cell culture supernatant and are transfected using the Amaxa ­Nucleofector® electroporation technology. Results:  Using this protocol, wild type P. berghei ANKA strain and marker-free ­PbmCherryHsp70-expressing P. berghei parasites were successfully transfected with DNA constructs designed for integration via single- or double-crossover homologous recombination. Conclusion:  An alternative protocol for Plasmodium transfection is hereby provided, which uses liver stage-derived P. berghei merozoites for transfection. This protocol has the potential to substantially reduce the number of mice used per transfection, as well as to increase the temporal flexibility and robustness of performing transfections, if mos‑ quitoes are routinely present in the laboratory. Transfection of liver stage-derived P. berghei parasites should enable generation of transgenic parasites within 8–18 days. Keywords:  Plasmodium berghei, Transfection, Liver stage-derived merozoites including successful transfection, which resulted in effi- ciencies of up to 30% [7]. Transfection efficiencies of rodent Plasmodium berghei parasites have increased up to 1:1000 as a result of implementing the highly effi- cient non-viral ­Nucleofector® technology [1, 8]. A major advantage of using the model organism P. berghei for Plasmodium research is the accessibility of the entire life cycle in vitro as well as in vivo, including the liver stage development. A further advantage is the availability of an almost complete genomic DNA library that originated from phage-based vectors, applicable for generation of knock-outs, and tagging of genes [9–11]. Methods for Generation of transgenic rodent malaria parasites by transfection of cell culture‑derived merozoites Gesine Kaiser1*  , Mariana De Niz1,3, Paul‑Christian Burda1,4, Livia Niklaus1,2, Rebecca Limenitakis Stanway1 and Volker Heussler1 Kaiser et al. Malar J (2017) 16:305 DOI 10.1186/s12936-017-1949-y Kaiser et al. Malar J (2017) 16:305 DOI 10.1186/s12936-017-1949-y Malaria Journal Open Access *Correspondence: gesine.kaiser@x100.de 1 Institute of Cell Biology, University of Bern, Bern, Switzerland Full list of author information is available at the end of the article Background Genetic manipulation of Plasmodium parasites Genetic manipulation of Plasmodium parasites In the past 15 years, Plasmodium parasites have become greatly accessible for genetic manipulation [1–3], facili- tated by the genome sequencing of human and rodent malaria parasites [4–6]. Advances continue and Plas- modium knowlesi parasites were recently successfully adapted for in  vitro culture in human red blood cells *Correspondence: gesine.kaiser@x100.de 1 Institute of Cell Biology, University of Bern, Bern, Switzerland Full list of author information is available at the end of the article Kaiser et al. Malar J (2017) 16:305 Kaiser et al. Malar J (2017) 16:305 Page 2 of 11 of animals used to achieve clonal transgenic parasite lines [17]. This work presents an established and opti- mized protocol for transfection of liver stage-derived schizonts and merozoites, which equally aims to reduce the number of animals used for the generation of trans- genic parasite lines. typical genetic manipulation, such as the generation of knock-outs and complemented parasites, fluorescent tagging of proteins and even conditional knock-outs, are available for both rodent and human Plasmodium para- sites [9, 12–14]. Classically, transfection of DNA con- structs into P. berghei parasites is performed into blood stage-derived schizonts and merozoites, and benefits from the fact that schizonts do not rupture in in vitro blood cultures and can thus be enriched and purified. Transfection of schizonts and free merozoites, compared to other asexual blood stages, is facilitated by the fact that DNA used for transfection has to cross only two or three membranes, namely the erythrocyte membrane (depending on whether or not merozoites have been released), the parasite plasma membrane (PM) and the nuclear membrane, instead of four, including the parasi- tophorous vacuole membrane [1]. The standard protocol for P. berghei transfection, requires the infection of two mice, which ideally should have a parasitaemia of about 3% usually achieved between day 5 and 7 after pre-infec- tion. Once the parasitaemia has reached about 3%, blood stage parasites are taken into culture for 16–18 h and fol- lowing this, schizonts are purified using a density gradi- ent. Purified schizonts and merozoites are subsequently transfected using the Amaxa ­Nucleofector® electropora- tion technology [1, 8]. This study took advantage of the fact that the merozoite stage of Plasmodium parasites is not restricted to the blood stage, but is also produced at the end of liver stage development. Culturing of HeLa cells HeLa cells (a gift from Robert Menard, Pasteur Institute, Paris) were grown in MEM (minimum essential medium) with Earle’s salts (Bioconcept, cat. no 1-31F01-I), supple- mented with 10% heat inactivated foetal calf serum—FCS (Bioswisstec, Cat. No. S4150), 1% penicillin/streptomycin (Bioconcept, Cat. No. 4-01F00-H) and 1% l-glutamine (Bioconcept, 5-10K00-H) in a humid incubator with 5% ­CO2 at 37  °C. Using accutase (Sigma-Aldrich, Cat. No. A6964), cells were passaged twice a week. Background The Plasmodium liver stage is characterized by an immense expansion of the parasite population. Intriguingly, a single sporozo- ite that has infected a host hepatocyte can mature into thousands of progeny merozoites [15]. At the end of exo-erythrocytic parasite development, merozoites are released from the parasitophorous vacuole (PV) into the hepatocyte cytoplasm. This leads to the detach- ment of the infected host cell from its neighbouring cells and in in  vitro cultures, to detachment of the infected cells, which then float freely in the culture supernatant. Merosomes, sacs containing infectious merozoites, are subsequently extruded from the detached cell and are also found in the cell culture supernatant [16, 17]. Sin- gle detached cells of in vitro-cultured P. berghei parasites were recently described to harbour an average of about 4500 individual merozoites [16, 18]. In a previous study by Stanway et al. individual merosomes or detached cells were collected and used for sub-cloning of transgenic parasites, thereby greatly contributing to the reduction Animal work statement Experiments were conducted in strict accordance with the guidelines of the Swiss Tierschutzgesetz (TSchG; Ani- mal Rights Laws) and approved by the ethical committee of the University of Bern (Permit Number: BE109/13). Balb/c mice used in experiments were between 6 and 10 weeks of age and were either bred in the central ani- mal facility of the University of Bern, or were supplied from Harlan Laboratories or Charles River. In vitro infection of HeLa cells with Plasmodium berghei detached cells/merosomes were present in the cell cul- ture supernatant. Detached cells and merosomes are characterized by their round shape. They contain thou- sands of small merozoites, which, in viable merosomes and detached cells, are constantly moving. If only few detached cells had been formed, infection duration was extended to up to 67 hpi. To harvest detached cells and merosomes, the supernatant of each well was gen- tly pipetted up and down three times and finally the medium of the three infected wells was pooled into one Eppendorf tube. Parasites were pelleted at 16,000g for 10 s, the direction of the tube was changed by 180° and the tube was spun again for 10 s at 16,000g. Successful centrifugation was verified by the presence of a small semi-transparent/white pellet visible at the bottom of the tube. If more than one transfection was planned, all sets of detached cells were prepared before the transfec- tion was performed. In vitro infection of HeLa cells with Plasmodium berghei Infection of HeLa cells with sporozoites required that mosquitoes infected with either P. berghei (ANKA strain) wild type parasites or any other selection-marker-free P. berghei parasite line were available. On the day of the infection, the parasites in these mos- quitoes were between day 16 to 24 post-blood feed. For one transfection (if more transfections were intended cells were seeded accordingly), HeLa cells were seeded into three wells of a 24-well plate (Greiner Bio-one, Cat. No. 662160) with 2 × 104 cells per well and cultured in complete MEM culture medium. On the following day at about 16.00, 3–5 infected mosquitoes per transfection were anesthetized with chloroform vapour. Anesthetized mosquitoes were briefly dipped into 70% (vol/vol) etha- nol and then transferred to PBS. For removal of salivary glands, mosquitoes were suc- cessively put into 200 µl of infection medium [complete MEM culture medium containing 2.5  μg  ml−1 ampho- tericin B (Sigma-Aldrich, Cat. No. A2942)] on a slide and the head was carefully removed using surgical forceps. The two sets of each three salivary gland lobes were iso- lated from the head or mosquito body, and transferred into an Eppendorf tube containing 20  µl of infection medium, kept on ice until infection of HeLa cells. Sali- vary glands were mechanically disrupted using a pestle (Sigma-Aldrich, Cat. No. Z359947) driven by a Cord- less motor (Sigma-Aldrich, Cat. No. Z359971), using approximately 10 pulses of 1–2 s. Transfection of liver stage‑derived merozoitesh Transfection of liver stage‑derived merozoites The transfection procedure was adapted from the pre- viously published protocol by Janse et  al. [8]. Mice to be injected with transfected parasites were pre- warmed using an infrared lamp for about 5–10  min prior to the injection, to allow the tail vein to dilate. 5–10 µg of DNA (contained in a maximum volume of 10 µl) were added to 100 µl Nucleofector solution 88A6 (Lonza, Cat. No. VVPA-1002 KT) and the same was done for each of the constructs to be transfected. Next, using a 1000-µl pipette, the supernatant was carefully removed from one detached cell-containing Eppen- dorf tube, without touching the detached cell pellet. Subsequently, 100  µl of DNA-containing Nucleofec- tor solution 88A6 were added and the detached cells and merosomes were cautiously resuspended. Resus- pended parasites were transferred from the Eppendorf tube into an electroporation cuvette and placed into the Amaxa Nucleofector device. Parasites were trans- fected using the program U33 of the Amaxa Nucleo- fector device. Immediately after transfection, 50  µl of RPMI1640 culture medium (Bioconcept, Cat. No. 1-41F01-I) were added to the transfected parasites. Following this, the whole volume of the cuvette (about 150  µl) was transferred into an Eppendorf tube using the provided Pasteur plastic pipette. Using an insulin syringe, the entire suspension was injected into the tail vein of a naïve Balb/c mouse (6–10 weeks of age) and the previous steps were repeated until all constructs were transfected. g Per anticipated transfection, 50,000 sporozoites were used, contained in a volume of 600 µl infection medium. After removing the medium from the three wells to be infected, 200  µl of sporozoite-containing infection medium were added to each of the three wells. To allow fast settling of the sporozoites, the 24-well plate was spun for 1 min at 200g. Following 2 h of incubation time (at 37  °C with 5% ­CO2), infected cells were washed with 500  µl pre-warmed infection medium to remove mos- quito debris and sporozoites that had not been infected. Cells were then incubated with 500 µl infection medium in a humid incubator at 37  °C with 5% ­CO2. To avoid fungal and bacterial growth, the infection medium was exchanged at 24 and 48 h post infection (hpi). In vitro infection of HeLa cells with Plasmodium berghei Next, 300 µl of infec- tion medium were added and the number of sporozoites was estimated using a Neubauer chamber. Infection of mosquitoes with Plasmodium berghei A P. berghei blood stabilate was injected intraperitoneally into a naïve Balb/c mouse (referred to as ‘pre-infection mouse’). When this mouse had reached a parasitaemia of 1–4%, 50 µl of infected blood, diluted with 150 µl PBS, were injected intravenously into a mouse (referred to as ‘feed mouse’) that 3 days prior to that had been intraperi- toneally injected with 200 µl phenylhydrazine (6 mg ml−1 in PBS) (Sigma-Aldrich, Cat. No. 114715). When the feed mouse, had reached a parasitaemia of at least 7%, usually 3–4 days after infection, with most of the circulating par- asites being gametocytes, the mouse was anaesthetized and used to allow about 100–150 female Anopheles ste- phensi mosquitoes to feed for 1 h. Infected mosquitoes were kept at 20.5 °C and 80% humidity and fed with 8% fructose containing 0.2% PABA (Sigma-Aldrich, Cat. No. 100536). Infected mosquitoes were dissected to obtain sporozoites for the detached cell transfection protocol between day 16 and 24 post blood feed. Page 3 of 11 Kaiser et al. Malar J (2017) 16:305 Drug selection and collection of transgenic parasites Drug selection and collection of transgenic parasites Detection of parasites by luminescence (NanoLuc) Parasite detection by luminescence was performed as described before [18]. Briefly, a drop of blood from the infected mouse was added to 20 µl of PLB (1 × Passive Lysis Buffer; Promega, Cat. No. E1941) contained in one well of a black, flat-bottom, 96-well plate (Greiner Bio- One, Cat. No. 655900). As a negative control, blood was collected from a non-infected mouse. A 1:50 dilution of the NanoGlo substrate was prepared in ­NanoGloTM lucif- erase assay buffer (Promega, Cat. No. N1120), 50 µl of the dilution were added to the sample and the luminescence signal was read within 5 min. gg p p p Having established the optimal sporozoite number to use for infection, the effect of age of the parasite in the mosquito on the number of detached cells/merosomes formed and the corresponding merozoite number was determined (Fig. 3a). Detached cell formation was quan- tified, using sporozoites obtained from mosquitoes from day 16 to day 30 post-blood feed. Based on previ- ous experiments, it was found that the success rate and efficiency of detached cell transfection starts to drop considerably when using fewer than 500 detached cells/ merosomes (4 × 106 merozoites). The minimum was set to 500 detached cells/merosomes and beyond day 24 post- blood feed, numbers of detached cells formed dropped below this critical level. Accordingly, harvesting sporo- zoites from mosquitoes between day 16 and day 24 post- blood feed results in the sufficient formation of detached cells. Thus, sporozoites can be used over a period of nine consecutive days, during which transfections can be flex- ibly and robustly planned and performed, as the processes from seeding of cells to liver stage-derived merozoite transfection takes place over a period of only 4 days.il Resultsh One day after transfection, drugs suitable for selection of transgenic parasites were given to infected mice (depend- ing on the transfected construct), as previously published [8]. Drug selection was continued until transgenic para- sites had reached a parasitaemia of 2% or higher. From 5  days after the transfection onwards, the parasitaemia of mice injected with transfected parasites was checked by thin blood smear. If the transfected construct encoded fluorescent or luminescent markers expressed under a promoter active in the blood stage, fluorescence or lumi- nescence was also checked. This work presents an established protocol that allows efficient transfection of liver stage-derived merozoites in so-called detached cells and merosomes (Fig. 1). This pro- tocol is based on the fact that in vitro, P. berghei-infected cells detach from their neighbouring cells and float into the cell culture supernatant. Thus, they can be easily puri- fied by harvesting and then centrifuging the supernatant. After initially succeeding in transfecting detached cells/ merosomes, the next aim was to optimize the formation and yield of detached cells to quantify the corresponding number of merozoites used, and to standardize the pro- tocol in a user-friendly way. As a first step, the optimal number of sporozoites to be used for host cell infection to maximize the generation of detached cells was deter- mined (Fig. 2). As expected, with a constant number of host cells, the number of detached cells increases as the number of sporozoites used per infection increases. This increase is not linear and reaches a plateau with about 50,000 sporozoites. On average, using 50,000 sporozoites, about 550 detached cells were obtained when harvesting at 65 hpi (Fig. 2a), which is about 20% of the parasites that were present at 48 hpi (Fig. 2b). When higher sporozoite numbers were used, the number of detached cells formed also increased but the conversion rate of parasites present at 48 hpi to detached cells dropped considerably (Fig. 2b), which suggests sub-optimal parasite development. Harvesting detached cells and merosomes At about 62–65 hpi (about 09.00 at day 3 post-infec- tion) it was verified by bright-field microscopy that Page 4 of 11 Kaiser et al. Malar J (2017) 16:305 Page 4 of 11 Detection of parasites by fluorescence To stain the parasite DNA, a drop of blood from the infected mouse was added to 50  µl of PBS contain- ing 1  μg  ml−1 Hoechst 33342 (Sigma-Aldrich, Cat. No. B2261) in PBS. Parasites were analysed under a fluores- cence microscope using the appropriate filters and an increasing magnification of between 200× and 1000×. Genotyping of transfected parasites Genomic DNA of transfected parasites was purified. Integration PCR was performed according to the sche- matic in Fig. 6. DNA sequences of primers used are shown in Table 1. Table 1  Primers used for genotyping in Fig. 6 Primer Sequence (5′–3′) 1 GTGTAGTAACATCAGTTATTGTGTG 2 ATACTGTATAACAGGTAAGCTGTTATTGTG 3 TTTCCCAGTCACGACGTTG 4 CTTAGTGTTTTGTATTAATGTCGATT TG QCR2 AGGGGCAAATACCAAAGTTGTGT QCR1 ACGCATATTCACGAGTTTCACA GW2 CTTTGGTGACAGATACTAC Table 1  Primers used for genotyping in Fig. 6 For detached cell quantification, in addition to a fluo- rescence microscopy-based assay, a luminescence-based assay was adapted to determine the numbers of detached cells and merozoites (Fig. 3). Using this assay, it was Kaiser et al. Malar J (2017) 16:305 Page 5 of 11 calculated that about 4.5–8.5 × 106 merozoites are used cells and contained merozoites is key to successful tran Day 0-3 In vitro infection of HeLa cells with P. berghei (a-c) a f d e g h c b i k j l Fig. 1  Workflow and visualization of critical protocol steps. a Optimal density of seeded HeLa cells 24 h post seeding. b Representative images of dissection of mosquito salivary glands (red arrow) attached to the head (left) or removed (right). c HeLa cells infected with P. berghei sporozoites at a density representing sporozoite numbers used in the here described protocol. d HeLa cells infected with ­PbmCherryHsp70 65 hpi, prior to detached cell (red) collection. e Collection of detached cells at 65 hpi. f Representative image of collected ­PbmCherryHsp70 detached cells (red) at 65 hpi and uninfected HeLa cell debris (unlabelled). g ­Amaxa® ­Nucleofector® II device with transfection program U33. h Intravenous injection of transfected parasites into a naïve mouse. i Drug selection of transgenic parasites. j Checking the parasitaemia by Wright stain or fluorescence. k Monitor trans‑ genic parasites by fluorescence. l Preparation of blood stabilates. Scale bars in all images are 50 µm Day 0-3 In vitro infection of HeLa cells with P. berghei (a-c) c c c d f e g h g h j k l l k Fig. 1  Workflow and visualization of critical protocol steps. a Optimal density of seeded HeLa cells 24 h post seeding. b Representative images of dissection of mosquito salivary glands (red arrow) attached to the head (left) or removed (right). c HeLa cells infected with P. berghei sporozoites at a density representing sporozoite numbers used in the here described protocol. Genotyping of transfected parasites Values shown are the result of ten separate limiting dilutions from three independent experiments Fig. 2  Experimental optimization of sporozoite numbers for HeLa cell infection to obtain maximal numbers of detached cells/merosomes. a, b HeLa cells were infected with different numbers of ­PbmCherryHsp70 parasites and counted at 48 hpi. Detached cells and merosomes were counted in the supernatant at 65 hpi. a Total number of detached cells formed at 65 hpi depending on the number of sporozoites used for infection. b Per‑ centage of detached cells formed in relation to parasite count at 48 hpi was calculated. Shown are means with errors depicted as 95% confidence intervals, n = 3 in biological triplicates Fig. 2  Experimental optimization of sporozoite numbers for HeLa cell infection to obtain maximal numbers of detached cells/merosomes. a, b HeLa cells were infected with different numbers of ­PbmCherryHsp70 parasites and counted at 48 hpi. Detached cells and merosomes were counted in the supernatant at 65 hpi. a Total number of detached cells formed at 65 hpi depending on the number of sporozoites used for infection. b Per‑ centage of detached cells formed in relation to parasite count at 48 hpi was calculated. Shown are means with errors depicted as 95% confidence intervals, n = 3 in biological triplicates Fig. 2  Experimental optimization of sporozoite numbers for HeLa cell infection to obtain maximal numbers of detached cells/merosomes. a, b HeLa cells were infected with different numbers of ­PbmCherryHsp70 parasites and counted at 48 hpi. Detached cells and merosomes were counted in the supernatant at 65 hpi. a Total number of detached cells formed at 65 hpi depending on the number of sporozoites used for infection. b Per‑ centage of detached cells formed in relation to parasite count at 48 hpi was calculated. Shown are means with errors depicted as 95% confidence intervals, n = 3 in biological triplicates Fig. 3  Microscopic and luminescence-based determination of detached cell and merozoite number, dependent on the parasite age in infected mosquitoes. a The relation of the parasite age in infected mosquitoes to the number of detached cells obtained and the relative merozoite number was investigated from day 16 to day 30. A critical cut off (red line), in detached cell numbers obtained is at 500 detached cells (4 × 106 merozoites), as the success rate of detached cell transfections using fewer than 500 detached cells decreases. Genotyping of transfected parasites Seeding and infections were performed according to the present protocol, collected detached cells were counted using a fluorescent microscope at 65 hpi and merozoite number was determined using a luminescence based assay, n = 3. Error bars denote SD. b Determination of luminescence of detached cells obtained over time, using sporozoites from day 16 to day 30 post-blood feed. Detached cells of ­PbmCherryHsp70-NLuc parasites were obtained and collected according to the present protocol. Aside from quantification of detached cell numbers by fluorescence, luminescence was used to estimate the number of detached cells used for transfection from day 16 onwards, following mosquito feeds. Luminescence correlates with number of detached cells contained, and suggests that at days 16–23, the number of viable merozoites remains relatively constant, while a significant decrease in luminescence is identified from day 24 onwards (denoting less detached cells and less merozoites). Values shown are the result of three independent experiments repeated in triplicate. Error bars denote SD. c ­PbmCherryHsp70-NLuc detached cells were collected and disrupted to release merozoites. The total number of merozoites of a single detached cell was counted, and a limiting dilution was performed to achieve one merozoite, which was lysed and its luminescence measured. The radiance calculated was 9.9 × 104 p s−1 cm−2. Performing a limiting dilution from 1 to ­106 merozoites we defined that a positive, significant ­(R2 = 0.96) correlation exists between merozoite number and the ­log10 of the relative light units of luminescence. This in turn allowed us to estimate the total number of merozoites in single individual detached cells and of the complete population of detached cells used for transfection. Values shown are the result of ten separate limiting dilutions from three independent experiments Fig. 3  Microscopic and luminescence-based determination of detached cell and merozoite number, dependent on the parasite age in infected mosquitoes. a The relation of the parasite age in infected mosquitoes to the number of detached cells obtained and the relative merozoite number was investigated from day 16 to day 30. A critical cut off (red line), in detached cell numbers obtained is at 500 detached cells (4 × 106 merozoites), as the success rate of detached cell transfections using fewer than 500 detached cells decreases. Genotyping of transfected parasites Seeding and infections were performed according to the present protocol, collected detached cells were counted using a fluorescent microscope at 65 hpi and merozoite number was determined using a luminescence based assay, n = 3. Error bars denote SD. b Determination of luminescence of detached cells obtained over time, using sporozoites from day 16 to day 30 post-blood feed. Detached cells of ­PbmCherryHsp70-NLuc parasites were obtained and collected according to the present protocol. Aside from quantification of detached cell numbers by fluorescence, luminescence was used to estimate the number of detached cells used for transfection from day 16 onwards, following mosquito feeds. Luminescence correlates with number of detached cells contained, and suggests that at days 16–23, the number of viable merozoites remains relatively constant, while a significant decrease in luminescence is identified from day 24 onwards (denoting less detached cells and less merozoites). Values shown are the result of three independent experiments repeated in triplicate. Error bars denote SD. c ­PbmCherryHsp70-NLuc detached cells were collected and disrupted to release merozoites. The total number of merozoites of a single detached cell was counted, and a limiting dilution was performed to achieve one merozoite, which was lysed and its luminescence measured. The radiance calculated was 9.9 × 104 p s−1 cm−2. Performing a limiting dilution from 1 to ­106 merozoites we defined that a positive, significant ­(R2 = 0.96) correlation exists between merozoite number and the ­log10 of the relative light units of luminescence. This in turn allowed us to estimate the total number of merozoites in single individual detached cells and of the complete population of detached cells used for transfection. Values shown are the result of ten separate limiting dilutions from three independent experiments Fig. 3  Microscopic and luminescence-based determination of detached cell and merozoite number, dependent on the parasite age in infected mosquitoes. a The relation of the parasite age in infected mosquitoes to the number of detached cells obtained and the relative merozoite number was investigated from day 16 to day 30. A critical cut off (red line), in detached cell numbers obtained is at 500 detached cells (4 × 106 merozoites), as the success rate of detached cell transfections using fewer than 500 detached cells decreases. Genotyping of transfected parasites d HeLa cells infected with ­PbmCherryHsp70 65 hpi, prior to detached cell (red) collection. e Collection of detached cells at 65 hpi. f Representative image of collected ­PbmCherryHsp70 detached cells (red) at 65 hpi and uninfected HeLa cell debris (unlabelled). g ­Amaxa® ­Nucleofector® II device with transfection program U33. h Intravenous injection of transfected parasites into a naïve mouse. i Drug selection of transgenic parasites. j Checking the parasitaemia by Wright stain or fluorescence. k Monitor trans‑ genic parasites by fluorescence. l Preparation of blood stabilates. Scale bars in all images are 50 µm Fig. 1  Workflow and visualization of critical protocol steps. a Optimal density of seeded HeLa cells 24 h post seeding. b Representative images of dissection of mosquito salivary glands (red arrow) attached to the head (left) or removed (right). c HeLa cells infected with P. berghei sporozoites at a density representing sporozoite numbers used in the here described protocol. d HeLa cells infected with ­PbmCherryHsp70 65 hpi, prior to detached cell (red) collection. e Collection of detached cells at 65 hpi. f Representative image of collected ­PbmCherryHsp70 detached cells (red) at 65 hpi and uninfected HeLa cell debris (unlabelled). g ­Amaxa® ­Nucleofector® II device with transfection program U33. h Intravenous injection of transfected parasites into a naïve mouse. i Drug selection of transgenic parasites. j Checking the parasitaemia by Wright stain or fluorescence. k Monitor trans‑ genic parasites by fluorescence. l Preparation of blood stabilates. Scale bars in all images are 50 µm calculated that about 4.5–8.5 × 106 merozoites are used per transfection (Fig. 3a). cells and contained merozoites is key to successful trans- fection. Microscopic assessment is the fastest and most efficient way to assess detached cell morphology (Fig. 4). Transfection of detached cells had a better efficiency g However, experiments also showed that not only the quantity but also the viability and quality of detached Page 6 of 11 Kaiser et al. Malar J (2017) 16:305 Fig. 2  Experimental optimization of sporozoite numbers for HeLa cell infection to obtain maximal numbers of detached cells/merosomes. a, b HeLa cells were infected with different numbers of ­PbmCherryHsp70 parasites and counted at 48 hpi. Detached cells and merosomes were counted in the supernatant at 65 hpi. a Total number of detached cells formed at 65 hpi depending on the number of sporozoites used for infection. Genotyping of transfected parasites b Per‑ centage of detached cells formed in relation to parasite count at 48 hpi was calculated. Shown are means with errors depicted as 95% confidence intervals, n = 3 in biological triplicates Fig. 3  Microscopic and luminescence-based determination of detached cell and merozoite number, dependent on the parasite age in infected mosquitoes. a The relation of the parasite age in infected mosquitoes to the number of detached cells obtained and the relative merozoite number was investigated from day 16 to day 30. A critical cut off (red line), in detached cell numbers obtained is at 500 detached cells (4 × 106 merozoites), as the success rate of detached cell transfections using fewer than 500 detached cells decreases. Seeding and infections were performed according to the present protocol, collected detached cells were counted using a fluorescent microscope at 65 hpi and merozoite number was determined using a luminescence based assay, n = 3. Error bars denote SD. b Determination of luminescence of detached cells obtained over time, using sporozoites from day 16 to day 30 post-blood feed. Detached cells of ­PbmCherryHsp70-NLuc parasites were obtained and collected according to the present protocol. Aside from quantification of detached cell numbers by fluorescence, luminescence was used to estimate the number of detached cells used for transfection from day 16 onwards, following mosquito feeds. Luminescence correlates with number of detached cells contained, and suggests that at days 16–23, the number of viable merozoites remains relatively constant, while a significant decrease in luminescence is identified from day 24 onwards (denoting less detached cells and less merozoites). Values shown are the result of three independent experiments repeated in triplicate. Error bars denote SD. c ­PbmCherryHsp70-NLuc detached cells were collected and disrupted to release merozoites. The total number of merozoites of a single detached cell was counted, and a limiting dilution was performed to achieve one merozoite, which was lysed and its luminescence measured. The radiance calculated was 9.9 × 104 p s−1 cm−2. Performing a limiting dilution from 1 to ­106 merozoites we defined that a positive, significant ­(R2 = 0.96) correlation exists between merozoite number and the ­log10 of the relative light units of luminescence. This in turn allowed us to estimate the total number of merozoites in single individual detached cells and of the complete population of detached cells used for transfection. Genotyping of transfected parasites e Example of a fungal contamination of Plasmodium infected cells. Scale bars are 50 µm Fig. 4  Representative microscopic images of detached cells. a Bright field and a′ fluorescence merged image of a normal detached cell, b and b′ of a ruptured detached cell and c, d examples of detached cells with abnormal morphology, c, c′ of merofusosomes [25] and d of absent merozoite formation. e Example of a fungal contamination of Plasmodium infected cells. Scale bars are 50 µm experimental procedure and the potential to reduce the number of mice used per transfection. In addition to transfection of P. berghei blood stages, the new protocol provides an optimized usage of P. berghei parasites for transfection. Using liver stage-derived merozoites for transfection, mouse numbers used should be reduced most efficiently when the protocol is used for high num- bers of transfections. Transfection of liver stage-derived schizonts and merozoites of either wild type P. berghei ANKA or transgenic selection-marker-free P. berghei ANKA parasites has been proven successful for transfec- tion of DNA constructs facilitating integration into the parasite genome by both single- and double-crossover homologous recombination. Transfections performed with DNA constructs for integration by single homolo- gous recombination had a slightly higher success rate than transfections performed with DNA constructs that integrated by double crossover homologous recombina- tion. Most likely this is related to the mechanism of inte- gration and whether or not expression can be episomal, rather than being a consequence of the transfection pro- cedure itself [23, 24]. can introduce bacteria and fungi, contained in the mos- quito debris, to the cell culture (Fig. 3e). If contamination has occurred, transfection should not be performed, as contaminated detached cells must not be injected into mice. This protocol was successfully used for transfection of DNA constructs facilitating integration into the parasite genome by single crossover homologous recombina- tion (Figs. 5, 6; Table 2) to introduce DNA constructs for cytoplasmic GFP expression and expression of a lucif- erase reporter gene in addition to a previously introduced cytoplasmic mCherry expression cassette [18]. Moreover, DNA constructs facilitating integration into the parasite genome by double crossover homologous recombina- tion, were also successfully transfected, for example for the generation of knock-out and complemented parasites (Figs. 5, 6; Table  2). DNA constructs were successfully transfected into different parasite backgrounds, such as wild type parasites, selection-marker-free cytoplas- mic mCherry expressing parasites [20] and knock-out parasites. Genotyping of transfected parasites Seeding and infections were performed according to the present protocol, collected detached cells were counted using a fluorescent microscope at 65 hpi and merozoite number was determined using a luminescence based assay, n = 3. Error bars denote SD. b Determination of luminescence of detached cells obtained over time, using sporozoites from day 16 to day 30 post-blood feed. Detached cells of ­PbmCherryHsp70-NLuc parasites were obtained and collected according to the present protocol. Aside from quantification of detached cell numbers by fluorescence, luminescence was used to estimate the number of detached cells used for transfection from day 16 onwards, following mosquito feeds. Luminescence correlates with number of detached cells contained, and suggests that at days 16–23, the number of viable merozoites remains relatively constant, while a significant decrease in luminescence is identified from day 24 onwards (denoting less detached cells and less merozoites). Values shown are the result of three independent experiments repeated in triplicate. Error bars denote SD. c ­PbmCherryHsp70-NLuc detached cells were collected and disrupted to release merozoites. The total number of merozoites of a single detached cell was counted, and a limiting dilution was performed to achieve one merozoite, which was lysed and its luminescence measured. The radiance calculated was 9.9 × 104 p s−1 cm−2. Performing a limiting dilution from 1 to ­106 merozoites we defined that a positive, significant ­(R2 = 0.96) correlation exists between merozoite number and the ­log10 of the relative light units of luminescence. This in turn allowed us to estimate the total number of merozoites in single individual detached cells and of the complete population of detached cells used for transfection. Values shown are the result of ten separate limiting dilutions from three independent experiments (Fig. 4c, d). Furthermore, it is also important to micro- scopically confirm that cells have not been contaminated during the process of infection with sporozoites, which when most of the detached cells contained viable and motile merozoites (Fig. 4a, b; Additional file  1: Movie S1) and only few abnormal detached cells were present Kaiser et al. Malar J (2017) 16:305 Page 7 of 11 Fig. 4  Representative microscopic images of detached cells. a Bright field and a′ fluorescence merged image of a normal detached cell, b and b′ of a ruptured detached cell and c, d examples of detached cells with abnormal morphology, c, c′ of merofusosomes [25] and d of absent merozoite formation. Genotyping of transfected parasites In conclusion, all techniques used to transfect blood stage parasites can also be used to manipulate liver stage-derived merozoites. Transfection of liver stage-derived detached cells and merosomes should be successful for all genetic modifica- tions that can be achieved by single or double crossover homologous recombination, such as expression of pro- teins fused with fluorescent, luminescent or other mark- ers and interference with protein function by knock-out, Discussionh The protocol described here allows the generation of transgenic P. berghei parasites. If infected mosquitoes are routinely accessible, the protocol offers a flexible Page 8 of 11 Kaiser et al. Malar J (2017) 16:305 Fig. 5  Success rate of detached cell transfection of Plasmodium berghei parasites. Detached cells and merosomes of wild type or ­PbmCherryHsp70 parasites were transfected according to the present protocol. a Illustration of the total success rate of detached cell transfection independent of parasites used and constructs transfected. b Success rate of detached cell transfection using DNA constructs allowing integration via either single or c double crossover homologous recombination. d Example of successfully transfected ­PbmCherryHsp70 parasites with a construct allowing expression of cytoplasmic GFP. Scale bar is 10 µm Fig. 5  Success rate of detached cell transfection of Plasmodium berghei parasites. Detached cells and merosomes of wild type or ­PbmCherryHsp70 parasites were transfected according to the present protocol. a Illustration of the total success rate of detached cell transfection independent of parasites used and constructs transfected. b Success rate of detached cell transfection using DNA constructs allowing integration via either single or c double crossover homologous recombination. d Example of successfully transfected ­PbmCherryHsp70 parasites with a construct allowing expression of cytoplasmic GFP. Scale bar is 10 µm Fig. 5  Success rate of detached cell transfection of Plasmodium berghei parasites. Detached cells and merosomes of wild type or ­PbmCherryHsp70 parasites were transfected according to the present protocol. a Illustration of the total success rate of detached cell transfection independent of parasites used and constructs transfected. b Success rate of detached cell transfection using DNA constructs allowing integration via either single or c double crossover homologous recombination. d Example of successfully transfected ­PbmCherryHsp70 parasites with a construct allowing expression of cytoplasmic GFP. Scale bar is 10 µm Table 2  shows the individual detached cell transfections performed that  are summarized in  Fig. Discussionh A 990 bp PCR product is generated only, if the construct has integrated at the correct site of the parasite genome. The PCR results show integration of the hdhfr/ yfcu cassette into the PBANKA_1145300 gene locus. Transfected parasites are present in a mixed population of parasites having a PBANKA_1145300 KO genotype and PBANKA_1145300 WT genotype b WT genome KO genome QCR2 QCR1 QCR2 GW2 650bp 990bp hdhfr/yfcu gDNA Primers PbWT PbKO QCR2 + QCR1 PbWT PbKO QCR2 + GW2 PCR: WT PCR: Integration 650bp 990bp Double crossover integration Fig. 6  Integration PCR of parasite lines generated by detached cell transfection using a single or b double crossover integration. a P. berghei parasites were transfected with pL0017-NLuc [18], a construct that allows integration by single crossover into the P. berghei c-ssu-rRNA or d-ssu-rRNA b b Fig. 6  Integration PCR of parasite lines generated by detached cell transfection using a single or b double crossover integration. a P. berghei parasites were transfected with pL0017-NLuc [18], a construct that allows integration by single crossover into the P. berghei c-ssu-rRNA or d-ssu-rRNA locus. PCR amplification was used to verify integration of the plasmid into the P. berghei genome. The schematic shows the possible annealing regions of the primer pairs for the different parasite genotypes that could result. If the construct did not integrate into the parasite genome the primer pairs 1 + 4 for the c-ssu-rRNA or 2 + 4 for the d-ssu-rRNA locus generated a 3 kb PCR product. If the construct was successfully integrated, the primer pairs are too far apart (>14 kb) to generate a PCR product. Primer pairs 1 + 3 and 2 + 3 are designed to confirm integration, a PCR product of 3 kb is generated only if the construct has integrated. PCR results of the PbNLuc parasites show integration into the c-ssu-rRNA locus, as the band for primer pair 1 + 4 is absent and the primer pair 1 + 3 gives a 3 kb band, there is no integration into the d-ssu-rRNA locus, which shows the same PCR products as for the WT parasites. b P. berghei parasites were transfected with PbGEM-336027, the construct facilitates integration of the hdhfr/ yfcu cassette via double crossover into the PBANKA_1145300 gene locus, resulting in a knock-out (KO). The primer pair QCR2 + QCR1 results in a 650 bp fragment, if parasites have a PBANKA_1145300 WT genotype. Discussionh 5a–c with  informa- tion about the transfected construct, the parasite line that was transfected, the type of integration, the success rate, how often the transfection was performed and at which day after the transfection the mice became positive Construct Parasite line Integration Success rate Number of  transfections Day mice became positive PbSBP1KO (P0B90-GFP) [19] mCherryHsp70 [20] Double crossover 50% (2) 2 7 PbSBP1KO (P0B90-GFP) [19] mCherryHsp70FLucef1a [21] Double crossover 100% (1) 2 6 PbSBP1KO (P0B90-GFP) [19] WT Double crossover 100% (1) 1 7 PfSBP1 (PL0017-mCherry) [19] PbSBP1KO [19] Single crossover 100% (1) 1 8 PbGEM-336027 mCherryHsp70 [20] Double crossover 100% (1) 1 21 PbNLuc (PL0017) [18] WT Single crossover 100% (1) 1 5 PbNLuc (PL0017) [18] mCherryHsp70 [20] Single crossover 100% (2) 2 4 PbNLuc (PL0017) [18] mCherryHsp70FLucef1a [21] Single crossover 66.6% (3) 3 4–5 PL0017 GFP [22] mCherryHsp70 [20] Single crossover 100% (8) 8 5–12 ble 2  shows the individual detached cell transfections performed that  are summarized in  Fig. 5 n about the transfected construct, the parasite line that was transfected, the type of integration w often the transfection was performed and at which day after the transfection the mice became po Table 2  shows the individual detached cell transfections performed that  are summarized in  Fig. 5a–c with  informa- tion about the transfected construct, the parasite line that was transfected, the type of integration, the success rate, how often the transfection was performed and at which day after the transfection the mice became positive conditional knock-out, modification, over-expression or replacement. In addition, this protocol should also be suc- cessful when introducing secondary genetic modifications such as complementation of previously knocked-out genes, additional marker fused proteins and double knock-outs. conditional knock-out, modification, over-expression or replacement. In addition, this protocol should also be suc- cessful when introducing secondary genetic modifications such as complementation of previously knocked-out genes, additional marker fused proteins and double knock-outs. A major advantage of the protocol is that liver stage- derived merozoites can be collected and transfected in less than 20  min, which is time saving and presumably beneficial for parasite viability. This is mainly because mature detached cells/merosomes can be directly conditional knock-out, modification, over-expression or replacement. In addition, this protocol should also be suc- cessful when introducing secondary genetic modifications such as complementation of previously knocked-out genes, additional marker fused proteins and double knock-outs. Page 9 of 11 Kaiser et al. Discussionh Malar J (2017) 16:305 3.0 kb c-ssu-rRNA locus 1+4 1+3 2+4 2+3 PbNLuc 1+4 1+3 2+4 2+3 PbWT WT c-ssu-rrna 1 4 Integration pL0017 NLuc 1 3 4 d-ssu-rRNA locus Integration pL0017 NLuc 2 3 4 WT d-ssu-rrna 2 4 a b Single crossover integration WT genome KO genome QCR2 QCR1 QCR2 GW2 650bp 990bp hdhfr/yfcu gDNA Primers PbWT PbKO QCR2 + QCR1 PbWT PbKO QCR2 + GW2 PCR: WT PCR: Integration 650bp 990bp Double crossover integration Fig. 6  Integration PCR of parasite lines generated by detached cell transfection using a single or b double crossover integration. a P. berghei parasites were transfected with pL0017-NLuc [18], a construct that allows integration by single crossover into the P. berghei c-ssu-rRNA or d-ssu-rRNA locus. PCR amplification was used to verify integration of the plasmid into the P. berghei genome. The schematic shows the possible annealing regions of the primer pairs for the different parasite genotypes that could result. If the construct did not integrate into the parasite genome the primer pairs 1 + 4 for the c-ssu-rRNA or 2 + 4 for the d-ssu-rRNA locus generated a 3 kb PCR product. If the construct was successfully integrated, the primer pairs are too far apart (>14 kb) to generate a PCR product. Primer pairs 1 + 3 and 2 + 3 are designed to confirm integration, a PCR product of 3 kb is generated only if the construct has integrated. PCR results of the PbNLuc parasites show integration into the c-ssu-rRNA locus, as the band for primer pair 1 + 4 is absent and the primer pair 1 + 3 gives a 3 kb band, there is no integration into the d-ssu-rRNA locus, which shows the same PCR products as for the WT parasites. b P. berghei parasites were transfected with PbGEM-336027, the construct facilitates integration of the hdhfr/ yfcu cassette via double crossover into the PBANKA_1145300 gene locus, resulting in a knock-out (KO). The primer pair QCR2 + QCR1 results in a 650 bp fragment, if parasites have a PBANKA_1145300 WT genotype. The primer pair QCR2 + GW2 is designed to show integration. A 990 bp PCR product is generated only, if the construct has integrated at the correct site of the parasite genome. Discussionh The PCR results show integration of the hdhfr/ f h PB l f d d l f h PB 3.0 kb c-ssu-rRNA locus 1+4 1+3 2+4 2+3 PbNLuc 1+4 1+3 2+4 2+3 PbWT WT c-ssu-rrna 1 4 Integration pL0017 NLuc 1 3 4 d-ssu-rRNA locus Integration pL0017 NLuc 2 3 4 WT d-ssu-rrna 2 4 a Single crossover integration Single crossover integration a d-ssu-rRNA locus 3.0 kb 1+4 1+3 2+4 2+3 PbNLuc 1+4 1+3 2+4 2+3 PbWT b WT genome KO genome QCR2 QCR1 QCR2 GW2 650bp 990bp hdhfr/yfcu gDNA Primers PbWT PbKO QCR2 + QCR1 PbWT PbKO QCR2 + GW2 PCR: WT PCR: Integration 650bp 990bp Double crossover integration Fig. 6  Integration PCR of parasite lines generated by detached cell transfection using a single or b double crossover integration. a P. berghei parasites were transfected with pL0017-NLuc [18], a construct that allows integration by single crossover into the P. berghei c-ssu-rRNA or d-ssu-rRNA locus. PCR amplification was used to verify integration of the plasmid into the P. berghei genome. The schematic shows the possible annealing regions of the primer pairs for the different parasite genotypes that could result. If the construct did not integrate into the parasite genome the primer pairs 1 + 4 for the c-ssu-rRNA or 2 + 4 for the d-ssu-rRNA locus generated a 3 kb PCR product. If the construct was successfully integrated, the primer pairs are too far apart (>14 kb) to generate a PCR product. Primer pairs 1 + 3 and 2 + 3 are designed to confirm integration, a PCR product of 3 kb is generated only if the construct has integrated. PCR results of the PbNLuc parasites show integration into the c-ssu-rRNA locus, as the band for primer pair 1 + 4 is absent and the primer pair 1 + 3 gives a 3 kb band, there is no integration into the d-ssu-rRNA locus, which shows the same PCR products as for the WT parasites. b P. berghei parasites were transfected with PbGEM-336027, the construct facilitates integration of the hdhfr/ yfcu cassette via double crossover into the PBANKA_1145300 gene locus, resulting in a knock-out (KO). The primer pair QCR2 + QCR1 results in a 650 bp fragment, if parasites have a PBANKA_1145300 WT genotype. The primer pair QCR2 + GW2 is designed to show integration. Discussionh The primer pair QCR2 + GW2 is designed to show integration. A 990 bp PCR product is generated only, if the construct has integrated at the correct site of the parasite genome. The PCR results show integration of the hdhfr/ yfcu cassette into the PBANKA_1145300 gene locus. Transfected parasites are present in a mixed population of parasites having a PBANKA_1145300 KO genotype and PBANKA_1145300 WT genotype Kaiser et al. Malar J (2017) 16:305 Page 10 of 11 Page 10 of 11 collected from the cell culture supernatant and need no further purification by density gradient centrifugation as is needed for the purification of blood stage schizonts.h about 500–1000 detached cells, which is sufficient for one transfection. Accordingly, it is possible to perform about 27 transfections with the number of infected mos- quitoes that result from sacrificing a single feed mouse. Conversely, with the standard protocol used for transfec- tion of blood stage-derived schizonts [8], which in theory requires 1 mouse per 4–5 transfections, performance of the same amount of transfections would require five mice.h pi g The standard protocol for blood stage transfection published by Janse et al. recommends using a minimum of ­106 schizonts, corresponding to about 8–18  ×  106 single merozoites per transfection [8]. Using the here- described protocol for transfection of liver stage-derived merozoites, about 4.5–8.5  ×  106 merozoites are used per transfection, depending on the number of detached cells obtained. An important observation was that effi- cient parasite development leading to the formation of detached cells is highly dependent on the age of parasites in infected mosquitoes. Therefore, it is recommended that mosquitoes are used between days 16 and 24 after blood feeding. Being able to use infected mosquitoes for 9 consecutive days to obtain detached cells, parasite transfections can be performed more flexibly in terms of timing and can be planned more robustly than is pos- sible when performing blood stage transfections, which greatly depend on the parasitaemia of infected mice to be used for blood stage cultures. The liver stage develop- ment in vitro is independent of host circadian rhythms and accordingly, infections to obtain liver stage derived merozoites can be performed at any time of the day, offering flexible transfection conditions. i Therefore, the protocol presented here is highly ben- eficial when intending to perform transfections in large scale. Additional file Additional file 1: Movie S1. Motile merozoites within detached cells. Detached cells containing marker-free ­PbmCherryHsp70 parasites (red) were harvested at 65 hpi and analysed using a fluorescence microscope. Images were acquired every 5 s. Scale bar is 10 μm. Additional file 1: Movie S1. Motile merozoites within detached cells. Detached cells containing marker-free ­PbmCherryHsp70 parasites (red) were harvested at 65 hpi and analysed using a fluorescence microscope. Images were acquired every 5 s. Scale bar is 10 μm. Conclusions So far, only one stage of Plasmodium berghei parasites, blood stage schizonts, could be transfected. Here a trans- fection protocol is described using liver stage-derived merozoites of P. berghei, which can be generated in vitro and in contrast to transfection of blood stage schizonts do not require sophisticated purification steps. The protocol described here provides more flexibility in terms of when and how many transfections can be performed. Moreo- ver, transfection of liver stage-derived merozoites has the potential to result in a considerable reduction of mice used, if used by a lab that routinely has access to infected mosquitoes. During the optimization process of the protocol it was established that about 50,000 sporozoites are needed to obtain sufficient numbers of liver stage-derived merozo- ites per transfection. On average this sporozoite number can be obtained from three infected mosquitoes [18]. Normally, about 100–120 mosquitoes are fed on a sin- gle feed mouse. With a routine infection rate of about 70–80%, about 80 infected mosquitoes were obtained. Per transfection, three mosquitoes that on average har- bour about 50,000 sporozoites are dissected and used for the infection of HeLa cells. This infection results in Discussionh Transfection of liver stage-derived merozoites and schizonts has the potential to reduce the mouse usage by about fivefold in laboratories that have routine access to infected mosquitoes compared to the current proto- col based on blood stage transfection. Since the in vitro liver stage development of Plasmodium takes place in cell culture plates, it is very suitable for scaling up the experimental design. Potential critical points could be that sporozoites, following isolation, should be used suf- ficiently quickly to ensure efficient host cell infection. Later in the experimental procedure, the time between detached cell purification and transfection is critical for parasite survival. Therefore, it is proposed that host cell infection with sporozoites and transfection of liver stage-derived parasites should be performed in sets, with a maximum of five transfections per set. Nevertheless, multiple infections could be prepared at time intervals if many transfections are planned. fl To obtain sporozoites needed for liver stage cultiva- tion, a total of two mice is required: a pre-infection mouse in which infection is initially established from a blood stage stabilate of the parent P. berghei line, and a phenylhydrazine-treated recipient mouse (referred to as ‘feed mouse’), which will be infected with the donor’s blood, and thereafter used to feed the mosquitoes. Since only low blood volumes are passaged from the pre-infec- tion mouse into the feed mouse, several feed mice can be infected from a single pre-infection mouse, to increase the number of mosquitoes that can be infected and thus the number of sporozoites that can be generated. In fact, in laboratories that routinely perform mouse infections with P. berghei, an additional pre-infection mouse is not required further reducing the number of experimental animals needed for the transfection. References Adapta‑ tion of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes. Proc Natl Acad Sci USA. 2013;110:531–6. 7. Moon RW, Hall J, Rangkuti F, Ho YS, Almond N, Mitchell GH, et al. Adapta‑ tion of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes. Proc Natl Acad Sci USA. 2013;110:531–6. Competing interests 18. De Niz M, Stanway RR, Wacker R, Keller D, Heussler VT. An ultrasensitive NanoLuc-based luminescence system for monitoring Plasmodium berghei throughout its life cycle. Malar J. 2016;15:232. The authors declare that they have no competing interests. Abbreviations DNA: deoxyribonucleic acid; GFP: green-fluorescent protein; hpi: hours post infection; PABA: para-aminobenzoic acid; PM: plasma membrane; PV: parasito‑ phorous vacuole; PBS: phosphate-buffered saline. Page 11 of 11 Kaiser et al. Malar J (2017) 16:305 Kaiser et al. Malar J (2017) 16:305 Author details 1 Institute of Cell Biology, University of Bern, Bern, Switzerland. 2 Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzer‑ land. 3 Present Address: Wellcome Centre for Molecular Parasitology, University of Glasgow, Glasgow, UK. 4 Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany. 13. Lacroix C, Giovannini D, Combe A, Bargieri DY, Späth S, Panchal D, et al. FLP/FRT-mediated conditional mutagenesis in pre-erythrocytic stages of Plasmodium berghei. Nat Protoc. 2011;6:1412–28. 14. Philip N, Waters AP. Conditional degradation of Plasmodium calcineurin reveals functions in parasite colonization of both host and vector. Cell Host Microbe. 2015;18:122–31. Publisher’s Note g y 19. De Niz M, Ullrich A, Heiber A, Soares AB, Pick C, Lyck R, et al. Virulence is conserved between rodent and human malaria parasites. Nat Commun. 2016;7:1–12. 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. 20. Burda P-C, Roelli MA, Schaffner M, Khan SM, Janse CJ, Heussler VT. A Plasmodium phospholipase is involved in disruption of the liver stage parasitophorous vacuole membrane. PLoS Pathog. 2015;11:e1004760. Received: 23 February 2017 Accepted: 20 July 2017 Received: 23 February 2017 Accepted: 20 July 2017 Received: 23 February 2017 Accepted: 20 July 2017 21. Prado M, Eickel N, De Niz M, Heitmann A, Agop-Nersesian C, Wacker R, et al. Long-term live imaging reveals cytosolic immune responses of host hepatocytes against Plasmodium infection and parasite escape mecha‑ nisms. Autophagy. 2015;11:1561–79. Authors’ contributions 10. Ménard R. Malaria methods and protocols. In: Ménard R, editor. Life Sci. Totowa: Humana Press; 2013. GK, RRS, MDN, PCB, and VTH conceived and designed the experiments. GK, MDN, PCB, and LN performed experiments. GK and MDN analysed the data. GK, MDN and VTH drafted the manuscript. All authors read and approved the final manuscript. 11. Pfander C, Anar B, Schwach F, Otto TD, Brochet M, Volkmann K, et al. A scalable pipeline for highly effective genetic modification of a malaria parasite. Nat Methods. 2011;8:1078–82. 12. Stanway RR, Witt T, Zobiak B, Aepfelbacher M, Heussler VT. GFP-targeting allows visualization of the apicoplast throughout the life cycle of live malaria parasites. Biol Cell. 2009;101:415–30. References 22. Franke-Fayard B, Trueman H, Ramesar J, Mendoza J, van der Keur M, van der Linden R, et al. A Plasmodium berghei reference line that constitutively expresses GFP at a high level throughout the complete life cycle. Mol Biochem Parasitol. 2004;137:23–33. 1. Janse CJ, Franke-Fayard B, Mair GR, Ramesar J, Thiel C, Engelmann S, et al. High efficiency transfection of Plasmodium berghei facilitates novel selec‑ tion procedures. Mol Biochem Parasitol. 2006;145:60–70. 1. Janse CJ, Franke-Fayard B, Mair GR, Ramesar J, Thiel C, Engelmann S, et al. High efficiency transfection of Plasmodium berghei facilitates novel selec‑ tion procedures. Mol Biochem Parasitol. 2006;145:60–70. 1. Janse CJ, Franke-Fayard B, Mair GR, Ramesar J, Thiel C, Engelmann S, et al. High efficiency transfection of Plasmodium berghei facilitates novel selec‑ tion procedures. Mol Biochem Parasitol. 2006;145:60–70. 2. Crabb BS, Rug M, Gilberger T-W, Thompson JK, Triglia T, Maier AG, et al. Transfection of the human malaria parasite Plasmodium falciparum. Meth‑ ods Mol Biol. 2004;270:263–76. 2. Crabb BS, Rug M, Gilberger T-W, Thompson JK, Triglia T, Maier AG, et al. Transfection of the human malaria parasite Plasmodium falciparum. Meth‑ ods Mol Biol. 2004;270:263–76. 23. Menard R, Janse C. Gene targeting in malaria parasites. Methods. 1997;13:148–57. 24. Van Spaendonk RML, Ramesar J, Van Wigcheren A, Eling W, Beetsma AL, Van Gemert GJ, et al. Functional equivalence of structurally distinct ribosomes in the malaria parasite, Plasmodium berghei. J Biol Chem. 2001;276:22638–47. 3. Mota MM, Thathy V, Nussenzweig RS, Nussenzweig V. Gene targeting in the rodent malaria parasite Plasmodium yoelii. Mol Biochem Parasitol. 2001;113:271–8. 4. Hall N. A comprehensive survey of the plasmodium life cycle by genomic, transcriptomic, and proteomic analyses. Science. 2005;307:82–6. 4. Hall N. A comprehensive survey of the plasmodium life cycle by genomic, transcriptomic, and proteomic analyses. Science. 2005;307:82–6. 25. Eickel N, Kaiser G, Prado M, Burda P-C, Roelli M, Stanway RR, et al. Features of autophagic cell death in Plasmodium liver-stage parasites. Autophagy. 2013;9:568–80. 5. Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW, et al. Genome sequence of the human malaria parasite Plasmodium falcipa- rum. Nature. 2002;419:498–511. 6. Carlton JM, Angiuoli SV, Suh BB, Kooij TW, Pertea M, Silva JC, et al. Genome sequence and comparative analysis of the model rodent malaria parasite Plasmodium yoelii yoelii. Nature. 2002;419:512–9. y y 7. Moon RW, Hall J, Rangkuti F, Ho YS, Almond N, Mitchell GH, et al. Acknowledgements 15. Sturm A, Heussler V. Live and let die: manipulation of host hepatocytes by exoerythrocytic Plasmodium parasites. Med Microbiol Immunol. 2007;196:127–33. We thank Chris J. Janse and Shahid M Khan for providing the selection-marker- free mCherry and the mCherryLuc expressing parasites. We thank Tobias Spielmann for providing the PfSBP1 and PbSBP1KO constructs. Images were acquired on equipment supported by the Microscopy Imaging Centre of the University of Bern. Financial support came from the Swiss National Founda‑ tion (310030_140691/1) (VTH) and the EVIMalaR EU network (FP7/2007-2013) (MDN and VTH). 16. Sturm A, Amino R, van de Sand C, Regen T, Retzlaff S, Rennenberg A, et al. Manipulation of host hepatocytes by the malaria parasite for delivery into liver sinusoids. Science. 2006;313:1287–90. 17. Stanway R, Graewe S, Rennenberg A, Helm S, Heussler VT. Highly efficient subcloning of rodent malaria parasites by injection of single merosomes or detached cells. Nat Protoc. 2009;4:1433–9. Competing interests Submit your next manuscript to BioMed Central and we will help you at every step: 8. Janse CJ, Ramesar J, Waters AP. High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei. Nat Protoc. 2006;1:346–56. • 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 • 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 • We accept pre-submission inquiries 9. Pfander C, Anar B, Brochet M, Rayner JC, Billker O. Recombination- mediated genetic engineering of Plasmodium berghei DNA. In: Ménard R, editor. Malaria. Methods in molecular biology (Methods and protocols), vol. 923. Totowa, NJ: Humana Press; 2012. p. 127–38.
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Telemedicine supported by Augmented Reality: an interactive guide for untrained people in performing an ECG test
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* Correspondence: pabifulc@unina.it 1Department of Electrical Engineering and Information Technology, University of Naples “Federico II”, Naples, Italy Full list of author information is available at the end of the article RESEARCH Open Access Telemedicine supported by Augmented Reality: an interactive guide for untrained people in performing an ECG test Fabio Narducci2, Raffaele Vertucci3, Pasquale Ambruosi1, Mario Cesarelli1 and Maria Romano1 * Correspondence: pabifulc@unina.it 1Department of Electrical Engineering and Information Technology, University of Naples “Federico II”, Naples, Italy Full list of author information is available at the end of the article Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 http://www.biomedical-engineering-online.com/content/13/1/153 Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 http://www.biomedical-engineering-online.com/content/13/1/153 © 2014 Bifulco et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background Telemedicine refers to the use of telecommunications and information technologies for the delivery of medical services where is needed [1-3]. For many applications, the cor- rect usage of medical device at the point of need is essential to provide an appropriate service, but there are some practical situations that may require untrained or inexpert people to interact with medical devices and patients. Some examples are telemedicine services on transportation (e.g. aircrafts [4-6], boats [7,8], trains, etc.), application dur- ing military actions [9], on islands or remote areas [10,11], some emergency applica- tions [12,13], but also home care telemedicine supported by family members [14-16], elderly care [17,18], operators training and so on. In these cases, untrained or improvised (but necessary) actors can, involuntarily, use medical instruments in an inappropriate manner and/or make improper connection between the patient and the medical device seriously invalidating the telemedicine service. In these scenarios, tailored applications of Augmented Reality (AR) can offer a valid support by guiding non-trained people to a correct usage of medical devices at the point of need. Augmented reality basically consists of a live view of the real-world in which some elements of the scene are “augmented” (enriched, enhanced) by computer- generated information such as graphics, texts and sounds. The application domains for AR are numerous and extend in different fields such as training and support, design, medicine, entertainment and cultural heritage [19-21]. Recently, there is a growing interest about AR in medicine. The main applications of AR are in the field of surgery, rehabilitation and teaching/training. Interventional medi- cine, surgery [22,23], laparoscopy and other procedures (e.g. needle biopsy [24]) can be assisted by integrating preoperative and intraoperative anatomic and functional data improving the visual perception of the surgeon [20,25-27]. Obviously, surgery AR appli- cations require very accurate registration and camera calibration [28]. AR and virtual reality have long since found use in rehabilitation and particularly in neurorehabilita- tion, by guiding and aiding the patient to perform therapy [29,30]. Teaching and train- ing of students or physicians can take great advantage by AR, which can be further enriched with direct haptic and auditory feedback [31-33]. Superimposition in real time of images from US, CT or MR scans can also help in learning [20]. Abstract Background: In many telemedicine applications, the correct use of medical device at the point of need is essential to provide an appropriate service. Some applications may require untrained people to interact with medical devices and patients: care delivery in transportation, military actions, home care and telemedicine training. Appropriate operation of medical device and correct connection with patient’s body are crucial. In these scenarios, tailored applications of Augmented Reality can offer a valid support by guiding untrained people at the point of need. This study aims to explore the feasibility of using Augmented Reality in telemedicine applications, by facilitating an acceptable use of biomedical equipment by any unskilled person. In particular, a prototype system was built in order to estimate how untrained users, with limited or no knowledge, can effectively interact with an ECG device and properly placing ECG electrodes on patient’s chest. Methods: An Augmented Reality application was built to support untrained users in performing an ECG test. Simple markers attached to the ECG device and onto patient’s thorax allow camera calibration. Once objects and their pose in the space are recognized, the video of the current scene is enriched, in real-time, with additional pointers, text boxes and audio that help the untrained operator to perform the appropriate sequence of operations. All the buttons, switches, ports of the ECG device together with the location of precordial leads were coded and indicated. Some user’s voice commands were also included to improve usability. Results: Ten untrained volunteers, supported by the augmented reality, were able to carry out a complete ECG test first on a mannequin and then on a real patient in a reasonable time (about 8 minutes on average). Average positioning errors of precordial electrodes resulted less than 3 mm for the mannequin and less than 7 mm for the real patient. These preliminary findings suggest the effectiveness of the developed application and the validity of clinical ECG recordings. Conclusion: This application can be adapted to support the use of other medical equipment as well as other telemedicine tasks and it could be performed with a Tablet or a Smartphone. Keywords: Augmented Reality, Untrained user, ECG device operation, Electrode placement Keywords: Augmented Reality, Untrained user, ECG device operation, Electrode placement Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 http://www.biomedical-engineering-online.com/content/13/1/153 Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 http://www.biomedical-engineering-online.com/content/13/1/153 Page 2 of 16 Page 2 of 16 Background There are only very few examples of AR applications in telemedicine, among these there are systems of virtual reality supporting distance teaching of minimally invasive surgery and systems for interactive telemedicine in the operating theatre [34,35]; some low-cost peripherals to support telehealth, visualization, education and collaborative systems [36]; some applications of distance training for the restoration of motor func- tion, supported by virtual reality [37]. The capability of AR to provide live support to users in operating on instrumenta- tions is also of interest for this study. AR can support and guide workers in operating, actuate, disassembling and maintaining complex devices or systems. As example, it was proposed a mixed reality environment aimed to improve the effectiveness of servicing and repair procedures in mission critical systems, while reducing the time required for the intervention. Also technicians with no previous experience on specific, complex de- vices were able to perform an assigned maintenance task when supported by the AR application. In maintenance activities there are well-defined sequences of procedures to Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 http://www.biomedical-engineering-online.com/content/13/1/153 Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 http://www.biomedical-engineering-online.com/content/13/1/153 Page 3 of 16 Page 3 of 16 be done in a relatively static environment. These features allow a defined design space, supporting a wide variety of systems and technologies. It was proved effective in sup- porting workers for maintenance activities [38]. As feasibility study for new possible telemedicine services, an AR application was built to support untrained users in performing an ECG-test. In particular, the objective of this study was to analytical assess the benefits of exploiting augmented reality princi- ples in order to estimate how untrained users with limited or no knowledge can prop- erly interact with an ECG device and properly placing ECG electrodes on patient’s chest. The proposed AR application was assessed in terms of efficacy and clinical acceptability. In many clinical activities, 12-lead electrocardiogram is an essential medical investiga- tion. On the other hand, it should carefully carried out. Misplaced ECG electrodes can cause changes in ECG recordings, which could have an impact on clinical decisions [39]. Incorrect electrode cable connections, reversal of electrodes, inadequate place- ment of the electrodes are common source of error (changes the true ECG morph- ology) and can conceal or simulate different pathology such as, myocardial ischemia or infarction, arrhythmias, ventricular hypertrophy [40-43]. Overall system description The developed AR application guides the user to perform a predefined sequence of simple tasks on the ECG device (e.g. connect cables, press buttons, check indicator lights, etc.) and on the patient (e.g. connect electrodes in a specific position, etc.), driv- ing user’s attention from time to time to the relevant item. A flow-chart of activities was created to describe all possible sequences of simple tasks necessary to carry out an electrocardiographic test using that particular ECG de- vice. The developed AR application drives the user to perform each predefined task by presenting text, graphics and audio messages to him/her (see Figure 1). Specifically designed sets of markers were attached to the ECG device and to the pa- tient: this allows the AR engine to evaluate on real-time the 3D pose of these objects with respect to the user. This permits to indicate or highlights a specific point or a part of the objects (e.g. a button to be pressed) by superimposing opportune signs to the current scene. The op- erators worn a Head Mounted Display (HMD) coupled to a webcam in order to see the virtual contents that augment the current scene (future developments will involve the use of a single Tablet or Smartphone). Figure 1 presents a general scheme of the pro- posed AR application. Background It is also worth mentioning that untrained service providers are one of the key barriers to implementation of tele- medicine services. The augmented reality engine The Augmented Reality engine is in charge of user’s head tracking and scene augmen- tation/rendering. The tracking system of the developed application is based on the ARToolKit, an open source AR library [44]. The ARToolKit video tracking libraries compute the current camera position and orientation relative to physical markers in Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 http://www.biomedical-engineering-online.com/content/13/1/153 Page 4 of 16 Figure 1 System architecture. Scheme of the operations of the developed AR application to guide an untrained user to correctly perform an ECG test. Figure 1 System architecture. Scheme of the operations of the developed AR application to guide an untrained user to correctly perform an ECG test. real time. Very simple markers (i.e. black squares containing specific identification sym- bols) are used. Markers are rigidly attached to the objects of interest to estimate user’s perspective, i.e. the relative 3D pose of these objects with respect to the camera (cam- era calibration). For each captured frame, the AR engine performs a defined sequence of steps. First of all, it looks for known markers in the framed scene. A thresholding op- eration turns the coloured image into a binary one in order to make the detection of the markers easier and faster. Once detected all visible markers, the engine computes the user’s perspective view (i.e. user’s relative 3D positioning with respect to the marked objects). At this stage, the AR engine loads pre-recorded information about each marked object (e.g. its geometry, the internal spatial coordinates of its parts, etc.) and then is possible to superimpose virtual objects (e.g. labels, arrows, spots etc) to the real scene. The “augmented” scene is finally displayed to the user by means of proper dis- plays, such as the HMD. In normal environmental conditions (i.e. acceptable lighting of the scene, absence of strong reflections or excessive shadows, etc.), a single marker could be enough to achieve a reliable augmentation of the scene. However, the marker should be always in the central field of view of the camera and should be entirely and clearly visible to re- duce the risk of detection miss. In several contexts, arranging a marker in the middle of operational field could simply be unfeasible or it could even interfere with the opera- tions. Furthermore, object characterized by uneven surfaces or significant rotations of displacements can hinder a continuous and stable detection of the user’s perspective. The augmented reality engine Previous studies [45] have shown that the use of multiple markers (or a constellation of markers) for each object of interest can address most of the problems arising in practical situations, thus delivering an inherently more robust and more accurate track- ing of object, even by using small markers. A useful advantage of the multi marker ap- proach is that it is easily scalable. In fact, by adding other markers it is possible to easily widen the tracking volume as desired. The AR application includes a calibration function meant to measure and compensate the geometrical distortion generated by Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 http://www.biomedical-engineering-online.com/content/13/1/153 Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 http://www.biomedical-engineering-online.com/content/13/1/153 Page 5 of 16 Page 5 of 16 the lens of the camera. In addition, a specific procedure allows a manual fine tuning and coregistration between the camera and its virtual counterpart in charge of rendering the re- quired graphics. Each of the six degrees of freedom, including the focal length of the camera and also the threshold used for marker identification, can be precisely adjusted. This task is performed only once, generally, on the very first system start-up. An ad-hoc graphic user interface supports this fine tuning calibration procedure. This procedure can be recalled when the equipment configuration is changed. The AR application memorizes and ex- changes information using XML (eXtensible Markup Language) files. The language is widely used and it allows extensibility, unambiguously and large compatibility. All informa- tion of the accurate location of any relevant element of the objects and of the working envir- onment is memorized in a database of XML files. More technically, an XML object file defines the object’s points of interest. These include Cartesian coordinates and properties of augmented aids, such as the location of textual information, audio messages, and the inter- action region on the object surface to be highlighted. The AR engine builds up the virtual scene by means of a DOM (Document Object Model) XML parser. To find the required data in the application database, a XML-based XPath query language is used. Combining the information from tracker and from XML database, the AR engine is therefore able to lo- cate user’s perspective view in the real world and to extract all the required augmented con- tents from the repository to properly guide him/her during the intervention. AR application development and set-up The AR application has to support an untrained user to perform an ECG-test on a pa- tient. Starting from the established medical guidelines and technical instructions of the device exploited, each ECG procedure was carefully analyzed and subdivided in very simple tasks (or steps), achieving the whole formalization of the all possible sequences of steps. Once we obtained the whole flow chart, it was coded as a deterministic finite automaton (DFA) that has a finite number of possible states and precise rules for step- ping form one state to another, producing unique runs of the automaton. Each proced- ure is an automaton run that starts at the root and ends in a leaf. A particular state represents a precise ECG-test step and the links with other steps define the order of the execution of the entire procedure. DFA results particularly suited to model either simple or complex procedures in an easy and comprehensive way. The DFA representa- tion was also translated into several XML files where each one represents a run of the automaton or, on the other hand, a possible procedure. The syntax of this file is very simple, a tag < step > defines an elementary task of any procedure. Once the parser reaches the end of the XML document, the procedure is completed. User is invited to execute each task by an audio message, while a correspondent text-box is presented on the user’s screen. Simultaneously, real scene is augmented by adding graphics (i.e. pointers, spots, etc.) to drive user’s attention on a particular point of the objects (e.g. the button to be pressed, etc.). Once accomplished the task, user sends a vocal command to the AR application to step to the next task. Only very few vocal commands are allowed such as: “Go Next” to pass to the next step; “Go Back” to return to the previous step; “Redo Instruction” to listen to once again the voice prompt of the current step. Basically, the ECG-test procedure includes: connection of the electrodes to the pa- tient (including the six precordial leads V1-V6), connection of the patient cable to the Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 http://www.biomedical-engineering-online.com/content/13/1/153 Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 http://www.biomedical-engineering-online.com/content/13/1/153 Page 6 of 16 ECG device, a sequence of operation on the ECG device leading to record 12-leads ECG on a strip chart. AR application development and set-up Each marker used to augment an object is a black square (of 4 cm per side) contain- ing a unique identification symbol. Markers can be easily printed on paper or card- board and have to be attached on the object (markers should be rigid and fixed with respect to the object to be augmented). To allow more reliable and stable functioning, a costellation of three markers was associated to the ECG device and a different set of three to the patient’s thorax. The multi-markers approach significantly improves the ac- curacy of the augmentation [46] compared to single marker approaches [47,48], thus reducing the matching error between the rendering of the augmenting point and its physical location in the real scene. The portable ECG device considered in this applica- tion was a Cardiette Microruler 12/1. The three markers were arranged in line and horizontally aligned, spaced from each other by 2 cm and attached to the top of the front part of the ECG device (see Figure 2a). The geometrical localization of the follow- ing parts of the ECG device was accurately measured with respect to central point of the markers costellation (located in the geometrical center of the middle marker) and then recorded in XML file: the main switch (placed on the left lateral side of the de- vice), the connector of the patient cable (placed on the right lateral side), all the but- tons (eight in total, placed on the main panel), all the LEDs (twelve in total, placed on the main panel), the accessible parts of the chart printer (placed on the main panel). For the augmentation of the patient’s thorax, the three markers were arranged on the tips of a T-shaped stucture showed in Figure 2b. The T-structure was made flat and rigid, its horizontal and vertical segment of the T-structure measured 10 cm (empiric- ally chosen according the mean size of adult’s thorax [49]). The T-structure was applied with stickers onto the patient’s thorax, taking care to align the vertical segment along the sternum and positioning the upper side of the lower marker (recognizable in figure by its C-like symbol) in correspondence of the xiphoid process (the lower end of the sternum), which can be easily recognizable by touching (see Figure 2b). The anatomical landmarks for the standard precordial electrode positions were fixed in accordance with the current ECG international standards [50]. AR application development and set-up The precordial electrode positions are: V1 and V2 at the fourth intercostals space to the right and left sternal border, re- spectively; V4 at the fifth left intercostal space in the mid-clavicular line; V3 midway between V2 and V4; and V5 and V6 at the horizontal level of V4 in the anterior and midaxillary lines, respectively [39,51]. Coordinates x, y and z were obtained for each of the precordial electrodes with respect to the reference system fixed to the T-shape marker set (see Figure 2c) and opportunely coded in the XML file. The x-, y- and z- axis correspond to the latero-lateral, cranial-caudal and dorsal-ventral patient’s anatom- ical axes, respectively. The user (see Figure 3a and 3b) worn an HMD (Silicon Micro Display ST1080) offer- ing a 1920x1080 pixels video resolution at 60Hz, 45 degrees diagonal field of view, 24 bit stereo audio and a weight of 180 grams. To provide a vision in augmented reality through the HMD, it was coupled with a Logitech HD Pro Webcam C910 that offers a very wide diagonal field of view of 83 degrees, 4.3 mm focal length and a maximum video resolution of 1920x1080 pixels. However, in order to get the best results in term of augmentation of the scene, we preferred to use a 640x480 pixels video resolution that allows to work at 30 FPS. Page 7 of 16 Page 7 of 16 Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 g g http://www.biomedical-engineering-online.com/content/13/1/153 Figure 2 (See legend on next page.) Figure 2 (See legend on next page.) Page 8 of 16 Page 8 of 16 Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 http://www.biomedical-engineering-online.com/content/13/1/153 (See figure on previous page.) Figure 2 Portable ECG with markers. The portable ECG device adopted for this study with attached on the correspondent three markers. The side of each marker measures 4 cm (a). The rigid T-shaped frame en- closing the three markers placed on the thorax of a mannequin. The upper side of the lowest marker is placed in correspond to the xiphoid process (b). Schematic representation of the reference frame adopted in the trials and the relative positions of the precordial electrodes V1, V2, V3, V4, V5 and V6 (c). Experiments setting To assess the feasibility of the AR application and its benefit, two separated experimental tests were designed: the first on a life-size mannequin and the second on a real patient. The experimental tests were carried out involving two groups of 10 people each (14 men and 6 women in total, mean age of 31.3 and median age of 30.5) with no medical expertise, no ex- perience of ECG test and who never used an ECG device. At each tester was asked to wear the HMD and, after familiarizing with the system for few minutes, the experimental trial Figure 3 Untrained user on a mannequin and on the portable ECG. Pictures representing an untrained user while placing precordial electrodes on the mannequin (a) and operating the ECG-device (b). Figure 3 Untrained user on a mannequin and on the portable ECG Pictures representing an untrained Figure 3 Untrained user on a mannequin and on the portable ECG. Pictures representing an untraine user while placing precordial electrodes on the mannequin (a) and operating the ECG-device (b). Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 http://www.biomedical-engineering-online.com/content/13/1/153 Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 http://www.biomedical-engineering-online.com/content/13/1/153 Page 9 of 16 Page 9 of 16 was started. The first group of 10 people carried out complete ECG recording sessions interacting with the ECG device and placing electrodes on the mannequin (see Figure 4a-d). The second test was performed on a volunteer, male adult acting as patient who lay supine on a table and breathed normally to resemble a practical case (see Figure 4a-b, 4e-f). Similarly, the second group of 10 untrained people was asked to perform complete ECG recordings only supported by the AR application. The ability to perform the required operations, the time required to complete the ECG recording, the positioning errors of the electrodes and the user’s judgment were collected for all the tester. In particular, the three spatial components of the distance between an ex- pected (true) location of an electrode and its actual placement were recorded as the error committed by the untrained user. This information was used to quantitatively assess the efficacy and the clinical acceptability of the developed AR application. Results and discussion The AR application was perceived as in- tuitive and easy to use, the opportunity to interact with vocal commands was par- ticularly appreciated. The execution time over both tests was on average of 8 minutes (including 3 minutes of ECG recording in the automatic mode) that can be considered acceptable for prac- tical purposes. Bar plots in Figure 5 show the averages and standard deviations of the x, y and z components of errors (computed on all testers) in placement of the precordial elec- trodes on the mannequin (Figure 5a) and patient’s chest (Figure 5b). Averages can be considered as an indication of the accuracy, while standard deviations as a measure of the precision (repeatability) achieved during the trials. In addition and in a more concise way, Table 1 reports errors measured as distance be- tween the expected and the occurred position for each of the precordial electrodes V1-V6. The average, standard deviation and maximum of these displacement errors are available for both tests (mannequin and real patient). For the first test, the average errors in electrode positioning on the mannequin re- sulted less than or equal to 3 mm, while the standard deviation less than 5 mm. Involvement of the real patient instead of the mannequin led to a slight increase of the errors committed. This can be reasonably due to thorax motion due to breathing or other little movements of the patient. Nevertheless, even in the test of the real pa- tient the mispositioning of the electrodes resulted less than 7 mm on average and reached a maximum of 16 mm on V6, these data support the effectiveness of the AR procedure and the clinical acceptability of the recorded ECG. As a matter of fact, elec- trode malposition exceeding 25 mm is associated with potentially significant ECG changes [39]. Taking into account this threshold value and observing the results achieved during both tests (Figure 5a-b and Table 1), the average errors in electrode positioning resulted reasonably acceptable and comparable with placement errors usu- ally made by technicians and nurses in an emergency care department [52]. This sup- ports the clinical validity of the acquired ECG waveforms by means of the developed AR system. It is worth noting that in both tests only for V5 and V6 significant variations of posi- tioning were registered on the z-axis. Results and discussion Each of the untrained tester was able to carry out in an appropriate manner the ECG-test with the only support of the AR application. All the testers reported that Figure 4 Examples of augmented scenes. Examples of augmented real scenes: the power button of the ECG device is surrounded by a light green rectangle, while audio and text invite the user to press it (a); a little red spot indicates the LED to be checked by the users (b); the location of the V3 precordial electrode on the mannequin is highlighted with a red circle (c); the location of the V6 precordial electrode is highlighted with a red circle (d) please note the inclination with respect to the plane of the markers; the location of the V3 precordial electrode on a real patient is highlighted with a red circle (e); placement of the V5 precordial electrode on the patient’s thorax, the location of V5 is highlighted with a red circle (f). Figure 4 Examples of augmented scenes. Examples of augmented real scenes: the power button of the ECG device is surrounded by a light green rectangle, while audio and text invite the user to press it (a); a little red spot indicates the LED to be checked by the users (b); the location of the V3 precordial electrode on the mannequin is highlighted with a red circle (c); the location of the V6 precordial electrode is highlighted with a red circle (d) please note the inclination with respect to the plane of the markers; the location of the V3 precordial electrode on a real patient is highlighted with a red circle (e); placement of the V5 precordial electrode on the patient’s thorax, the location of V5 is highlighted with a red circle (f). Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 http://www.biomedical-engineering-online.com/content/13/1/153 Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 http://www.biomedical-engineering-online.com/content/13/1/153 Page 10 of 16 Page 10 of 16 they had not encountered any particular difficulty in interacting with the AR appli- cation and in carrying out all the requested actions. Some of them reported only minor problems in perception of distances in the direction perpendicular to their plane of view. This is due to the use of a single camera, which obviously fails to accurately render the depth of the scene. Results and discussion BioMedical Engineering OnLine 2014, 13:153 http://www.biomedical-engineering-online.com/content/13/1/153 Page 12 of 16 Table 1 Positioning errors for precordial electrodes Precordial lead Test V1 V2 V3 V4 V5 V6 Mannequin Average error [cm] 0.30 0.18 0.23 0.24 0.22 0.27 ± SD of the error [cm] ± 0.10 ± 0.10 ± 0.13 ± 0.19 ± 0.11 ± 0.41 Max error [cm] 0.45 0.40 0.45 0.54 0.41 1.42 Patient Average error [cm] 0.40 0.27 0.44 0.46 0.49 0.69 ± SD of the error [cm] ± 0.07 ± 0.20 ± 0.20 ± 0.22 ± 0.18 ± 0.28 Max error [cm] 0.50 0.73 0.78 0.81 0.79 1.56 Average, standard deviation and maximum of the displacement errors done by the ten users of the developed AR application in placing precordial electrodes (V1-V6) during the tests on mannequin and real patient. All data are expressed in centimeters. The displacement errors were computed as the square root of the sum of the square of the three Cartesian components (x, y, and z) of the displacement vector. Table 1 Positioning errors for precordial electrodes Average, standard deviation and maximum of the displacement errors done by the ten users of the developed AR application in placing precordial electrodes (V1-V6) during the tests on mannequin and real patient. All data are expressed in centimeters. The displacement errors were computed as the square root of the sum of the square of the three Cartesian components (x, y, and z) of the displacement vector. when manually operating on the electrocardiograph, the distance between the camera and markers cannot be greater than the length of operator’s arm, nor the angle of gaze be particularly tilted. Hence, the need for manual interaction with objects marked se- verely restricts the space in which augmented reality operates. Due to the perspective of the scene taken by the camera, the greatest pose error of an AR marker lies in the direction that connects the marker with the camera. However, in this specific application (e.g. in placing the electrodes V1 to V4) the error made on the z-axis of Figure 2c (coincident with the most probable line of sight of user) results in practice negligible for the precordial electrodes V1 to V4: the user’s hand must stops on patient’s skin. This, obviously, does not hold for the electrodes V5 and V6 because of their intrinsic positions. Results and discussion This is inherent to the specific positions of these two electrodes and their relative positioning with respect to the markers on the thorax. When the user directs his gaze towards V5 or V6, the plane on which lies the marker (x- and y-axes represented in Figure 2c) results significantly angled and then the 3D pose errors increase [48]. This mainly occurs because of the natural curvature of the human thorax. Indeed, many studies have shown that the accuracy of positioning vir- tual object on the real scene mainly depends on camera distance and viewing angle with respect to the markers and also on other factors (e.g. size of the marker, focal length, field of view, pixel resolution, etc.) [48,53,54]. However, in the specific case, the distance and the relative angle between camera and markers are actually limited. Indeed, when placing the electrodes onto a patient or Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 http://www.biomedical-engineering-online.com/content/13/1/153 Page 11 of 16 Page 11 of 16 Figure 5 Average x, y and z components of positioning errors for precordial electrodes. Averages (bars) and Standard Deviations (segments) of the errors (distance from the expected location reported in centimeters for each of the x, y and z axes of the space) in the placement of the precordial electrodes on the mannequin (a) and on the patient’s chest (b) by the ten untrained users. V1, V2, V3, V4, V5 and V6 are the six precordial locations; Figure 5 Average x, y and z components of positioning errors for precordial electrodes. Averages (bars) and Standard Deviations (segments) of the errors (distance from the expected location reported in centimeters for each of the x, y and z axes of the space) in the placement of the precordial electrodes on the mannequin (a) and on the patient’s chest (b) by the ten untrained users. V1, V2, V3, V4, V5 and V6 are the six precordial locations; Figure 5 Average x, y and z components of positioning errors for precordial electrodes. Averages (bars) and Standard Deviations (segments) of the errors (distance from the expected location reported in centimeters for each of the x, y and z axes of the space) in the placement of the precordial electrodes on the mannequin (a) and on the patient’s chest (b) by the ten untrained users. V1, V2, V3, V4, V5 and V6 are the six precordial locations; Bifulco et al. Results and discussion Consequently, the placement of these electrodes suffers from greater errors (an higher component on z axis, see Figure 5a-b, and consequently an increase in the positioning error, see Table 1). Lastly, it is also interesting to note that the errors are consistent with data reported by the developers of the system ARToolKit [48]. Indeed, if we consider that usually the oper- ator is located at approximately 30-40 cm from the patient (and therefore from the markers) with an angle between 0° and 45° the errors predicted by previous studies [48,53] are contained within +/- 5 mm; while for a greater angle (e.g. 45°-80°) they increase to ap- proximately +/-12 mm. In the extreme case in which the inclination of the user’s gaze line reaches or exceeds 90 degrees with respect to the marker plane, the markers are no longer in sight making impossible to coherently augment the scene. Even if this event did not occur during the trials, it is possible and should be taken into account. Conclusion This study has highlighted the possibility of using Augmented Reality to support un- trained user while performing an ECG-test. The developed AR application is at the proof-of-concept stage and can certainly be improved. Furthermore, it can be easily generalized to the use of other medical equipment. AR can have a relevant and positive impact on various telemedicine applications. For example, telemedicine applications involving acquisition of diagnostic signals onboard of a plane or a ship by an untrained crew member or even passengers, as well as in the battlefield by untrained soldiers where it can obtain very beneficial effect. Also other home-care solutions, where a relative may be request to interact with medical device or emergency application (e.g. use of a defibrillator by an inexpert user), can take advantage Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 http://www.biomedical-engineering-online.com/content/13/1/153 Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 http://www.biomedical-engineering-online.com/content/13/1/153 Page 13 of 16 Page 13 of 16 of such AR applications. More appropriate and reliable usage of medical device can be ob- tained, but also opens new horizons for novel and/or more advanced telemedicine applications. However, some issues and question should be fully addressed before moving to clin- ical applications. While no particular difficulties arose in using AR with medical device (mainly because they are rigid and their 3D geometry known and fixed), problems occur when “augmenting” a generic, alive, human body: large anatomical variations and patient’s motion can be accurately taken into account. It is worth to remember that even if some parts of the human body can be roughly considered rigid, change in pro- portions should be accounted, e.g., between adults of infants, but also between different body types. Some parts of the body are in continuous motion such as the chest during breathing. However, in the conducted trials, with the patient breathing normally, this phenomenon did not significantly affect the results. These considerations lead to the use of non-rigid constellations of markers (e.g. proportionally scalable in geometric proportions) to take into account different body types, or even to a marker-less recog- nition of the body (however, this requires the use of sophisticated software and can ad- versely affect the real-time operation). The final AR application should also consider and properly instruct the operator on how to face with special situation (e.g. in the case of very hairy patients). Competing interest RV i l d h Competing interest RV is employed at the Selex ES, an international leader in electronic and information technologies for defense systems and aerospace, the other authors declare no competing interests. Authors’ contributions ll h d h All authors conceived the study and planned research activity. FN and PA developed the necessary software application. All the authors planned the practical tests carried out by FN and PA. PA, PB and MR drafted the manuscript. All authors contributed to revise it and approved its final version. Conclusion The flexibility of the XML language offers the possibility to eas- ily enrich or to customize the set of operations and warnings. For example, a simple re- minder for the operator to shave the skin or clip small patches of hair for proper sticking of electrodes, whether it is the case, can be included in the final AR application. This study, being essentially a proof of concept, does not provide solutions to these problems. Only a small variation in the markers T-structure has been designed to over- come the problem of excessive user’s slant. An improved version was obtained by add- ing two extra markers placed adjacently to the left marker (that indicated by the trapezoidal symbol) and the lower marker (that indicated by the C-like symbol), but ar- ranged at 90 degree with respect to the T-structure plane (i.e. laying in anatomical Figure 6 An improved marker set. Picture of the improved T-structure including the two extra markers oriented perpendicularly to the patient’s coronal plane. Figure 6 An improved marker set. Picture of the improved T-structure including the two extra markers oriented perpendicularly to the patient’s coronal plane. Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 Page 14 of 16 http://www.biomedical-engineering-online.com/content/13/1/153 Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 Page 14 of 16 Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 http://www.biomedical-engineering-online.com/content/13/1/153 Figure 7 Possible AR application on smartphones. Possible, future scenario involving the use of the proposed AR application on smartphones. Figure 7 Possible AR application on smartphones. Possible, future scenario involving the use of the proposed AR application on smartphones. sagittal planes). Figure 6 shows the new marker constellation for the patient’s body. Very preliminary results seem to confirm the expectations. Further developments of this study are oriented towards the realization of this AR appli- cation on mobile devices, i.e. tablets or smartphones (see Figure 7). These devices intrin- sically incorporate a camera, a relatively large screen, a processor capable of running the software, and other multimedia interfaces such as speakers and microphone. Possibly, the software can be also downloaded, at the very moment, from the Internet. The use of tab- lets or smartphones, today widely disseminated, could bring further benefits. Consent Written informed consent was obtained from the patient for the publication of this re- port and any accompanying images. Acknowledgements g Authors are very thankful to Selex ES to provide strong support to this study. 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Author details 1 1Department of Electrical Engineering and Information Technology, University of Naples “Federico II”, Naples, Italy. 2VRLab, University of Salerno, Salerno, Italy. 3Selex ES, Giugliano, Italy. Received: 27 March 2014 Accepted: 16 October 2014 Published: 21 November 2014 Received: 27 March 2014 Accepted: 16 October 2014 Published: 21 November 2014 Page 15 of 16 Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 http://www.biomedical-engineering-online.com/content/13/1/153 Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 http://www.biomedical-engineering-online.com/content/13/1/153 References Shah MN, Morris D, Jones CM, Gillespie SM, Nelson DL, McConnochie KM, Dozier A: A qualitative evaluation of a telemedicine-enhanced emergency care program for older adults. J Am Geriatr Soc 2013, 61(Suppl 4):571–576. 19. Thomas B, Close B, Donoghue J, Squires J, De Bondi P, Morris M, Piekarski W: ARQuake: An Outdoor/Indoor Augmented Reality First Person Application. 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BioMedical Engineering OnLine 2014 13:153. Bifulco et al. BioMedical Engineering OnLine 2014, 13:153 http://www.biomedical-engineering-online.com/content/13/1/153 Submit your next manuscript to BioMed Central and take full advantage of: y p 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 g • 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 • Convenient online submission
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The hidden burden of adult allergic rhinitis: UK healthcare resource utilisation survey
Clinical and translational allergy
2,015
cc-by
9,998
© 2015 Price et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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:  The affliction of allergic rhinitis (AR) has been trivialised in the past. Recent initiatives by the European Academy of Allergy & Clinical Immunology and by the EU parliament seek to rectify that situation. The aim of this study was to provide a comprehensive picture of the burden and unmet need of AR patients. Methods:  This was a cross-sectional, online, questionnaire-based study (June–July 2011) including symptomatic seasonal AR (SAR) patients (≥18 years) from a panel. SAR episode pattern, severity, medication/co-medication usage, residual symptoms on treatment, number of healthcare visits, absenteeism and presenteeism were collected. Results:  One thousand patients were recruited (mild: n = 254; moderate/severe: n = 746). Patients with mod- erate/severe disease had significantly more symptomatic episodes/year (8.0 vs 6.0/year; p = 0.025) with longer episode-duration (12.5 vs 9.8 days; p = 0.0041) and more commonly used ≥2 AR therapies (70.5 vs 56.1 %; OR 1.87; p = 0.0001), looking for better and faster nasal and ocular symptom relief. The reported symptom burden was high irrespective of treatment, and significantly (p < 0.0001) higher in the moderate/severe group. Patients with moder- ate/severe AR were more likely to visit their GP (1.61 vs 1.19 times/year; OR: 1.49; p = 0.0061); due to dissatisfaction with therapy in 35.4 % of cases. Patients reported SAR-related absenteeism from work on 4.1 days/year (total cost to UK: £1.25 billion/year) and noted presenteeism for a mean of 37.7 days/year (vs 21.0 days/year; OR 1.71; p = 0.0048). Asthma co-morbid patients reported the need to increase their reliever- (1 in 2 patients) and controller-medication (1 in 5 patients) if they did not take their rhinitis medication. Conclusions:  This study differentiated between patients with mild and moderate/severe AR, demonstrating a bur- den of poorly controlled symptoms and high co-medication use. The deficiency in obtaining symptom control with what are currently considered firstline treatments suggests the need for a novel therapeutic approach. Keywords:  Allergic rhinitis, UK, Symptom episode, Co-medication, Absenteeism, Presenteeism to highlight the profound impact this prevalent condition has on the quality of life (QoL) of AR sufferers and their families [8, 9]. Furthermore, the Polish presidency of the EU has highlighted the importance of early diagnosis and management of allergic diseases to promote active and healthy ageing [10], and made this an EU priority [11, 12]. All of these initiatives represent a fundamental shift in the perception of AR. The hidden burden of adult allergic rhinitis: UK healthcare resource utilisation survey David Price1*, Glenis Scadding2, Dermot Ryan3,4, Claus Bachert5, G. Walter Canonica6, Joaquim Mullol7, Ludger Klimek8, Richard Pitman9, Sarah Acaster10, Ruth Murray11 and Jean Bousquet12,13,14,15 Price et al. Clin Transl Allergy (2015) 5:39 DOI 10.1186/s13601-015-0083-6 Price et al. Clin Transl Allergy (2015) 5:39 DOI 10.1186/s13601-015-0083-6 Open Access *Correspondence: david@respiratoryresearch.org 1 University of Aberdeen, Aberdeen, UK Full list of author information is available at the end of the article Recruitment, patients and data collection Potential participants from a UK patient panel database (Opinion Health) were contacted about taking part in the study. This is an extensive database of patients with a variety of medical conditions, who gave prior consent to be contacted for research purposes. Patients are recruited into the Opinion Health panel from various channels, including direct mailing, bespoke telephone recruitment, peer/healthcare provider referral, magazine/newspaper advertising, and from  relevant  charities/associations/ communities. The wide range of recruitment methods employed has led to a strong and nationally representa- tive sample of the general population of which 18 % are aged over 65 years (30 % who are 55+ years), over 35 % are from lower household income bands with 17 % from Social Grade D or E.h Clinical trials assess patients with the most severe symptoms with insufficient information from observa- tional studies to understand the differences in burden between mild and moderate/severe rhinitis. To date, many surveys on the burden of AR have been conducted in Europe [2–5, 25] and in the US [6, 23, 37] but no cross- sectional questionnaire-based study, has assessed sea- sonal AR (SAR) episode pattern and duration, medication and co-medication usage (and the reasons for co-medi- cating), characterized residual symptoms on treatment nor provided information on healthcare visits, impact on asthma medication usage, absenteeism and presenteeism in a single study, stratified by disease severity (i.e. mild and moderate/severe). These potential participants were provided with the survey address and unique identifier, which they could use to access the online survey. Participants who fol- lowed the link were presented with a study screening form to assess their eligibility. Patients (≥18 years of age), currently residing in the UK, with a self-reported clini- cal diagnosis by a medical professional of SAR and cur- rently experiencing rhinitis symptoms, were recruited after informed consent. Currently symptomatic patients were selected to minimize recall error, enabling patients to draw on current symptomatic experience. Patients who experienced AR symptoms all year round (i.e. per- ennial allergic rhinitis) with no seasonal flare-ups were excluded.h The aim of this study was to describe the burden and unmet need of AR in one study, stratifed by disease severity. AR patients have been included in hundreds of clinical trials without a true understanding of the real burden of this disease, the way patients experience their symptoms and how they and their health care provider manage their disease in real-life. Methods St d d i activities [6, 13], well-being, cognitive functioning [14] even mood [15] and sleep [16]. Most AR patients attend- ing their healthcare provider have persistent disease, with many using multiple therapies [17]. AR imposes a high socioeconomic burden, particularly in terms of indirect costs, including absenteeism and presenteeism (i.e. pro- ductivity loss or under-performance at work and school) [18–21]. It has also been associated with poor asthma control; patients reporting severe rhinitis exhibit poorer asthma control than those with mild disease, with a nega- tive impact equivalent to that of smoking [22]. Study designh This was a cross-sectional, online, questionnaire-based study designed to collect representative views of peo- ple diagnosed with SAR. It was carried out in the UK between June and July 2011. The survey content was informed by experts (see Additional file  1). Experts contributed to all aspects of the survey from item and response level development and provision of key con- cepts to explore to provision of full UK AR medication listings. Ethics approval was obtained from Independ- ent Investigational Review Board Inc., (Florida, USA). Concept elicitation interviews with five patients were conducted prior to the start of the study to establish the most effective way to capture data with the least patient burden. These interviews were designed to ensure patient comprehension of the questions asked. Additional infor- mation to describe terms included in the survey were included based on patient advice. Most AR patients visiting their physician have mod- erate/severe disease with persistent symptoms [2, 17, 23–25]. Insufficient symptom control by currently considered firstline therapies has been identified as a major concern [2, 4, 17], a situation which has not improved over time [6, 7]. Co-medication is common; patients self-medicate and doctors co-prescribe (anti- histamines and intransal corticosteroids (INS) predom- inantly) [2, 3, 23, 26, 27] despite lack of evidence for this strategy in the literature [28–30]. AR patients have high expectations from their treatment [31], but most are dissatisfied with the results [32, 33]. Up to 40 % of patients have residual moderate/severe symptoms even after specialized treatment [17]. Management is often complicated by polysensitization [13, 34], the presence of allergic and non-allergic disease in the same patient (i.e. mixed rhinitis) [35] and confounded by phenotypes such as severe chronic upper airway disease (SCUAD) [36]. Background Allergic rhinitis (AR) has been trivialized over the years, despite its prevalence, chronicity and the burden it imposes on individuals and society [1–7]. Fortunately, the burden of AR is now being recognised both by the European Academy of Allergy & Clinical Immunology (EAACI) as well as at the EU parliament level, in order Reports in the literature already tell us that the daily burden of AR symptoms can be intrusive and debilitat- ing, negatively impacting patients’ QoL [4, 5], normal *Correspondence: david@respiratoryresearch.org 1 University of Aberdeen, Aberdeen, UK Full list of author information is available at the end of the article *Correspondence: david@respiratoryresearch.org 1 University of Aberdeen, Aberdeen, UK Full list of author information is available at the end of the article Page 2 of 12 Page 2 of 12 Page 2 of 12 Price et al. Clin Transl Allergy (2015) 5:39 Methods activities [6, 13], well-being, cognitive functioning [14] even mood [15] and sleep [16]. Most AR patients attend- ing their healthcare provider have persistent disease, with many using multiple therapies [17]. AR imposes a high socioeconomic burden, particularly in terms of indirect costs, including absenteeism and presenteeism (i.e. pro- ductivity loss or under-performance at work and school) [18–21]. It has also been associated with poor asthma control; patients reporting severe rhinitis exhibit poorer asthma control than those with mild disease, with a nega- tive impact equivalent to that of smoking [22]. Surveys All eligible participants were granted online access to the main survey to be completed at their own pace. Patients next completed symptom severity and socio-demo- graphic/healthcare utilisation questionnaires (see Addi- tional file  1). Symptom severity was assessed by EMA and FDA endorsed efficacy endpoints 12 h reflective total nasal symptom score (rTNSS; consisting of nasal conges- tion, itching, rhinorrhea and sneezing) and 12 h reflective total ocular symptom score (rTOSS; comprising ocular itch, redness and watering). These reflective scores assess symptom severity for the previous 12  h. Patients rated all symptoms as ‘none = 0’, ‘mild = 1’, ‘moderate = 2’ or ‘severe = 3’, both for symptoms ‘today’ and for symptoms ‘at their worst’. Socio-demographic Information collected included patients’ age, gender, ethnicity and educational level. The healthcare resource utilisation survey included questions on duration and number of SAR symptom epi- sodes, SAR medication usage, GP visits, impact on co- morbid asthma, absenteeism and presenteeism. These latter two items were based on the Work Productivity and Activity Impairment (WPAI) questionnaire. The full WPAI questionnaire was not used in order to minimise participant burden. Symptom episode was defined for patients as ‘an episode is a period of time when you expe- rience symptoms (or need to take medication to treat symptoms) continuously’. Episode pattern and duration Patients with moderate/severe AR experienced signifi- cantly more symptomatic episodes/year than those with mild disease (median 8.0 vs 6.0; p  =  0.025) with each of these episodes lasting significantly longer (12.5 vs 9.8 days; p = 0.0041; Table 1). Recruitment, patients and data collection A secondary aim was to use the data obtained to inform future AR clinical trial design and result relevancy. The survey was sent to 1300 potential participants. The aim was to recruit 1000 SAR participants, 200 mild and 800 moderate/severe. For the purpose of screening, Page 3 of 12 Price et al. Clin Transl Allergy (2015) 5:39 disease severity was graded using the ARIA-defined cri- teria of sleep disturbance, impairment of daily activities including leisure/sports, impairment of work/study and presence of troublesome symptoms [1]. severities, for parametric and non-parametric data, respectively. Results are presented with means and stand- ard deviations, unless significant skew was observed in the outcome, in which case medians are presented. Chi-squared tests and Fisher’s exact tests (where cell fre- quency was less than 5) were used to compare categori- cal outcomes to investigate differences between the two SAR severities and results presented as frequencies and percentages. Odds ratios were calculated for moderate/ severe versus mild SAR for a given exposure with refer- ence to no exposure. For all analyses p values <0.05 were judged to be statistically significant. Sensitization pattern Grass and tree pollen were the most commonly reported sensitizing allergens, but indoor allergen (e.g. to animal dander, mites) and mould sensitization was also com- mon. A high level of polysensitization was apparent particularly in the moderate/severe group (Table 1). Sig- nificantly (p < 0.001) more patients with moderate/severe disease were aware of their sensitizing allergen (Table 1). Participants received £10 upon completion of the sur- vey. All subjects were free to withdraw from participation in this study at any time, and for any reason. Statistics Statistical analyses were conducted in STATA 12 to compare baseline characteristics and exposures for mild disease to moderate/severe disease. For the pur- pose of analysis, participants with moderate/severe AR were defined as those who scored a rTNSS ≥8 out of 12, including a congestion score ≥2/3, when describing their ‘worst symptoms’. These rTNSS and nasal congestion score cut-offs were chosen in order to align with moder- ate/severe definitions from a recently conducted clinical trial [38]. Participants with mild disease were the remain- ing patients. The number of patients with mild and mod- erate/severe AR in both groups was very similar whether severity was classified according to rTNSS and conges- tion scores or according to the ARIA definition. Demographic and socioeconomic characteristics Demographic and socioeconomic characteristics One thousand SAR patients were recruited (mild: n = 254; moderate/severe: n = 746). The average age was 42.6 [standard deviation (SD) 12.1] years, with female gender and white ethnicity predominating (Table  1). Most participants were in full or part-time employ- ment or self-employed (69.1 %), with over three quarters (76.9 %) educated to A-level standard (i.e. international baccalaureate level or above). Survey responseh The survey was sent to 1300 potential participants. Data collection was stopped once 1000 patients completed the survey. Medication usage Almost all patients reported taking medication to treat their rhinitis symptoms (90.6 and 96.2  % of patients with mild and moderate/severe AR, respectively). Oral H1-antihistamines were the medications most com- monly reported, followed by INS (Table 2). Patients with moderate/severe AR were more likely to report nasal spray use (66.7 %) than those with mild disease [58.3 %; i Student t tests and Wilcoxon rank-sum tests were used to compare continuous outcomes for the two SAR Price et al. Clin Transl Allergy (2015) 5:39 Page 4 of 12 Table 1  Participant demographic and baseline data Table 1  Participant demographic and baseline data SAR severity: participants with moderate/severe AR were defined as those who scored a rTNSS ≥8 out of 12, including a congestion score ≥2/3, when describing their ‘worst symptoms’. Participants with mild AR included all remaining patients SD standard deviation Allergic rhinitis severity p value Mild (n = 254) Moderate/severe (n = 746) Age, mean (sd) 44.1 (13.0) 42.1 (11.8) 0.0274 Gender, n (%) female 175 (68.9) 503 (67.4) 0.665 Ethnicity, n (%) White 226 (89.0) 666 (89.3) 0.894 Asian 16 (6.3) 41 (5.5) 0.633 Black 3 (1.2) 25 (3.4) 0.070 Mixed 5 (2.0) 5 (0.7) 0.072 No response 4 (1.6) 9 (1.2) 0.654 Allergen sensitivity (self-reported) Grass pollen 165 (65.0) 579 (77.6) <0.001 Tree pollen 119 (46.9) 462 (61.9) <0.001 Weed pollen 56 (22.0) 259 (34.7) <0.001 Animals 57 (22.4) 231 (31.0) <0.001 Mites 29 (11.4) 163 (21.8) <0.001 Moulds 25 (9.8) 152 (20.4) <0.001 Not sure 57 (22.4) 96 (12.9) <0.001 Other 25 (9.8) 83 (11.1) 0.569 No. symptom episodes/year, median 6.0 8.0 0.025 No. days/episode Mean (SD) 9.8 (18.1) 12.5 (20.2) 0.0041 Median 4.0 5.0 0.013 Asthma diagnosis, n (%) 70 (30.4) 257 (35.8) 0.1368 SAR severity: participants with moderate/severe AR were defined as those who scored a rTNSS ≥8 out of 12, including a congestion score ≥2/3, when describing their ‘worst symptoms’. Participants with mild AR included all remaining patients SD standard deviation groups (Table  2). This was in line with the proportion of patients who reported ocular medication use (mild: 31.3 %; moderate/severe: 38.3 %). The search for faster response also drove AR treatment choice, with almost 35 % of patients with moderate/severe AR citing this as their reason for co-medicating (Table 2). odds ratio (OR) 1.44; 95 % confidence interval (CI) 1.05– 1.97; p = 0.0196]. Medication usage One-third of patients in both groups used ocular medication (Table 2). Only 0.9 and 1.7 % of patients with mild or moderate/severe disease, respec- tively, reported use of injections (either immunotherapy or systemic corticosteroids) to treat their AR. Most patients reported the use 2 or more AR medi- cations (56.1 % of patients with mild AR and 70.5 % of patients with moderate/severe AR), but were nearly twice as likely to do so if they had moderate/severe dis- ease (OR: 1.87; 95 % CI 1.36–2.56; p = 0.0001) (Table 2). The search for better nasal symptom relief, was the most common reason reported by patients for taking 2 or more AR medications. This was particularly evident in the moderate/severe group, where 58.3 % of patients cited the need for more effective nasal treatment as the reason for co-medicating compared to 42.6  % of those with mild AR (OR 1.88; 95 % CI 1.25–2.84; p = 0.0014) (Table  2). More effective ocular symptom relief was another important determinant governing co-prescrib- ing behaviour, reported by over 40 % of patients in both Symptom burden Th b The symptom burden reported by these patients was high, even though over 90  % of them were tak- ing an AR medication. On the day of assessment, par- ticipants in both severity groups reported significant nasal and ocular symptoms. However, this burden (both nasal and ocular) was significantly higher in those with moderate/severe disease (Fig. 1). Patients with moderate/severe disease also reported a signifi- cantly (p < 0.0001) higher overall nasal symptom bur- den when symptoms were at their worst (10.0 [SD 1.5] vs 5.9 [SD 1.9]). On the day of assessment (June–July 2011), many patients were experiencing ‘moderate’ or ‘severe’ nasal itch, Page 5 of 12 Page 5 of 12 Price et al. Clin Transl Allergy (2015) 5:39 Table 2  Medication usage in mild and moderate/severe seasonal allergic rhinitis patients SAR severity: paricipants with moderate/severe AR were defined as those who scored a rTNSS ≥8 out of 12, including a congestion score ≥2/3, when describing their ‘worst symptoms’. Symptom burden Th b Participants with mild AR included all remaining patienti SAR severity Mild (n = 254) Moderate/severe (n = 746) Odds ratio (95 % CI) P value Taking medication, n (%) 230 (90.6) 718 (96.2) 2.68 (1.45, 4.89) 0.0004 Oral medications, n (%) 184 (80.0 %) 605 (84.3 %) 1.34 (0.89, 1.98) 0.1322 Cetirizine 82 (44.6) 313 (51.7) 1.33 (0.94, 1.89) 0.0885 Loratadine 61 (33.2) 195 (32.2) 0.96 (0.67, 1.39) 0.8153 Chlorphenamine 61 (33.2) 178 (29.4) 0.84 (0.58, 1.22) 0.3349 Pseudoephedrine 14 (7.6) 92 (15.2) 2.18 (1.19, 4.25) 0.0081 Phenylephrine 7 (3.8) 33 (5.5) 1.46 (0.62, 3.97) 0.3716 Acrivastine 20 (10.9) 82 (13.6) 1.29 (0.75, 2.28) 0.3420 Levocetirizine 0 (0) 19 (3.1) – 0.011 Fexofenadine 10 (5.4) 38 (6.3) 1.17 (0.56, 2.68) 0.6741 Desloratadine 3 (1.6) 24 (4.0) 2.49 (0.74, 13.06) 0.1651 Other 17 (9.2) 57 (9.4) 1.02 (0.57, 1.93) 0.9408 Nasal sprays, n (%) 134 (58.3 %) 479 (66.7 %) 1.44 (1.05, 1.97) 0.0196 Fluticasone propionate 96 (71.6) 338 (70.6) 0.95 (0.60, 1.47) 0.8083 Beclomethasone 33 (24.6) 110 (23.0) 0.91 (0.57, 1.48) 0.6875 Mometasone 4 (3.0) 31 (6.5) 2.25 (0.77, 8.92) 0.1241 Fluticasone furoate 4 (3.0) 12 (2.5) 0.89 (0.26, 3.84) 0.8401 Flunisolide 1 (0.8) 12 (2.5) 3.42 (0.50, 147.15) 0.2116 Budesonide 2 (1.5) 10 (2.1) 1.41 (0.29, 13.36) 0.6602 Ipratropium bromide 0 (0) 5 (1.0) – 0.29 Other 18 (13.4) 48 (10.0) 0.72 (0.39, 1.36) 0.2600 Oxymetazoline 9 (6.7) 39 (8.1) 1.23 (0.57, 2.97) 0.5871 Azelastine 25 (18.7) 106 (22.1) 1.23 (0.75, 2.10) 0.3860 Ocular medications, n (%) 72 (31.3 %) 275 (38.3 %) 1.36 (0.98, 1.90) 0.0552 Sodium cromoglicate 14 (19.4) 82 (29.8) 1.76 (0.91, 3.61) 0.0798 Antazoline 12 (16.7) 50 (18.2) 1.11 (0.54, 2.44) 0.7651 Xylometazoline 9 (12.5) 36 (13.1) 1.05 (0.47, 2.62) 0.8943 Azelastine 3 (4.2) 13 (4.7) 1.14 (0.30, 6.41) 0.8400 Olopatadine 3 (4.2) 17 (6.2) 1.52 (0.42, 8.29) 0.5137 Lodoxamide trometamol 1 (1.4) 9 (3.3) 2.40 (0.32, 106.74) 0.3950 Other 33 (45.8) 98 (35.6) 0.65 (0.37, 1.15) 0.1121 Co-medicating, n (%) 129 (56.1) 506 (70.5) 1.87 (1.36, 2.56) 0.0001 Reported reason for co-medicating, n (%)  More effective nasal treatment 55 (42.6) 295 (58.3) 1.88 (1.25, 2.84) 0.0014  More effective ocular treatment 54 (41.9) 209 (41.3) 0.98 (0.65, 1.48) 0.9089  Faster nasal response 22 (17.1) 116 (22.9) 1.45 (0.86, 2.52) 0.1490  Faster ocular 13 (10.1) 57 (11.3) 1.13 (0.59, 2.33) 0.7007  Other 18 (19.0) 48 (9.5) 0.65 (0.35, 1.23) 0.1378 Table 2  Medication usage in mild and moderate/severe seasonal allergic rhinitis patients bothersome nasal symptom; with 61.5  % of participants with moderate/severe AR rating its severity as ‘moderate’ or ‘severe’ on the day of assessment compared to 33.5 % of those with mild disease (Fig. Discussionh This study provides a comprehensive view of the AR bur- den and unmet need in the UK. A complete dataset has been collected from a medically- diagnosed, symptomatic, SAR patient population (of similar disease severity to those included in a recent SAR study) [39] including information on SAR episode pattern and duration, medication/co-medi- cation usage, reasons for co-medication, residual symptoms on treatment, number of healthcare visits, absenteeism and productivity loss in patietns with mild and moderate/severe AR. It, therefore, represents a complete assessment of AR burden and unmet need in a single survey. Health care visits Participants with moderate/severe AR reported visit- ing their GP for their SAR more frequently than those with mild AR (1.61 vs 1.19 times/year; OR 1.49; 95 % CI 1.11–2.01; p  =  0.0061). In both groups, dissatisfaction with treatment was a primary reason for the visit; 28 % of visits for patients with mild AR versus 35 % of visits for those with moderate/severe disease, with patients with moderate/severe AR being significantly more likely to report treatment dissatisfaction than those in the mild group (OR 1.49; 95 % CI 1.06–2.13; p = 0.0194). This was a relatively large survey, including 1000 AR patients with wide representation of age, educational level and employment status. Survey content was broad and informed by several world-renowned experts in the field of AR. As this was an online survey, there was no interviewer bias. Responders were free to answer the questions in a time convenient to them and at their own pace. Patients were initially screened for severity using the Allergic Rhinitis and its Impact on Asthma (ARIA) severity classification system yielding 200 patients with mild AR and 800 patients with moderate/severe AR to ensure adequate representation of patients with moder- ate/severe AR in the survey (i.e. patients most likely to Symptom burden Th b 2). Ocular itch was the most both- ersome ocular symptom; 59.4  % patients with moderate/ congestion, rhinorrhea and sneezing as well as ocular itch, watering and redness, despite treatment, with significantly more patients with moderate/severe AR experiencing greater symptom severity for each nasal and ocular symp- tom (Table 3; Fig. 2). Congestion appeared to be the most Page 6 of 12 Price et al. Clin Transl Allergy (2015) 5:39 Fig. 1  Nasal and ocular symptom burden reported by seasonal aller- gic rhinitis patients with mild (n = 254) or moderate/severe disease (n = 746) on the day of assessment. Over 90 % of these patients in both groups were taking AR medication (see Table 2). Data are pre- sented as mean and standard deviation. rTNSS: reflective total nasal symptom score (max = 12); rTOSS: reflective total ocular symptom score (max = 9). *p < 0.0001 vs mild AR 95  % CI 1.01–3.68; p  =  0.0303). Similarly, 15.7  % of patients with mild AR with co-morbid asthma reported the need to increase their controller medication if they failed to take their AR medication, rising to 19.5 % of patients in the moderate severe group (OR 2.04; 95 % CI 0.86–5.03; p = 0.0781). Absenteeism and presenteeism Patients with moderate/severe AR reported absenteeism from work due to their SAR on 4.1 (SD 16.4) days/year compared to 2.5 (SD 7.7) days/year for patients in the mild group (OR: 1.34; 95 % CI: 0.87-2.11; p = 0.1708). This was significantly more likely for patients with moderate/severe AR who reported 37.7 (SD 53.0) days/year when their productivity was affected by their SAR symptoms, almost double that noted by patients with mild disease (21.0 days [SD 29.9]; OR: 1.71; 95 % CI: 1.15-2.54; p = 0.0048). Participants with mild AR did report some negative impact on their productivity, clustered predominantly at the lower impact end of the productivity scale (i.e. < 50 % impact). The negative impact on participant-reported work productivity due to SAR symptoms was much more apparent for those with moderate/severe disease. These patients were almost 4 times more likely to experi- ence > 50 % negative impact on their work productivity than those with mild disease (32.8 % vs 12.2 %; OR: 3.52; 95 % CI: 2.10-6.13; p < 0.0001) (Fig. 3). Fig. 1  Nasal and ocular symptom burden reported by seasonal aller- gic rhinitis patients with mild (n = 254) or moderate/severe disease (n = 746) on the day of assessment. Over 90 % of these patients in both groups were taking AR medication (see Table 2). Data are pre- sented as mean and standard deviation. rTNSS: reflective total nasal symptom score (max = 12); rTOSS: reflective total ocular symptom score (max = 9). *p < 0.0001 vs mild AR severe AR rated its severity as ‘moderate’ or ‘severe’ on the day of assessment compared to 39.7 % of those with mild AR (Fig. 2). Impact on asthma Many AR participants reported co-morbid asthma; 30.4 and 35.8 % of participants with mild and moder- ate/severe AR, respectively, and reported modifying their asthma medication (both reliever and controller) if they failed to take their AR medication. Patients with moderate/severe AR were twice as likely to describe this behaviour. For asthma reliever medication, 45.7 % of patients with mild AR with co-morbid asthma (n = 70) reported increased use compared to 53.7 % of patients with moderate/severe AR (n = 257) (OR 1.93; Page 7 of 12 Page 7 of 12 Price et al. Clin Transl Allergy (2015) 5:39 Table 3  Nasal and ocular symptom burden of patients with mild and moderate/severe AR on the day of assessment SAR severity: paricipants with moderate/severe AR were defined as those who scored a rTNSS ≥8 out of 12, including a congestion score ≥2/3, when describing their ‘worst symptoms’. Impact on asthma Participants with mild AR included all remaining patients Symptom severity: Assessed by individual symptom scores of the rTNSS and rTOSS; 0 = none, 1 = mild, 2 = moderate, 3 = severe SAR seasonal allergic rhinitis, rTNSS reflective total nasal symptom score, rTOSS reflective total ocular symptom score SAR severity: paricipants with moderate/severe AR were defined as those who scored a rTNSS ≥8 out of 12, including a congestion score ‘worst symptoms’. Participants with mild AR included all remaining patients Symptom severity: Assessed by individual symptom scores of the rTNSS and rTOSS; 0 = none, 1 = mild, 2 = moderate, 3 = severe SAR seasonal allergic rhinitis, rTNSS reflective total nasal symptom score, rTOSS reflective total ocular symptom score Symptom severity: Assessed by individual symptom scores of the rTNSS and rTOSS; 0 = none, 1 = mild, 2 = moderate, 3 = se SAR seasonal allergic rhinitis, rTNSS reflective total nasal symptom score, rTOSS reflective total ocular symptom score Although the data relates to the UK in terms of aller- gen exposure, as well as treatment and referral patterns, the results also have a broader relevance for clinical trial design in general. For example, knowledge of the dura- tion of a typical mild and moderate/severe SAR symp- tom episode could inform trial duration decisions and also encourage contextualization of efficacy endpoints with a temporal focus. A potential limitation of this sur- vey was that patients were recruited from a patient panel. These panels include a varied and heterogeneous patient population. Panel patients are not subjected to stringent visit their healthcare provider). However, to align with moderate/severe definition commonly employed in AR clinical trials, severity was also classified using rTNSS and congestion score cut offs for the purpose of data analysis. Very similar numbers were reported using this method of categorization; 254 and 746 for patients with mild and moderate/severe AR, respectively. This con- firms the robustness of the ARIA severity definition as a quick, simple and accurate method of severity categori- zation, and also that the moderate/severe definition used in the present analysis largely conforms to ARIA. visit their healthcare provider). However, to align with moderate/severe definition commonly employed in AR clinical trials, severity was also classified using rTNSS and congestion score cut offs for the purpose of data analysis. Impact on asthma Participants with mild AR included all remaining patients Symptom severity: Assessed by individual symptom scores of the rTNSS and rTOSS; 0 = none, 1 = mild, 2 = moderate, 3 = severe SAR seasonal allergic rhinitis, rTNSS reflective total nasal symptom score, rTOSS reflective total ocular symptom score Symptom Symptom severity SAR severity P value Mild (n = 254) Moderate/severe (n = 746) Nasal symptoms of the rTNSS Nasal itch, n (%) None 63 (24.8) 87 (11.7) <0.001 Mild 128 (50.4) 298 (39.9) 0.004 Moderate 54 (21.3) 283 (37.9) <0.001 Severe 9 (3.5) 78 (10.5) 0.001 Nasal congestion, n (%) None 67 (26.4) 61 (8.2) <0.001 Mild 102 (40.2) 226 (30.3) 0.004 Moderate 67 (26.4) 312 (41.8) <0.001 Severe 18 (7.1) 147 (19.7) <0.001 Rhinorrhea, n (%) None 82 (32.3) 111 (14.9) <0.001 Mild 102 (40.2) 241 (32.3) 0.023 Moderate 56 (22.0) 279 (37.4) <0.001 Severe 14 (5.5) 115 (15.4) <0.001 Sneezing, n (%) None 55 (21.7) 68 (9.1) <0.001 Mild 108 (42.5) 256 (34.3) 0.019 Moderate 75 (29.5) 281 (37.7) 0.019 Severe 16 (6.3) 141 (18.9) <0.001 Ocular symptoms of the rTOSS Ocular itch, n (%) None 51 (20.1) 97 (13.0) 0.006 Mild 102 (40.2) 206 (27.6) <0.001 Moderate 74 (29.1) 276 (37.0) 0.023 Severe 27 (10.6) 167 (22.4) <0.001 Ocular watering, n (%) None 70 (27.6) 154 (20.6) 0.022 Mild 102 (40.2) 220 (29.5) 0.002 Moderate 64 (25.2) 239 (32.0) 0.040 Severe 18 (7.1) 133 (17.8) <0.001 Ocular redness, n (%) None 91 (35.8) 183 (24.5) <0.001 Mild 106 (41.7) 316 (42.4) 0.861 Moderate 51 (20.1) 209 (28.0) 0.013 Severe 6 (2.4) 38 (5.1) 0.067 symptom burden of patients with mild and moderate/severe AR on the day of assessment Table 3  Nasal and ocular symptom burden of patients with mild and moderate/severe AR on the day of assessment Table 3  Nasal and ocular symptom burden of patients with mild and moderate/severe AR on the day of assessment P value SAR severity: paricipants with moderate/severe AR were defined as those who scored a rTNSS ≥8 out of 12, including a congestion score ≥2/3, when describing the ‘worst symptoms’. Impact on asthma Very similar numbers were reported using this method of categorization; 254 and 746 for patients with mild and moderate/severe AR, respectively. This con- firms the robustness of the ARIA severity definition as a quick, simple and accurate method of severity categori- zation, and also that the moderate/severe definition used in the present analysis largely conforms to ARIA. Page 8 of 12 Price et al. Clin Transl Allergy (2015) 5:39 Fig. 2  Proportion of patients with mild (n = 254) or moderate severe AR (n = 746) scoring a ‘2’ (moderate) or ‘3’ (severe) for individual nasal and ocular symptom scores on the day of assessment. Over 90 % of these patients in both groups were taking AR medication (see Table 2). Significance values for mild vs moderate/severe groups are given for each level of symptom severity in Table 3 intermittent/persistent). These classifications are not interchangeable [1], and whilst the SAR/PAR classifica- tion is still widely used in primary care, the newer (and more therapeutically relevant) ARIA classification sys- tem should be promoted at both the patient and physi- cian level. By design, most patients included in the survey had moderate/severe disease and so represent the type of patients who present to physicians [2, 4, 17, 23]. Also, patients were included in this survey based on a reported medical diagnosis of SAR, rather than a medically-con- firmed diagnosis. No data were collected on irritant exposure or smoking history. It would have been inter- esting to examine their impact on symptom burden and therapeutic response. As with all surveys of this nature there was a reliance on patient recall. Variability was noted for some responses as evidenced by large stand- ard deviations around the mean. Where this occurred, median values were used. Fig. 2  Proportion of patients with mild (n = 254) or moderate severe AR (n = 746) scoring a ‘2’ (moderate) or ‘3’ (severe) for individual nasal and ocular symptom scores on the day of assessment. Over 90 % of these patients in both groups were taking AR medication (see Table 2). Significance values for mild vs moderate/severe groups are given for each level of symptom severity in Table 3 Fig. 2  Proportion of patients with mild (n = 254) or moderate severe AR (n = 746) scoring a ‘2’ (moderate) or ‘3’ (severe) for individual nasal and ocular symptom scores on the day of assessment. Impact on asthma Over 90 % of these patients in both groups were taking AR medication (see Table 2). Significance values for mild vs moderate/severe groups are given for each level of symptom severity in Table 3 The survey found that patients experienced several symptomatic bursts throughout the year, each lasting for some days, with participants with moderate/severe AR reporting significantly greater symptom episode frequency and duration than their milder counterparts. There was a clear symptom burden shift from patients with mild to those with moderate/severe AR, the latter, more likely to report more and longer episodes/year. These facts were previously unrecognised. The symptom burden shift pro- vides evidence of the quality of the survey data and its inclusion/exclusion criteria and have a relaxed ecol- ogy of care making the information they provide more indicative of the real world. Conversely, AR patients recruited into randomized controlled trials (RCTs) are poorly representative of those seen in primary care [40]. In the present study AR was classified according to time of year when symptoms appeared (i.e. SAR) rather than the ARIA classification based on symptom longevity (i.e. Fig. 3  Presenteeism due to SAR reported by patients with mild disease (n = 164) and those with moderate/severe disease (n = 521). *p ≤ 0.0093 vs mild AR. Patients with moderate/severe AR significantly (OR 3.52; CI 2.10–6.13; p < 0.0001) more likely than those with mild AR to have a >50 % impairment in their work productivity due to their SAR symptoms Fig. 3  Presenteeism due to SAR reported by patients with mild disease (n = 164) and those with moderate/severe disease (n = 521). *p ≤ 0.0093 vs mild AR. Patients with moderate/severe AR significantly (OR 3.52; CI 2.10–6.13; p < 0.0001) more likely than those with mild AR to have a >50 % impairment in their work productivity due to their SAR symptoms Fig. 3  Presenteeism due to SAR reported by patients with mild disease (n = 164) and those with moderate/severe disease (n = 521). *p ≤ 0.0093 vs mild AR. Patients with moderate/severe AR significantly (OR 3.52; CI 2.10–6.13; p < 0.0001) more likely than those with mild AR to have a >50 % impairment in their work productivity due to their SAR symptoms Page 9 of 12 Price et al. Clin Transl Allergy (2015) 5:39 moderate/severe disease and were difficult to control with mono or multiple therapies. Impact on asthma This result showed that monotherapy provides insufficient symptom relief for a substantial proportion of patients with mild AR too, suggesting that they may underestimate the true severity of their disease and/or rely on over-the-counter AR medications, being resistant to attending their physi- cian in order to receive a more effective treatment option, or indeed a more accurate severity diagnosis. Others have confirmed that co-mediation prescribing behaviour has been steadily rising in the UK in the last 2 decades; dual therapy has doubled since 1992, whilst use of triple ther- apy has increased eight-fold [41]. i y p This survey also serves to highlight the large indirect burden of AR in the UK; the hidden costs associated with this disease are substantial. Many patients with AR also have asthma, with failure to control one having a detri- mental effect on control of the other [1]. In the present survey, asthma medication usage (both reliever and con- troller) was likely to be increased by participants if they failed to use their AR medication, and more likely to occur in those with moderate/severe AR. Other indirect costs reported included absenteeism and presenteeism. On average, patients with moderate/severe AR reported 4 days/year absent from work due to their SAR. Assum- ing an average cost of £71 for each lost day [44], this amounts to £1.14 billon/year in the UK alone. This figure does not take presenteeism into consideration, which was reportedly negatively impacted on 38 days/year and car- ries a substantial indirect cost [19]. Knowledge of AR symptom patterns is vital when con- sidering relevancy of clinical trial data and appropriate- ness of clinical trial design. Patients with intermittent AR (as categorized by ARIA) experience symptoms for <4  days/week or for less than 4 consecutive weeks [1]. Based on the results presented here, we now have cor- responding information for SAR (i.e. average symptom episode lasts 9.8 days for mild SAR and 12.5 days for mod- erate/severe SAR). Therefore, SAR trials of 14 days dura- tion are sufficiently long to assess the clinical efficacy of medications in most patients; since this timeframe spans a single episode, and thus reflects the real-world situation. Additionally, any improvements afforded by AR medica- tions in patients with moderate/severe AR should now be contextualized and assessed for clinical relevancy within a 12.5 day time frame. Impact on asthma A similar pattern of mono- and multiple-therapy insufficiency has also been observed in other countries [4]. There is, therefore, a clear need for a faster and more effective AR treatment option with wide symptomatic and pathologic coverage, which provides more complete and rapid symptom con- trol. MP29-02, comprising azelastine hydrochloride, flu- ticasone propionate and a novel formulation in a single spray, is the newest addition to the AR treatment arse- nal and is promising in this regard [39, 42]. Allergen- specific immunotherapy should be strongly considered for patients who fail to respond to symptomatic therapy, particularly for those patients for whom symptoms are predominantly caused by one allergen [43], and may sig- nificantly reduce the burden of AR in these patients. sensitivity to discriminate according to disease symptom severity. Knowledge of duration and frequency of AR symptom episodes is important to know when assessing the symptomatic and economic burden of AR, and when considering treatment choice. It indicates that rapid relief of symptoms is important to control the disease. The extent to which patients co-medicate is underes- timated by physicians and payers alike, since over the counter medications are frequently added to prescrip- tion medications. This finding has also been observed in Spain and France [3, 26, 27]. The majority of participants who took part in this survey reported using 2 or more AR medications (most commonly an INS plus an oral H1-anti- histamine) in an attempt to achieve better and faster nasal and ocular symptom relief. This was true for both the par- ticipants with moderate/severe disease (70.5 %) and those with mild AR (56.1 %), although significantly more likely in those with more severe disease. Therefore, the direct cost of AR may be higher than previously thought, as patients supplement with multiple treatments, driven by their search for better efficacy. This search for a faster and more effective nasal therapy was more in evidence as a driver for those patients with moderate/severe AR emphasizing the higher symptom burden of this group, not only in terms of symptom severity, but also in terms of episode frequency and duration. The fact that over half of patients with mild AR co-medicated was an unexpected finding. Authors’ information DP is Professor of Primary Care Respiratory Medicine at the University of Aberdeen, co-founder of the Respiratory Effectiveness Group (http://www. effectivenessevaluation.org) and Director of Observational and Pragmatic Research Institute Singapore. GS is Hon. Consultant Rhinologist and Allergist at the Royal National TNE Hospital, London and Hon. Senior Lecturer at University College, London. DR has had a career-long interest in respiratory allergy and was twice chairman of the UK Primary Care Respiratory Society. He is a mem- ber of ARIA and current Chairman of the Primary care Interest Group of EAACI. CB is an ENT specialist and allergologist at the Ghent University Hospital, and runs the Upper Airways Research Laboratory, Ghent University. He is also affili- ated with the Karolinska Institute in Stockholm, Sweden. GWC is Professor of Respiratory Medicine and Director of Allergy & Respiratory Disease Clinic, Dept Internal Medicine, University of Genoa, IRCCS AOU San Martino, Genoa Italy. JM is an ENT specialist and Director of the Rhinology Unit and Smell Clinic, ENT Department, Hospital Clínic de Barcelona; and Professor of Research and Head of the Laboratory Clinical and Experimental Respiratory Immunoallergy at IDIBAPS. Barcelona, Catalonia, Spain. LK is Director of the Center for Rhinol- ogy and Allergology, Wiesbaden, Germany (http://www.Allergiezentrum.org), Vice-President of the German Academy of Allergology and Clinical Immunol- ogy, Vice-President of the German Union of Allergologists and Professor at Hei- delberg University, Germany. RP is an employee of Icon plc. SA is the director of a Clinical Outcomes Assessment research consultancy. RM is the director of a Medical and Scientific research consultancy. JB is a Professor Emeritus at the University of Montpellier in France. He is recognized as past chairman of the Global Initiative for Asthma (GINA) and as the founder and Chairman of ARIA (Allergic Rhinitis and its Impact on Asthma), in collaboration with the World Health Organization. Prof Bousquet is also past Chairman of the WHO Global Alliance Against Chronic Respiratory Diseases (GARD), Director of the WHO Collaborating Centre for Asthma and Rhinitis in Montpellier, and coordinator of several projects of the European Union in research, health and ICT. Professor Bousquet’s current interests lie with the European Innovation Partnership on Active and Health Aging, and updating how chronic diseases like allergic rhinitis are managed using an integrated care pathway. Conclusionsh This cross-sectional online questionnaire-based study represents a comprehensive assessment of the burden and unmet need of AR in the UK in a large patient popu- lation. Knowledge of the results of study should be used to inform clinical trial design and relevancy of clinical findings, and to assess the potential impact of AR treat- ments on the true burden and unmet need in this highly prevalent condition. Abbreviations AR: allergic rhinitis; ARIA: allergic rhinitis in asthma; CI: confidence interval; INS: intranasal corticosteroid; OR: odds ratio; RCT: randomized controlled trial; rTNSS: reflective total nasal symptom score; rTOSS: reflective total ocular symptom score; SAR: seasonal allergic rhinitis; SCUAD: severe chronic upper airway disease; SD: standard deviation; WPAI: work productivity and activity impairment. Author details 1 1 University of Aberdeen, Aberdeen, UK. 2 The Royal National Throat, Nose and Ear Hospital, London, UK. 3 Woodbrook Medical Centre, Loughborough, UK. 4 University of Edinburgh, Edinburgh, UK. 5 Upper Airways Research Laboratory, Ghent University Hospital, Ghent, Belgium. 6 Allergy and Respira- tory Clinic, IRCCS AOU S. Martino, Genoa, Italy. 7 Hospital Clínic, IDIBAPS, CIBERES, Barcelona, Catalonia, Spain. 8 Center for Rhinology and Allergology, Wiesbaden, Germany. 9 ICON, Oxford, UK. 10 Acaster Consulting, London, UK. 11 Medscript Ltd, Dundalk, Ireland. 12 University Hospital, Montpellier, France. 13 MACVIA-LR, Contre les Maladies Chronique spour un Vieillissement Actif en Languedoc Roussilon, European Innovation Partnership on Active and Healthy Ageing Reference Site, Montpellier, France. 14 INSERM, VIMA : Ageing and Chronic Diseases, Epidemiological and Public Health Approaches, U1168, Paris, France. 15 UVSQ, UMR-S 1168, Université Versailles St-Quentin-en- Yvelines, Versailles, France. Authors’ contributions All authors have been involved in the analysis and interpretation of data. SA and RP also contributed to the conception and design of the survey. All authors were involved in the drafting of the manuscript, critically revised each draft and gave their final approval for publication. All authors agree to be accountable for all aspects of this work and have participated sufficiently to take public responsibility for the content. All authors read and approved the final manuscript. Impact on asthma It is also clear that direct head-to- head trials of active comparators are needed, not simply comparisons versus placebo, since the vast majority of patients with moderate/severe AR are treated, and most are co-medicating. Therefore, studies versus placebo However, co-medication does not appear to pro- vide the symptom relief, which AR patients seek. Logi- cally, one would assume that use of several medications from different classes would provide improved patho- logic coverage leading to better symptom control. But, this does not appear to be the case. The present survey results confirm the results obtained in randomized clini- cal trials [29, 30]. Both patients with mild and moderate/ severe AR included in this survey remained sympto- matic, with those with more severe disease more likely to be so, even though > 90 % of patients were on AR treat- ment, and many were co-medicating. In other words, patients’ symptoms were still of moderate severity, on average, despite treatment. Nasal congestion and ocular itching remained problematic for 60 % of patients with Page 10 of 12 Page 10 of 12 Page 10 of 12 Price et al. Clin Transl Allergy (2015) 5:39 only, in those patients with moderate/severe disease are not clinically-relevant, may provide a distorted view of the effectiveness of active comparators, and are likely to increase the number of insufficiently effective drugs reg- istered, failing to meet patient expectations of treatment. The results of our study support the request of ARIA to conduct clinical trials against gold standard therapy in order to show clinically relevant improvements that will lower the burden of AR and improve its management. A recently published state of the art analysis of a new AR therapy, is an important first step in this direction [39]; (1) patients included in the trial had moderate/severe dis- ease, representing the type of patient commonly seen in practice, (2) first-line AR medications were used as active comparators (in addition to placebo), (3) results were contextualised within a typical symptom episode win- dow and (4) data were analysed to show not only superior efficacy to established first line therapies but also a faster response, which is what patients want [33, 45]. Authors’ information The impact of patients’ attitudes on their AR health outcomes and their decision processes when considering which AR medication to take are interesting avenues for additional research. More information on patient knowl- edge (both about the disease and available treatments) as well as incidence of co-morbidities (e.g. food allergy, asthma, atopic dermatitis) would also provide a more global look at burden of care. Finally, patients should be empowered to take responsibility for their own AR con- trol, encouraged to improve their disease awareness and knowledge of AR therapeutic options and improve con- cordance with their treatment regimen. In this regard, the importance of a common AR control concept and language (for both patients and physicians) has been rec- ognized [46]. MACVIA ARIA has recently launched an app, called Allergy Diary, which uses a simple visual ana- logue scale (VAS) to assess control and will use this same VAS in an app for health care providers (called Allergy Diary Companion) and in the updated guideline to guide AR treatment decisions [46]. Competing interests 5. Canonica GW, Mullol J, Pradalier A, Didier A. Patient perceptions of aller- gic rhinitis and quality of life: findings from a survey conducted in europe and the United States. World Allergy Organ J. 2008;1:138–44. DB has Board Membership with Aerocrine, Almirall, Amgen, AstraZeneca, Boehringer Ingelheim, Chiesi, Meda, Mundipharma, Napp, Novartis, and Teva. Consultancy: A Almirall, Amgen, AstraZeneca, Boehringer Ingelheim, Chiesi, GlaxoSmithKline, Meda, Mundipharma, Napp, Novartis, Pfizer, and Teva; Grants and unrestricted funding for investigator-initiated studies from UK National Health Service, British Lung Foundation, Aerocrine, AKL Ltd, Almirall, AstraZeneca, Boehringer Ingelheim, Chiesi, Eli Lilly, GlaxoSmith- Kline, Meda, Merck, Mundipharma, Napp, Novartis, Orion, Pfizer, Respira- tory Effectiveness Group, Takeda, Teva, and Zentiva; Payments for lectures/ speaking: Almirall, AstraZeneca, Boehringer Ingelheim, Chiesi, Cipla, GlaxoS- mithKline, Kyorin, Meda, Merck, Mundipharma, Novartis, Pfizer, SkyePharma, Takeda, and Teva; Payment for manuscript preparation: Mundipharma and Teva; Patents (planned, pending or issued): AKL Ltd.; Payment for the development of educational materials: GlaxoSmithKline, Novartis; Stock/ Stock options: Shares in AKL Ltd which produces phytopharmaceuticals and owns 80 % of Research in Real Life Ltd and its subsidiary social enter- prise Optimum Patient Care; received Payment for travel/accommodations/ meeting expenses from Aerocrine, Boehringer Ingelheim, Mundipharma, Napp, Novartis, and Teva; Funding for patient enrolment or completion of research: Almirral, Chiesi, Teva, and Zentiva; and Peer reviewer for grant committees: Medical Research Council (2014), Efficacy and Mechanism Evaluation programme (2012), HTA (2014). GS has received research grants from GSK and ALK as well as honoraria for articles, consulting, lectures/ chairing and/or advisory boards from ALK, Bausch & Lomb, Church & Dwight, Circassia, GSK, Groupo Uriach, Meda, Merck, Ono, Shionogi and Stallergenes. DS has been paid consultancy fees by Stallergenes, Uriach and TEVA. He has lectured on behalf of MEDA, GSK, AZ, Chiesi, Thermo- Fisher, Boehringer, Novartis and Almirall. He is Director of Health Strategy at Optimum Patient Care. CB is on the speaker’s bureau for Meda. GWC has received honoraria for lectures or scientific advisory boards: ALK, Allergy Therapeutics, AstraZeneca, Boston Scientific, Bruschettini, Chiesi, Circassia, Faes, GSK, Meda, Menarini, Mundifarma, Novartis, Recordati, Roche, Sanofi- Aventis, Uriach, Stallergènes, Thermo Fisher, Teva and Valeas. JM is or has been a member of national and international scientific advisory Boards (consulting), received fees for lectures, or grants for research projects from ALK-Abelló, Boheringer-Ingelheim, FAES, GSK, Hartington Pharmaceuticals, Hyphens, Johnson & Johnson, MEDA Pharma, Menarini, MSD, Novartis, Pierre Fabre, Sanofi, UCB, and Uriach Group. Competing interests LK has received research grants from ALK-Abelló, Allergopharma, Bionorica, Dr. Pfleger, Stallergenes, HAL, Artu Biologicals, Allergy Therapeutics/Bencard, Hartington, Lofarma, MEDA, MSD, Novartis/Leti, ROXALL, GSK, Essex-Pharma, Cytos, Curalogic, and has served on the speaker’s bureau for the above mentioned pharmaceuti- cal companies. RP has no conflict of interest to report. SA was employed by Oxford Outcomes who were commissioned by Meda to conduct this research. SA now works for Acaster Consulting Ltd, which receives fees for research and consultancy from Meda. RM has received consultancy fees from GSK, Meda, MACVIA-ARIA and Research in Real life. JB has received honoraria for: Scientific and advisory boards: Almirall, Meda, Merck, MSD, Novartis, Sanofi-Aventis, Takeda, Teva, Uriach. Lectures during meetings: Almirall, AstraZeneca, Chiesi, GSK, Meda, Menarini, Merck, MSD, Novartis, Sanofi-Aventis, Takeda, Teva, Uriach. Board of Directors: Stallergènes. 6. Meltzer EO, Gross GN, Katial R, Storms WW. Allergic rhinitis substantially impacts patient quality of life: findings from the Nasal Allergy Survey Assessing Limitations. J Fam Pract. 2012;61:S5–10. 7. Nathan RA. The burden of allergic rhinitis. Allergy Asthma Proc. 2007;28:3–9. 8. European Academy of Allergy and Clinical Immunology (EAACI). Beware of Allergy Campaign. http://www.bewareofallergy.com. Accessed Aug 2015. 9. Antonescu E, Childres N, Gardini E, Grossetete F, Juvin P, Parvanova A, et al. Written declaration on recognising the burden of allergic disease. European Parliament. http://www.eaaci.org. Accessed June 2015. 10. Samolinski B, Fronczak A, Kuna P, Akdis CA, Anto JM, Bialoszewski AZ, et al. Prevention and control of childhood asthma and allergy in the EU from the public health point of view: Polish Presidency of the European Union. Allergy. 2012;67:726–31. 11. Bousquet J, Addis A, Adcock I, Agache I, Agusti A, Alonso A, et al. Inte- grated care pathways for airway diseases (AIRWAYS-ICPs). Eur Respir J. 2014;44:304–23. 12. Bousquet J, Michel J, Standberg T, Crooks G, Iakovidis I, Gomez M. The European Innovation Partnership on Active and Healthy Aging: the Euro- pean Geriatric Medicine introduces the EIP on AHA Column. Eur Geriatr Med. 2014;5:361–2. 13. Valovirta E, Myrseth SE, Palkonen S. The voice of the patients: allergic rhinitis is not a trivial disease. Curr Opin Allergy Clin Immunol. 2008;8:1–9. 14. Kremer B, den Hartog HM, Jolles J. Relationship between allergic rhinitis, disturbed cognitive functions and psychological well-being. Clin Exp Allergy. 2002;32:1310–5. 15. Braido F, Baiardini I, Scichilone N, Musarra A, Menoni S, Ridolo E, et al. 1. Bousquet J, Khaltaev N, Cruz AA, Denburg J, Fokkens WJ, Togias A, et al. Allergic Rhinitis and its impact on asthma (ARIA) 2008 update (in col- laboration with the World Health Organization, GA(2)LEN and AllerGen). Allergy. 2008;63(Suppl 86):8–160. Previous data presentation at conferences Allergol Clin Immunol. 2009;19:27–34. 4. Bousquet PJ, Demoly P, Devillier P, Mesbah K, Bousquet J. Impact of aller- gic rhinitis symptoms on quality of life in primary care. Int Arch Allergy Immunol. 2013;160:393–400. Some of the data presented within this article have been presented at the European Academy of Allergy Clinical Immunology Congress (2012 and 2013). European Academy of Allergy Clinical Immunology Congress (2012 and 2013). Funding d f 2. Canonica GW, Bousquet J, Mullol J, Scadding GK, Virchow JC. A survey of the burden of allergic rhinitis in Europe. Allergy. 2007;62(Suppl 85):17–25. 3. Mullol J. A survey of the burden of allergic rhinitis in Spain. J Investig Allergol Clin Immunol. 2009;19:27–34. Competing interests Ill- ness perception, mood and coping strategies in allergic rhinitis: are there differences among ARIA classes of severity? Rhinology. 2014;52:66–71. f 16. Green RJ, Davis G, Price D. Concerns of patients with allergic rhinitis: the Allergic Rhinitis Care Programme in South Africa. Prim Care Respir J. 2007;16:299–303. 17. Mullol J, Bartra J, del CA, Izquierdo I, Munoz-Cano R, Valero A. Specialist- based treatment reduces the severity of allergic rhinitis. Clin Exp Allergy. 2013;43:723–9. 18. Lamb CE, Ratner PH, Johnson CE, Ambegaonkar AJ, Joshi AV, Day D, et al. Economic impact of workplace productivity losses due to allergic rhinitis compared with select medical conditions in the United States from an employer perspective. Curr Med Res Opin. 2006;22:1203–10. 19. Hellgren J, Cervin A, Nordling S, Bergman A, Cardell LO. Allergic rhinitis and the common cold–high cost to society. Allergy. 2010;65:776–83. 20. Small M, Piercy J, Demoly P, Marsden H. Burden of illness and quality of life in patients being treated for seasonal allergic rhinitis: a cohort survey. Clin Transl Allergy. 2013;3:33. 21. Walker S, Khan-Wasti S, Fletcher M, Cullinan P, Harris J, Sheikh A. Seasonal allergic rhinitis is associated with a detrimental effect on examination performance in United Kingdom teenagers: case-control study. J Allergy Clin Immunol. 2007;120:381–7. 22. Clatworthy J, Price D, Ryan D, Haughney J, Horne R. The value of self- report assessment of adherence, rhinitis and smoking in relation to asthma control. Prim Care Respir J. 2009;18:300–5. 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Clinically relevant effect of a new intranasal therapy (MP29-02) in allergic rhinitis assessed by responder analysis. Int Arch Allergy Immunol. 2013;161:369–77. 28. Anolik R, Mometasone Furoate Nasal Spray With Loratadine Study Group. Clinical benefits of combination treatment with mometasone furoate nasal spray and loratadine vs monotherapy with mometasone furoate in the treatment of seasonal allergic rhinitis. Ann Allergy Asthma Immunol. 2008;100:264–71. 40. Costa DJ, Amouyal M, Lambert P, Ryan D, Schunemann HJ, Daures JP, et al. How representative are clinical study patients with allergic rhinitis in primary care? J Allergy Clin Immunol. 2011;127:920–6. 29. Ratner PH, van Bavel JH, Martin BG, Hampel FC Jr, Howland WC III, Rogenes PR, et al. A comparison of the efficacy of fluticasone propionate aqueous nasal spray and loratadine, alone and in combination, for the treatment of seasonal allergic rhinitis. J Fam Pract. 1998;47:118–25. 41. 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Scadding GK, Durham SR, Mirakian R, Jones NS, Leech SC, Farooque S, et al. BSACI guidelines for the management of allergic and non-allergic rhinitis. Clin Exp Allergy. 2008;38:19–42. 32. Ciprandi G, Incorvaia C, Scurati S, Puccinelli P, Soffia S, Frati F, et al. Patient-related factors in rhinitis and asthma: the satisfaction with allergy treatment survey. Curr Med Res Opin. 2011;27:1005–11. y 44. UK Healthcare: The case for health benefits. http://www.ukhealthcare.org. uk/the-case-for-health-benefits. Accessed June 2015. 33. Valovirta E, Ryan D. Patient adherence to allergic rhinitis treatment: results from patient surveys. Medscape J Med. 2008;10:247. 45. Acaster S, Ali S, Breheny K, Bachert C, Bousquet J, Price D. Treatment preferences in patients with moderate/severe seasonal allergic rhinitis: findings of a discrete choice experiment. Allergy. 2012;67:A891. 34. Mosges R, Klimek L. Today’s allergic rhinitis patients are different: new factors that may play a role. Allergy. 2007;62:969–75. i 46. Bousquet J, Schunemann HJ, Fonseca J, Samolinski B, Bachert C, Canonica GW et al. MACVIA-ARIA Sentinel NetworK for allergic rhinitis (MASK-rhini- tis): The new generation guideline implementation. Allergy. 2015 [Epub f 35. Settipane RA, Lieberman P. Update on nonallergic rhinitis. Ann Allergy Asthma Immunol. 2001;86:494–507. g ahead of print]. 36. Bousquet PJ, Bachert C, Canonica GW, Casale TB, Mullol J, Klossek JM, et al. Uncontrolled allergic rhinitis during treatment and its impact on quality of life: a cluster randomized trial. J Allergy Clin Immunol. 2010;126:666–8. 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
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ПЕРЕВАГИ САМООСВІТНЬОЇ ДІЯЛЬНОСТІ МАЙБУТНІХ МАГІСТРІВ МЕДСЕСТРИНСТВА В УМОВАХ ДИСТАНЦІЙНОГО НАВЧАННЯ
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I. M. Melnychuk, S. O. Yastremska І. Ноrbachevsky Ternopil State Medical University THE ADVANTAGES OF SELF-EDUCATIONAL ACTIVITIES FOR THE FUTURE MASTERS OF NURSING WITHIN THE DISTANCE LEARNING Мета роботи – виявлення переваг, що сприяють формуванню самоосвітньої компетенції у майбутніх магістрів медсестрин- ства засобами дистанційного навчання. Основна частина. Аналіз наукових інтерпретацій поняття “самоосвіта” дозволив нам визначити самоосвітню компетентність як інтегровану характеристику особистості, що поєднує знання, вміння, способи і досвід самоосвіти і виявляється в потребі, здатності й готовності до реалізації такого виду діяльності, спрямованої на досягнення особистісної, професійної, соціальної самореалізації особистості. У статті відображено основні теоретичні підходи до визначення поняття “самоосвітня компетент- ність майбутніх магістрів медсестринства”, представлено результати діагностики цього особистісно-професійного утворення. В результаті наукової розвідки ми виокремили чотири компоненти в структурі самоосвітньої компетентності студентів медичних закладів вищої освіти. д щ Наголошено на необхідності нових підходів до організації навчального процесу в магістратурі медсестринського спрямування, і, передусім, це стосується організації самоосвітньої діяльності майбутніх магістрів медсестринства засобами дистанційного навчання. Висновок. Доведено, що потенціал електронного інформаційно-освітнього середовища створює необхідні умови для проек- тування студентами індивідуальної траєкторії самоосвіти, створення електронного кейса багажу знань, підвищує рівень емо- ційного сприйняття інформації, здійснює самоконтроль результатів навчальної діяльності, активізує самостійну пізнавальну ініціативність, мотивує студентів до самонавчання. Ключові слова: майбутні магістри медсестринства; самоосвіта; самоосвітня компетентність; самоконтроль; електронне інформаційно-освітнє середовище; засоби дистанційного навчання. The aim of the work – to identify the bene ts that contribute to the formation of self-educational competence in future masters of nursing by means of distance learning. The main body. The analysis of scienti c interpretations of the concept of “self-education” allowed us to de ne self-educational competence as an integrated personality characteristic that combines knowledge, skills, methods and experience of self-education and manifests itself in the need, ability and readiness for implementation of such kind of activity aimed at achieving personal, professional, and social self-realization. The article re ects the main theoretical approaches to the de nition of the concept of “self-educational competence of future masters of nursing”; the results of diagnostics of this person-professional phenomenon are presented. As a result of scienti c research, we have identi ed four components in the structure of self-educational competence of students of Medical Insti- tution of Higher Education. g The necessity of new approaches to the organization of the educational process in the magistracy of nursing is emphasized. First of all, this concerns the organization of self-education of future masters of nursing by means of distance learning. Conclusion. ДОСВІД З ОРГАНІЗАЦІЇ НАВЧАЛЬНОЇ РОБОТИ ДОСВІД З ОРГАНІЗАЦІЇ НАВЧАЛЬНОЇ РОБОТИ УДК 378.22:614.253.52:371.333:37.041 DOI 10.11603/me.2414-5998.2019.1.10091 УДК 378.22:614.253.52:371.333:37.041 DOI 10.11603/me.2414-5998.2019.1.10091 I. M. Melnychuk, S. O. Yastremska І. Ноrbachevsky Ternopil State Medical University THE ADVANTAGES OF SELF-EDUCATIONAL ACTIVITIES FOR THE FUTURE MASTERS OF NURSING WITHIN THE DISTANCE LEARNING It is proved that the potential of the digital informational and educational environment creates the necessary conditions for students to design an individual trajectory of self-education and create a digital portfolio of knowledge; it increases the level of emotional perception of information, caries out self-control of the results of educational activities, activates an independent cognitive initiative, and motivates students to self-study. Key words: future masters of nursing; self-education; self-educational competence; self-control; digital information environment; means of distance learning. самоосвіта є однією з головних умов успішного розвитку людського суспільства, світу в цілому і, звичайно, умовою життєвого успіху кожної окре- мо взятої особистості [1]. Особливого значення самоосвіта є однією з головних умов успішного розвитку людського суспільства, світу в цілому і, звичайно, умовою життєвого успіху кожної окре- мо взятої особистості [1]. Особливого значення У І. М. Мельничук, С. О. Ястремська Вступ. У сучасному інформаційному суспіль- стві динамічно зростають вимоги до професійних якостей фахівців. У цьому контексті безперервна Вступ. У сучасному інформаційному суспіль- стві динамічно зростають вимоги до професійних якостей фахівців. У цьому контексті безперервна У І. М. Мельничук, С. О. Ястремська ISSN 1681-2751. МЕДИЧНА ОСВІТА. 2019. № 1 96 ISSN 1681-2751. МЕДИЧНА ОСВІТА. 2019. № 1 96 ISSN 1681-2751. МЕДИЧНА ОСВІТА. 2019. № 1 96 ДОСВІД З ОРГАНІЗАЦІЇ НАВЧАЛЬНОЇ РОБОТИ І. Наумченко, Г. Сухобскої та ін. Шляхи та засо- би формування потреби в самоосвіті досліджені Г. Гнездиловим, Б. Райським, І. Редковець та ін. набуває самоосвіта в контексті навчання протягом життя, при якому основним суб’єктом планування, організації, регулювання і здійснення освіти може виступати тільки сама людина, а процес освіти пе- реростає в процес самоосвіти. Водночас такі інноваційні тенденції вітчизняної освіти, як: розвиток інформаційних технологій (С. Гончаренко, В. Красільнікова, Є. Полат та інші), зміна ролі самоосвіти на сучасному етапі розвитку суспільства, впровадження багаторівневої системи в практику МЗВО, призводять до виникнення й роз- ширення протиріч між зростанням вимог суспіль- ства до постійного підвищення рівня освіченості та конкурентоспроможності особистості й непід- готовленістю більшості студентів вищої школи підвищувати цей рівень у процесі самоосвіти [3]. Існує протиріччя й між необхідністю формування самоосвітньої компетентності студентів МЗВО в процесі вивчення дисциплін професійного спря- мування і нерозробленістю змісту і педагогічних умов формування цієї компетенції в освітньому процесі МЗВО. Разом з тим, більшість студентів медичних за- кладів вищої освіти (МЗВО) не готові і не вміють ефективно організувати процес самоосвітньої ді- яльності. Отже, в сучасних умовах актуалізується проблема формування самоосвітньої компетентності студен- тів МЗВО, вирішення якої сприятиме підвищенню якості підготовки майбутніх фахівців у контексті глобалізації освіти і підвищення їх конкуренто- спроможності на ринку праці [2]. У сучасній науково-педагогічній літературі на- громаджено достатньо інформації, що може бути основою для вирішення проблеми дослідження. Питання компетентнісного підходу в професій- ній освіті розглянуті в працях вітчизняних та за- рубіжних науковців: В. Антипової, В. Бездухова, Н. Бібік, В. Болотова, Ю. Варданяна, В. Вербиць- кого, Ф. Гоноболіна, І. Зимньої, І. Зязюна, І. Колес- нікової, Н. Кузьміної, Г. Лежніної, О. Локшиної, М. Лук’янової, А. Маркової, О. Овчарук, Г. Пахо- мової, О. Пометун, М. Філатова, Р. Хайгерті, А. Ху- торського та ін. Мета роботи – виявлення переваг, що сприяють формуванню самоосвітньої компетенції у майбут- ніх магістрів медсестринства засобами дистанцій- ного навчання. Основна частина. Нині необхідні нові підходи до організації навчального процесу в магістратурі медсестринського напряму, і, передусім, це стосу- ється самостійної роботи та самоосвітньої діяль- ності магістрантів. Застосування такого ресурсу, як самоосвіта (самоосвітня діяльність), відкриває для майбутніх магістрів медсестринства додаткові переваги, відображені на рисунку 1. ДОСВІД З ОРГАНІЗАЦІЇ НАВЧАЛЬНОЇ РОБОТИ ДОСВІД З ОРГАНІЗАЦІЇ НАВЧАЛЬНОЇ РОБОТИ Організація ефективного процесу освіти і само- освіти значною мірою визначається методами самоосвітньої діяльності в інформаційному се- редовищі [8], дидактичними властивостями реа- лізації яких є: можливість зберігання, переробки і передачі інформації будь-якого виду й обсягу та доступу до різних джерел інформації, організація Internet-конференцій, можливість використання сучасного програмного забезпечення професійного призначення. Одним із таких методів самоосвітньої діяльності є сучасні інформаційні засоби – сервіси Всесвітньої мережі “Інтернет”, які поєднують без- ліч світових інформаційних ресурсів [6, 9]. Зокрема, в роботах американських дослідників С. Меріана та Р. Кафелла (S. Merriam, R. Caffarella) самоосвітня діяльність трактується як форма на- вчання, в якій студенти несуть основну відповідаль- ність за планування, виконання та оцінювання сво- го досвіду навчання [4]. Тому пропонуємо розгляда- ти самоосвіту майбутніх магістрів медсестринства як специфічний вид діяльності, який здійснюється особистістю на добровільних засадах з метою за- доволення пізнавальних потреб або покращення власних особистісних властивостей і здібностей. У такому розумінні самоосвіти підкреслюється її діяльнісний складник [5], який реалізується від- повідно до задуму самої особистості (тобто цілі самоосвіти визначаються особистістю або сприй- маються нею як власні) [6]. Отже, суть самоосвіт- ньої діяльності полягає в тому, що особистість є суб’єктом своєї діяльності щодо досягнення вну- трішньо засвоєних нею цілей, які становлять основ- ний мотив самоосвіти. Для організації самоосвітньої роботи майбутніх магістрів медсестринства у МЗВО засобами дис- танційного навчання також використовуються елек- тронні навчальні курси, підручники, навчальні по- сібники, тренажери, лабораторні віртуальні практи- куми, стенди, мережеві проекти, телеконференції, тематичні каталоги з окремих мед сестринських сфер та ін. Тому нині ми розглядаємо можливості використання дистанційної самоосвіти, в межах якої студент та інформаційні джерела розділені в процесі навчання простором і часом. Освітній про- цес здійснюється з використанням комп’ютерних технологій та відеоконференцій, різних засобів отримання навчального матеріалу за допомогою web-сторінки, електронної пошти; дискусій учас- ників у web-форумах [10]. Отже, у системі профе- сійної підготовки майбутніх магістрів медсестрин- ства у МЗВО засобами дистанційного навчання використання всесвітньої інформаційної мережі сприяє встановленню атмосфери постійної само- освіти студентів, їхнього перетворення в дієвих суб’єктів пізнання і спілкування. З огляду на зміни в цільових характеристиках професійної підготовки майбутніх магістрів медсе- стринства, вважаємо, що самоосвітня діяльність – це цілеспрямована, самостійна, пізнавальна, практико- зорієнтована діяльність щодо розширення наявних знань і компетенцій та отримання нових в одній або декількох сферах медсестринської діяльності; це про- цес самовдосконалення особистості майбутніх магі- стрів медсестринства. Отже, довготривале та якісне отримання та структурування знань відбувається в результаті самоосвітньої діяльності студентів. Вступ. У сучасному інформаційному суспіль- стві динамічно зростають вимоги до професійних якостей фахівців. У цьому контексті безперервна Історичні аспекти проблеми самоосвіти відобра- жені в працях А. Айзенберга, А. Громцева, П. Ка- мерева, Б. Райського та ін. Сутність, особливості та функції самоосвіти, її місце в професійній ді- яльності фахівця визначені в роботах Н. Кузьміної, Рис. 1. Переваги самоосвітньої діяльності майбутніх магістрів медсестринства. Рис. 1. Переваги самоосвітньої діяльності майбутніх магістрів медсестринства. ISSN 1681-2751. МЕДИЧНА ОСВІТА. 2019. № 1 97 ДОСВІД З ОРГАНІЗАЦІЇ НАВЧАЛЬНОЇ РОБОТИ Самоосвітня діяльність є ключовою в системі підготовки майбутніх магістрів медсестринства у МЗВО засобами дистанційного навчання, оскільки цей процес організовується з метою суб’єктного розвитку студента й охоплює послідовний збір, пе- реробку й інтерпретацію професійно значущої ін- формації та є супутнім освітній діяльності в межах дистанційного навчання. Індивідуально-особис- тісний процес перетворення інформації в знання (самоосвіта) стає провідним видом діяльності в сучасному інформаційному суспільстві. Водночас проблема підвищення ефективності вищої медсестринської освіти полягає в пошуку шляхів вирішення протиріч між зростаючим обся- гом навчальної інформації та тенденцією до скоро- чення нормативних термінів освоєння цієї інфор- мації. У такому випадку досить дієвими є Інтернет і дистанційна самоосвіта. Тому для вирішення цієї проблеми у межах дослідження ми пропонуємо: Однією з форм самоосвітньої діяльності є само- стійна робота. Її відмінність від самоосвіти поля- гає в тому, що самостійна робота в основному по- роджується і керується ззовні, тоді як самоосвітня діяльність – внутрішніми мотивами, які, на думку Н. Воропай, виходять за межі навчальних [6]. Ін- коли самостійна робота і самоосвітня діяльність настільки тісно переплітаються, що їх складно розмежувати [7]. – планувати освітній процес на основі раціональ- ного використання можливостей Інтернету, що уможливлює навчання майбутніх магістрів мед- сестринства в оптимальному для кожного темпі та за індивідуальною освітньою траєкторією; – використовувати інформаційні технології (розробка програм дистанційного навчання) для контро лю якості виконаної самоосвітньої роботи. ISSN 1681-2751. МЕДИЧНА ОСВІТА. 2019. № 1 8 ISSN 1681-2751. МЕДИЧНА ОСВІТА. 2019. № 1 98 ДОСВІД З ОРГАНІЗАЦІЇ НАВЧАЛЬНОЇ РОБОТИ Характеризуючи самоосвітню діяльність майбут- ніх магістрів медсестринства як вид діяльності, що спрямований на реалізацію потреб особистості в соціалізації, підвищенні освітнього рівня, отриман- ня задоволення від здобутого самостійним шляхом знання, ми відзначаємо доступні, інформаційні, практико зорієнтовані можливості Інтернету в са- моосвітній діяльності студента. зультати констатувального етапу експерименталь- ної роботи показали, що в більшості студентів як контрольних, так і експериментальних груп само- освітня компетентність сформована недостатньо: на низькому рівні – 47 % (ННІ) і 52 % (ЖМІ), на середньому рівні – 45 % (ННІ) і 42 % (ЖМІ), на високому рівні – 8 % (ННІ), 6 % (ЖМІ). Формування компонентів самоосвітньої ком- петентності у студентів експериментальної гру- пи здійснювалося шляхом застосування засобів дистанційного навчання (електронні підручники, електронні бібліотеки, комп’ютерні навчальні і тестуючі системи, слайд-лекції (відео-, аудіолек- ції), тематичні сайти в локальній мережі (Інтра- нет), електронні навчально-методичні комплекси) і створення електронного інформаційного освіт- нього середовища. ДОСВІД З ОРГАНІЗАЦІЇ НАВЧАЛЬНОЇ РОБОТИ Аналіз різних інтерпретацій понять “самоосвіта” і “компетентність” дозволив нам визначити самоос- вітню компетентність як інтегровану характеристику особистості, що включає в себе знання, вміння, спо- соби й досвід самоосвіти, яка виявляється в потребі, здатності і готовності до реалізації даного виду дія- льності, спрямованої на досягнення особистісної, професійної, соціальної самореалізації особистості [11]. В результаті аналізу психолого-педагогічної літератури нами виділено чотири компоненти в структурі самоосвітньої компетентності студентів- магістрантів спеціальності “Медсестринство”. Використовувалася схема трифазного дистанцій- ного навчання. На першій очній фазі відбувалось ознайомлення студентів із сайтом; присвоєння кожному студенту власного логіна і пароля, за до- помогою яких уможливлюється вхід у систему; ін- структаж стосовно організації навчання та контро- лю; апробація студентами входу в систему та орга- нізації комунікативної взаємодії з викладачами. Перший компонент – мотиваційно-ціннісний, свідчить про сформованість потреби студентів до постійного саморозвитку та самовдосконалення, стійкого інтересу, що забезпечує позитивне став- лення до самоосвітньої діяльності, прагнення до постійної самоосвіти, усвідомлення її особистої і соціальної значущості. Друга – дистанційна фаза – передбачала само- стійну роботу студентів з електронними освіт- німи ресурсами, розміщеними на порталі ННІ медсестриства ТДМУ за електронною адресою: http://dl.tdmu.edu.ua. Під час дистанційної фази навчання використовувалися мережеві технології (автономні мережеві курси з використанням ме- режі “Інтернет”) та кейс-технології, засновані на комплектуванні наборів (кейсів) мультимедійних навчально-методичних матеріалів на електронних та інших носіях. За способом організації занять використовувалися дві форми дистанційного на- вчання – синхронне і асинхронне. Когнітивно-комунікативний компонент характе- ризує рівень знань студентів про сутність, форми, прийоми, способи самоосвіти, рівень володіння загальноосвітніми, предметними і професійними знаннями, здатність до безперервної особистої са- моосвіти шляхом застосування сучасних інформа- ційно-телекомунікаційних технологій. Операційно-діяльнісний компонент передбачає володіння сукупністю конструктивно-проекту- вальних, організаторських, комунікативних умінь, умінь самостійно організовувати свою самоосвітню діяльність, самостійно працювати з інформацією, проводити дослідження, виконувати міждисциплі- нарні проекти тощо. Третя, очна фаза, передбачала державний іспит – публічний захист виконаної магістерської роботи. До захисту магістерської роботи студент допус- кався, якщо рейтинг за результатами підсумкового контролю становив не менше 50 %. Четвертий компонент – особистісно-рефлексив- ний – передбачає аналіз і самооцінку студентами своєї готовності до самоосвіти, адекватну оцінку своїх досягнень, потребу в рефлексії самоосвіти. Динаміку змін у рівнях сформованості само- освітньої компетентності можна охарактеризува- ти як рух від керованої викладачем самоосвітньої діяльності студента до самостійної самоосвітньої діяльності майбутнього фахівця, які викликані внут рішніми мотивами і потребами. На констатувальному етапі експерименту, який проводився на базі Навчально-наукового інституту (ННІ) медсестринства ТДМУ та КВНЗ “Житомир- ський медичний інститут” (ЖМІ), нами виділені три рівні сформованості самоосвітньої компетент- ності студентів: низький, середній та високий. ДОСВІД З ОРГАНІЗАЦІЇ НАВЧАЛЬНОЇ РОБОТИ Ре- На заключному етапі ми провели повторні заміри рівнів сформованості самоосвітньої компетентнос- ISSN 1681-2751. МЕДИЧНА ОСВІТА. 2019. № 1 99 ДОСВІД З ОРГАНІЗАЦІЇ НАВЧАЛЬНОЇ РОБОТИ ті студентів МЗВО в контрольних і експеримен- тальних групах, підсумки яких показали позитивні результати. Динаміка рівнів сформованості само- освітньої компетентності наведена на рисунку 2. Рис. 2. Динаміка рівнів сформованості самоосвітньої компетентності майбутніх магістрів медсестринства. 45 40 35 30 25 20 15 10 5 0 Творчий Продуктивний Конструктивний Базовий Творчий Продуктивний Конструктивний Базовий Рис. 2. Динаміка рівнів сформованості самоосвітньої компетентності майбутніх магістрів медсестринства. Аналіз результатів показує однозначне підвищен- ня рівня сформованості самоосвітньої компетент- ності в експериментальних групах, у той час як у контрольних групах зміни в позитивну сторону також відбулися, але незначні. Так, збільшилася кількість магістрантів з творчим рівнем на 7,7 % у КГ і на 29,3 % в ЕГ; збільшилася кількість студентів із продуктивним рівнем на 5,8 % у КГ і на 8,0 % в ЕГ; зменшилася кількість магістрантів з конструк- тивним та базовим рівнем, відповідно, на 1,9 % та 14,7 % у КГ та на 11,6 % і 22,6 % в ЕГ. Висновок. Таким чином, потенціал електронно- го інформаційно-освітнього середовища створює необхідні умови для проектування індивідуальної траєкторії самоосвіти, створення електронного кей- са накопичених знань, підвищує рівень емоційно- го сприйняття інформації, здійснює самоконтроль студентів за результатами навчальної діяльності, активізує самостійну пізнавальну ініціативність, стимулює мотивацію до самонавчання. 5. Hayes C. Beyond the American Dream. Lifelong learning and the search for meaning in a postmodern world / C. Hayes. – Wasilla : Autodidactic Press, 2008. – 365 p. 4. Merriam S. B. Learning in Adulthood. A comprehensive guide / S. B. Merriam, R. S. Caffarella. – San Francisco : Jossey-Bass, 2011. – 229 р. 6. Воронова Л. И. Реформа медсестринского обра- зования. Медсестринский процесс. Основные этапы / Л. И. Воронова, Л. П. Попова // Головна медична сестра. 2005. – № 5. – С. 46–49. 4. Merriam S. B. Learning in Adulthood. A comprehensive guide / S. B. Merriam, R. S. Caffarella. – San Francisco : Jossey-Bass, 2011. – 229 р. 5. Hayes C. Beyond the American Dream. Lifelong learning and the search for meaning in a postmodern world / C. Hayes. – Wasilla : Autodidactic Press, 2008. – 365 p. 6. Воронова Л. И. Реформа медсестринского обра- зования. Медсестринский процесс. Основные этапы / Л. И. Воронова, Л. П. Попова // Головна медична сестра. 2005. – № 5. – С. 46–49. 7. Пелещишин А. М. Методи та алгоритми моделюван- ня Web-систем / А. М. Пелещишин. – URL : http://disser. com.ua/contents/31604.html (дата звернення 12.10.2015). 8. Топол І. Впровадження дистанційної форми навчан- ня у навчальний процес медичних вишів / І. Топол // Список літератури Сучасний стан та перспективи розвитку освітнього простору: глобальний вимір : матеріали Міжнар. наук.- практ. Інтернет-конф. (Запоріжжя, 25 листоп. 2016 р.). – Запоріжжя : ЗДІА, 2016. – С. 308–311. 9. Кисельова О. Б. Формування компетентності са- моосвіти у майбутніх педагогів в умовах інформацій- но-навчального середовища : автореф. дис. на здобуття наук. ступеня канд. пед. наук / О. Б. Кисельова. – Х., 2011. – 20 с. 11. Modeling and measuring competencies in higher education professional and vet learning / In: Sigrid Blömeke, Olga Zlatkin-Troitschanskaia, Christiane Kuhn, Judith Fege – eds. – 2014. – Vol. 1. – 231 p. 11. Modeling and measuring competencies in higher education professional and vet learning / In: Sigrid Blömeke, Olga Zlatkin-Troitschanskaia, Christiane Kuhn, Judith Fege – eds. – 2014. – Vol. 1. – 231 p. 11. Modeling and measuring competencies in higher education professional and vet learning / In: Sigrid Blömeke, Olga Zlatkin-Troitschanskaia, Christiane Kuhn, Judith Fege – eds. – 2014. – Vol. 1. – 231 p. 9. Кисельова О. Б. Формування компетентності са- моосвіти у майбутніх педагогів в умовах інформацій- но-навчального середовища : автореф. дис. на здобуття наук. ступеня канд. пед. наук / О. Б. Кисельова. – Х., 2011. – 20 с. Список літератури 4. Merriam S. B. Learning in Adulthood. A comprehensive guide / S. B. Merriam, R. S. Caffarella. – San Francisco : Jossey-Bass, 2011. – 229 р. 1. Овчарук О. Компетентності як ключ до оновлення змісту освіти / О. Овчарук // Стратегія реформування освіти в Україні: рекомендації з освітньої політики. – К. : К.І.С, 2003. – 225 с. 1. Овчарук О. Компетентності як ключ до оновлення змісту освіти / О. Овчарук // Стратегія реформування освіти в Україні: рекомендації з освітньої політики. – К. : К.І.С, 2003. – 225 с. 2. Коростіль Л. А. Самоосвіта особистості як соціаль- не та педагогічне явище / Л. А. Коростіль // Педагогічні науки : зб. наук. праць. – Суми : Видавництво СумДПУ, 2009. – № 1. – С. 138–145. 2. Коростіль Л. А. 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Пелещишин. – URL : http://disser. com.ua/contents/31604.html (дата звернення 12.10.2015). 8. Топол І. Впровадження дистанційної форми навчан- ня у навчальний процес медичних вишів / І. Топол // ISSN 1681-2751. МЕДИЧНА ОСВІТА. 2019. № 1 100 ISSN 1681-2751. МЕДИЧНА ОСВІТА. 2019. № 1 ДОСВІД З ОРГАНІЗАЦІЇ НАВЧАЛЬНОЇ РОБОТИ 10. Запорожан В. М. Взаємозв’язок мотивації досяг- нень та успіхів у навчанні у студентів медичного факуль- тету / В. М. Запорожан, Л. Р. Нікогосян, К. В. Аймедов // Медична освіта. – 2011. – № 2. – С. 108–113. Сучасний стан та перспективи розвитку освітнього простору: глобальний вимір : матеріали Міжнар. наук.- практ. Інтернет-конф. (Запоріжжя, 25 листоп. 2016 р.). – Запоріжжя : ЗДІА, 2016. – С. 308–311. References 7. Peleshchyshyn, A.M. (2015). Metody ta alhorytmy modeliuvannia Web-system [Methods and algorithms for modeling Web-systems]. Retrieved from: http://disser.com. ua/contents/31604.html (application date dated 12.10.2015). [in Ukrainian]. 7. Peleshchyshyn, A.M. (2015). Metody ta alhorytmy modeliuvannia Web-system [Methods and algorithms for modeling Web-systems]. Retrieved from: http://disser.com. ua/contents/31604.html (application date dated 12.10.2015). [in Ukrainian]. 1. Ovcharuk, O. (2003). Kompetentnosti yak kliuch do onovlennia zmistu osvity [Competencies as a key to updating the content of education]. Stratehiia reformuvannia osvity v Ukraini: rekomendatsii z osvitnoi polityky – Strategy for reforming education in Ukraine: recommendations for edu- cational policy. Kyiv: “K.I.S” [in Ukrainian]. 8. Topol, I. (2016). Vprovadzhennia dystantsiinoi formy navchannia u navchalnyi protses medychnykh vyshiv [Imple mentation of distance learning in the educational pro- cess of medical universities]. Suchasnyi stan ta perspektyvy rozvytku osvitnoho prostoru: hlobalnyi vymir: materialy Mizhnar. nauk.-prakt. Internet-konf. – Current State and Prospects of Educational Space Development: Global Di- mension: Materials of International Scienti c Practical Internet Conference. November, 25. Zaporizhzhia: ZDIA [in Ukrainian]. 2. Korostil, L.A. (2009). Samoosvita osobystosti yak sotsialne ta pedahohichne yavyshche [Self-education of the individual as a social and pedagogical phenomenon]. Pedahohichni nauky: zb. nauk. prats. – Pedagogical sciences: Collection of Scienti c works. Sumy: SumSPU Publishing House [in Ukrainian]. 3. Yastremska, S.O. (2017). Innovatsiinyi osvitnii protses pidhotovky maibutnikh mahistriv sestrynskoi spravy [Innovative educational process of preparing future masters of nursing]. Onovlennia zmistu, form ta metodiv navchannia i vykhovannia v zakladakh osvity: zb. nauk. prats [Naukovi zapysky Rivnenskoho derzhavnoho humanitarnoho universytetu]. Rivne – Improvement of content, forms and methods of education and education in educational insti- tutions: Collection of Scient. Works [Scienti c Notes of Rivne State Humanitarian University], 16 (59), 190-193 [in Ukrainian]. 3. Yastremska, S.O. (2017). Innovatsiinyi osvitnii protses pidhotovky maibutnikh mahistriv sestrynskoi spravy [Innovative educational process of preparing future masters of nursing]. Onovlennia zmistu, form ta metodiv navchannia i vykhovannia v zakladakh osvity: zb. nauk. prats [Naukovi zapysky Rivnenskoho derzhavnoho humanitarnoho universytetu]. Rivne – Improvement of content, forms and methods of education and education in educational insti- tutions: Collection of Scient. Works [Scienti c Notes of Rivne State Humanitarian University], 16 (59), 190-193 [in Ukrainian]. 9. Kyselova, O.B. (2011). Formuvannia kompetentnosti samoosvity u maibutnikh pedahohiv v umovakh informatsiino-navchalnoho seredovyshcha [Formation of competency of self-education of future teachers in conditions of informational and educational environment]. Candidate’s Extended abstract [in Ukrainian]. 10. Zaporozhan, V.M., Nikohosian, L.R., & Aime- dov, K.V. (2011). References Vzaiemozviazok motyvatsii dosiahnen ta uspikhiv u navchanni u studentiv medychnoho fakultetu [The relationship between the motivation of achievements and the success of studying in students of the medical faculty]. Medychna osvita – Medical Education, 2, 108-113 [in Ukrainian]. 10. Zaporozhan, V.M., Nikohosian, L.R., & Aime- dov, K.V. (2011). Vzaiemozviazok motyvatsii dosiahnen ta uspikhiv u navchanni u studentiv medychnoho fakultetu [The relationship between the motivation of achievements and the success of studying in students of the medical faculty]. Medychna osvita – Medical Education, 2, 108-113 [in Ukrainian]. 4. Merriam, S.B., & Caffarella, R. S. (2011). Learning in adulthood. A comprehensive guide. San Francisco: Jossey- Bass. 4. Merriam, S.B., & Caffarella, R. S. (2011). Learning in adulthood. A comprehensive guide. San Francisco: Jossey- Bass. 4. Merriam, S.B., & Caffarella, R. S. (2011). Learning in adulthood. A comprehensive guide. San Francisco: Jossey- Bass. 5. Hayes, C. (2008). Beyond the American dream. Lifelong learning and the search for meaning in a postmodern world. Wasilla: Autodidactic Press. 11. Sigrid Blömeke, Olga Zlatkin-Troitschanskaia, Christiane Kuhn, & Judith Fege (Eds.). (2014). Modeling and measuring competencies in higher education profes- sional and vet learning. 11. Sigrid Blömeke, Olga Zlatkin-Troitschanskaia, Christiane Kuhn, & Judith Fege (Eds.). (2014). Modeling and measuring competencies in higher education profes- sional and vet learning. 6. Voronova, L.I., & Popova, L.P. (2005). Reforma medsestrynskogo obrazovaniya. Medsestrynskiy protsess. Osnovnye etapy [Nursing education reform. Nursing pro- cess. Basic steps]. Holovna medychna sestra – Chief Nurse, 5, 46-49 [in Russian]. Отримано 25.01.19 Отримано 25.01.19 Електронна адреса для листування: yastremska@tdmu.edu.ua Електронна адреса для листування: yastremska@tdmu.edu.ua ISSN 1681-2751. МЕДИЧНА ОСВІТА. 2019. № 1 101
https://openalex.org/W2763180071
http://www.scielo.br/pdf/ac/v17n4/1678-8621-ac-17-04-0009.pdf
English
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Adoption of environmental practices on construction sites
Ambiente Construído/Ambiente construído
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THOMAS, N. I. R.; COSTA, D. B. Adoption of environmental practices on construction sites. Ambiente Construído, Porto Alegre, v. 17, n. 4, p. 9-24, out./dez. 2017. ISSN 1678-8621 Associação Nacional de Tecnologia do Ambiente Construído. http://dx.doi.org/10.1590/s1678-86212017000400182 Abstract ustainability on construction sites and its impacts on the environment have become increasingly relevant. Large quantities of materials, water and energy, among other resources of various types and origins, are consumed on construction sites during the production activities and by the temporary facilities. This paper aims at presenting a set of criteria for the adoption of sustainable management practices on construction sites aiming at mitigating environmental impacts produced during the construction phase. An in- depth case study was carried out on the new building of the Construction Chamber for the state of Bahia, Brazil. Different environmental impacts for each construction activity specific to the construction site were studied and an economic feasibility study of each solution was performed, followed by the implementation of these practices. Monitoring data was collected through checklists, indicators and periodic project management meetings. Participant observation and document analysis were also used as sources of evidence. The main findings refer to the identification of solutions, main difficulties in adopting such solutions, the development of indicators for monitoring low environmental impact on construction sites, as well as a set of recommendations for the deployment of sustainable practices on construction sites. S S Keywords: Sustainability. Construction site. Environmental practices. Management. Palavras-chave: Sustentabilidade. Canteiro de obra. Práticas ambientais. Gestão. 7 7 Dayana Bastos Costa Departamento de Construção e Universidade Federal da Bahia Salvador - BA - Brasil Resumo A sustentabilidade nos canteiros de obra e seus impactos no meio ambiente vêm se tornando bastante relevantes. Grande quantidade de materiais, água e energia, entre outros recursos de diversos tipos e origens, é consumida durante a execução das atividades de construção, o que inclui as instalações provisórias. Este artigo visa apresentar um conjunto de critérios para a adoção de práticas gerenciais sustentáveis em canteiros de obra visando à mitigação de impactos ambientais produzidos durante a fase de execução. O estudo de caso foi realizado na nova sede do Sindicato da Indústria da Construção Civil da Bahia, Brasil. Foram estudados diferentes impactos ambientais para cada atividade de construção do canteiro estudado, bem como se realizou uma análise de viabilidade para cada solução, seguida da implementação das práticas. Foram coletados dados relativos ao monitoramento da implementação das práticas por meio de listas de verificação, indicadores e reuniões periódicas com os gestores. As principais fontes de evidência foram observação participante e análise de documentos. Os principais resultados encontrados estão relacionados à identificação das soluções, às principais dificuldades na adoção dessas soluções, ao desenvolvimento de indicadores para monitoramento nos canteiros e a um conjunto de recomendações para a adoção de práticas sustentáveis nos canteiros de obra. Natasha Ilse Rothbucher Thomas Dayana Bastos Costa Departamento de Construção e Universidade Federal da Bahia Salvador - BA - Brasil Dayana Bastos Costa Departamento de Construção e Universidade Federal da Bahia Salvador - BA - Brasil Introduction Noise emission is also an important issue, as the construction site activities temporarily increase noise pollution, and are often set in the urban environment (BALLESTEROS et al., 2010). The construction process itself causes different environmental impacts (KILBERT, 2005, UNITED..., 2006, GANGOLELLS et al., 2011, ABDUL-RAHMAN et al., 2016). Bulk materials, water and electricity among other resources of various types and origins are consumed on construction sites during the different production activities and by the temporary facilities. These activities generate solid waste and particle emissions causing a concern with soil contamination, air and water pollution and soil erosion (GANGOLELLS et al., 2014). Noise emission is also an important issue, as the construction site activities temporarily increase noise pollution, and are often set in the urban environment (BALLESTEROS et al., 2010). Recebido em 13/05/17 Aceito em 30/06/17 Recebido em 13/05/17 Aceito em 30/06/17 Palavras-chave: Sustentabilidade. Canteiro de obra. Práticas ambientais. Gestão. 9 Ambiente Construído, Porto Alegre, v. 17, n. 4, p. 9-24, out./dez. 2017. Thomas, N. I. R.; Costa, D. B. 10 Introduction On a global scale, the construction industry is responsible for about 50% of CO2 emitted into the atmosphere, 20-50% of all natural resources consumed and 50% of all solid waste produced, some of which occurs during the construction phase of the building’s life cycle, causing many environmental impacts (PULASKI, 2004; KHASREEN; BANFILL; MENZIES, 2009; PROBERT et al., 2010). The growth of the construction industry and its subsequent environmental impacts highlight the importance of the need for sustainable construction processes and actual ways to manage measures of sustainability (TAM et al., 2006; DING, 2008). Greater recognition within the architecture-engineering- construction (AEC) industry of the increase opportunity to influence the sustainability of project outcomes could lead to significant sustainability improvements in the AEC industry (HOLLOWAY; PARRISH, 2015). However, there is a lack of knowledge on the available solutions, technologies and procedures involved with the employment of sustainable practices on construction sites (GANGOLLES et al., 2009). It is necessary to know the legislations, resources and minimum conditions required to deploy solutions after performing a technical and economic feasibility study based on the particularities of the construction site, specific production activities, logistics, financial constraints and human resources. In addition, there is a need to develop tools for measuring and monitoring the implementation of sustainable practices on site during the execution phase. The objective of this paper is to develop a set of criteria for the adoption of environmental practices and assessing sustainable construction site aimed at mitigating the environmental impacts produced during the construction process. This study is based on an in-depth case study on the New Headquarters of the Construction Chamber for the state of Bahia (Sinduscon-BA), Brazil. In order to propose these criteria, information was obtained from a literature review on the guidelines suggested by the environmental assessment methods and from a previous work (GUIMARÃES, 2013; THOMAS; COSTA; GUIMARÃES, 2013), which aimed to identify priority needs for sustainable solutions for construction site developments. The construction process itself causes different environmental impacts (KILBERT, 2005, UNITED..., 2006, GANGOLELLS et al., 2011, ABDUL-RAHMAN et al., 2016). Bulk materials, water and electricity among other resources of various types and origins are consumed on construction sites during the different production activities and by the temporary facilities. These activities generate solid waste and particle emissions causing a concern with soil contamination, air and water pollution and soil erosion (GANGOLELLS et al., 2014). Environmental practices in construction sites The different environmental assessment methods for sustainable buildings present a series of good practice guidelines to be applied during the construction phase. Table 1 presents the contribution from BREEAM (Building Research Establishment Environmental Assessment Methodology (2009)), LEED (Leadership in Energy and Environmental Design (UNITED…, 2009)), AQUA-HQE process, an environmental assessment method derived from the French Haute Qualité Environnementale (HQE) method adapted by the Alberto Vanzolini Foundation for Brazil (FUNDAÇÃO…, 2010), and Selo Casa Azul, which was developed by the Brazilian Government owned bank, the CAIXA Econômica Federal, being a social-environmental classification instrument used for residential building projects (JOHN; PRADO, 2010). Therefore, the environmental impact of construction has become of high relevance. However, the identification and monitoring of these environmental impacts have to date received little attention. It is very important to predict what the environmental impacts of construction are and identify how they can be prevented before starting an activity (IRIZARRY et al., 2012). Furthermore, many of the impacts can be controlled and mitigated by the project management team during the design and pre-construction of a building (KILBERT, 2005) thus improving the sustainability of the construction phase of a building. Ambiente Construído, Porto Alegre, v. 17, n. 4, p. 9-24, out./dez. 2017. Table 1 - Contributions towards sustainable construction sites by EAM Generic Topics Environmental Aspects Environmental Assessment Methods (EAM) AQUA Process BREEAM LEED SELO CASA AZUL Resource Consumption Material Resources X X X X Energy Efficiency X X Water Management X X X X Emissions and Solid Waste Waste X X X X Air Pollution X X X Water and Soil Pollution X X X X Interface with External Environment Urban/Environment Quality X X X Noise Pollution X X X Visual Pollution X X X Intrinsic Quality of the Construction Site Health and Safety X X Temporary Installations X X X X Innovation X X Source: Thomas, Costa and Guimarães (2013). Table 1 - Contributions towards sustainable construction sites by EAM Although all assessment methods show differences within their methods of criteria evaluation and their categories, it can be seen that all criteria fall under similar themes, forming a flow system related to inputs and outputs (consumptions and emissions) within the physical, biotic and anthropic context of the construction enterprise, and its relationship with the external and internal environment, according to Degani’s (2003) classification. Environmental practices in construction sites Therefore, the discussion on sustainable construction sites will be focused on the following four elements: Resource Consumption, Emissions and Solid Waste, Interface with External Environment and Intrinsic Quality of the Construction Site. By adopting good practices, many of the environmental aspects and their associated impacts can be reduced or almost completely mitigated. construction site activities, can be adopted to minimize risks from air pollution on the occupational health of site workers and the neighborhood (KUKADIA; UPTON; HALL, 2003, UNITED…, 2006; COUNCIL…, 2010; ARAÚJO; COSTA; MORAES, 2014). Wastewater produced during site activities can also cause groundwater contamination and natural watercourse pollution, and therefore requires wastewater management plans that control infiltration and surface run-off (KIM et al., 2005; NETREGS…, 2012). Best practices are therefore required to minimize such risks (PULASKI, 2004). The interface with external environment is another important aspect and may involve urban and environmental quality, noise pollution and visual pollution. The distress and annoyances, resulting from site traffic and machinery arising directly or indirectly from the production activities, cause visual and noise pollution (GEHLEN, 2008). Abdul-Rahman et al. (2016) recommends that possible visual and noise pollution from the construction project and the effects on the local climate should be investigated. The environmental aspects related to resource consumption involve material resources, energy efficiency and waste management. Those aspects related to resource consumption can be reduced through rational resource use, coordinated and modular design principles and adopting practices that reduce material losses and selecting materials, products and construction systems with low environmental impacts and low embodied energy considering its whole life cycle process (BERGE, 2009). The generation of waste products during the different site production activities can be reduced through adequate planning and management. The waste can potentially be valorized as a raw material or energy source (RAO; JHA; MISRA, 2007; GANGOLELLS et al., 2014). It is also important to control the intrinsic quality of the construction site, such as health and safety, temporary installations and innovation. The impacts caused on the health, safety and welfare of site workers should be controlled (HINZE; GODFREY; SULLIVAN, 2013; GANGOLELLS et al., 2013) and must also be considered by installing appropriate collective and individual protections during the different activities (PULASKI, 2004). Improvements on the internal air quality, comfort and environmental performance of temporary facilities may also be required (ARAÚJO, 2009). Adoption of environmental practices on construction sites Environmental practices in construction sites In addition, innovative technologies can provide significant improvements Waste, air pollution, and water and soil pollution are environmental aspects related to Emissions and Waste. Best practices and technologies, aimed at controlling particle emissions during the different 11 Ambiente Construído, Porto Alegre, v. 17, n. 4, p. 9-24, out./dez. 2017. on the performance of site equipment and temporary facilities. important and very important (I & VI), as well as the practices (PA) less applied by the respondents (see Table 2). Based on an online survey responded by 66 high- level professionals from development and construction companies from four metropolitan regions of Brazil, reported by Guimarães (2013) and Thomas, Costa e Guimarães (2013), a set of priorities was established and categorized in four elements: Resource Consumption, Emissions and Solid Waste, Interface with External Environment, and Intrinsic Quality of the Construction Site. Those authors classified as research priorities the technology solutions and practices considered According to Thomas, Costa e Guimarães (2013), the technological solutions and practice needs identified in Table 2 can help governments, research academies, entity classes and private industries in prioritizing funding and resources for further research. Especially in this work, this list guided the deployment of environmental good practices to be selected and analyzed based on the context and resource required. Table 2 - Technological solutions and practices identified as research priorities (Continues...) Technological Solutions and Practices Research Priority I&VI (%) PA (%) Resource Consumption Reduce material losses during the carrying out of services, deliveries, internal transport, storage, and handling of materials on construction sites. 100 58 Development of materials, products and construction systems selection criterion from responsible sources that include life cycle analysis information (origin, processing, use e maintenance, durability e disposal) and material properties like technical performance. 95 52 Improvements of temporary installations with the aim of reducing potable water and electric energy consumption, as also in equipment, lighting and air conditioning systems on construction sites 86 29 Reduction of electric energy consumption during the production activities on construction sites 85 18 Development of a supplier analysis system formalizing aspects related to federal revenues, use of environmental operating licenses, social responsibility, correct use of codes and standards, etc. Thomas, N. I. R.; Costa, D. B. 12 Environmental practices in construction sites 85 39 Rainwater use on construction sites 59 5 Use of alternative energy sources including renewable energy 48 0 Greywater use on construction sites 44 2 Emissions and Solid Waste Control of generation, quantification, separation, storage and disposal of construction waste on construction sites including Take-back scheme (return of waste products to manufacturers) 97 39 Development of decontamination technologies and disposal solutions for dangerous construction waste products such as paints, solvents, oils or other contaminated waste products arising from demolition, renovations, radiological clinical repairs, industrial installations among others (Class D) 94 9 Exploitation and use of construction waste on construction sites 91 23 Development of technologies and economically viable applications of construction waste materials that still have no recycling or recuperation solutions (Class C) 89 17 Vegetation preservation methods in construction site areas 85 33 Sewage catchment and treatment systems and minimization of risks resulting from drainage on construction sites 85 21 Identification of risks of particle emissions arising from production activities on construction sites and towards the neighborhood 85 21 Control of groundwater levels, contamination and minimization of risks resulting from its management 85 21 Characterization, monitoring e control of particle material emissions 74 12 Table 2 - Technological solutions and practices identified as research priorities (Continues...) Resource Consumption Ambiente Construído, Porto Alegre, v. 17, n. 4, p. 9-24, out./dez. 2017. Table 2 - Technological solutions and practices identified as research priorities (continuation) Table 2 - Technological solutions and practices identified as research priorities (continuation) Technological Solutions and Practices Research Priority I&VI (%) PA (%) Interface with External Environment Reduce the erosive processes and risks of collapse/subsidence 98 53 Characterization of sound emitting activities and plans to mitigating noise nuisance 92 27 Management of site access, pedestrian flow, equipment, loading and loading on construction sites 92 55 Reduction of the site interferences with local traffic, street conservation and pavements to guarantee accessibility 91 47 Identification of risks of particle emissions derived from production activities on the neighborhood. 88 30 Definition of demolition processes and contingency plans 80 29 Intrinsic Quality of the Construction Site (IQ) - Health and Safety Collective protection equipment against falling from heights on construction sites 98 77 Protections against people handling equipment 98 76 Protections against electric shocks 98 76 Safeguards for material handling equipment 97 71 Machine and equipment protection 97 68 Protection of excavations 95 70 National certification system for Industrialized Collective Protection Equipment, similar to existing certification system for Personal Protective Equipment (PPE) on construction sites 79 41 Intrinsic Quality of the Construction Site (IQ) -Temporary Installations on Construction Sites Improvements of work safety conditions inside the temporary installations 97 55 Improvements of the health and hygiene conditions inside the temporary installations 97 62 Reuse of temporary installation components and construction systems 92 42 Improvements in the lighting, ventilation and air quality conditions of temporary installations 89 41 External visual communication and signaling 86 67 I – Important; VI – Very Important; PA – Practices Adopted Souce: Thomas Costa e Guimarães (2013) Adoption of environmental practices on construction sites 13 Research method The identification of possible best practices to be implemented was based on the literature review on the different environmental assessment methods (previously presented in Table 1), and from the findings of a survey on sustainable best practices on construction sites applied in Brazil by Guimarães (2013) and Thomas, Costa and Guimarães (2013) presented in Table 2. Also, good practices on solid waste were based on the National Environment Council (CONAMA) Resolution 307, which establishes directives, criteria and procedures for the management of waste deriving from construction in Brazil (BRASIL, 2002). Health and Safety practices were based on the NR-18 standards (Regulatory Standard -18 - Working conditions and the Environment in the Construction Industry in Brazil) (BRASIL, 2015). Air pollution practices were based on the London Council publication (COUNCILS..., 2010). This work was developed using the in-depth case study strategy, which was conducted on the construction site of the new headquarters of Sinduscon-BA. This case was selected because it was the first sustainable commercial building developed in the city of Salvador and due to the opportunity to be involved from the conceptual design to handover of the project. In order to adopt best practices for a low environmental impact construction site, this study was divided into three stages: (a) identification of best practices; (b) selection and Implementation of best practices; and (c) monitoring, actions and learning (Figure 1). (d) Identification of best practices 13 Adoption of environmental practices on construction sites 1 Ambiente Construído, Porto Alegre, v. 17, n. 4, p. 9-24, out./dez. 2017. Figure 1 – Research design Figure 1 – Research design Before selecting the good practices, it was necessary to characterize the construction site in terms of its localization, construction method, ground conditions and topography, presence of water courses, water table level, presence of fauna and flora, neighborhood conditions such as nearby roads, parking, current noise levels, surrounding building types and use, such as hospitals, schools, residential housing and neighborhood habits Other characteristics were also evaluated, such as external risks, overhead electricity cables and pylons, wind direction, natural risks (flooding, landslides), local expectations of neighborhood and waste disposal areas, to mention a few. Selection and implementation of best practices The selection of good practices and solutions to mitigate the environmental impacts of the building was carried out through weekly progress meetings during the first six months of the project with the project team, including the project engineer, general contractor, the project and environmental manager and the owners (Chamber CEO and Project Director) (Table 3). Then, monthly meetings and videoconferences with the AQUA- HQE consultancy company were held to validate the selected practices. The following evaluation criteria were adopted to assist in the selection of practices and solutions: Based on Araújo (2009), a matrix was created in an Excel sheet listing all of the construction site activities in the left hand vertical column and then, the different environmental aspects for each theme were listed in the top horizontal line (Table 3). For each environmental aspect, an analysis was made marking an “X” for all of the construction activities imposing a high potential risk of occurrence and an “O” for low risk of occurrence. Squares were left blank if there was no correlation between activity and environmental aspect. This way, the most important environmental aspects could be prioritized for each construction activity allowing for the selection of good practices according to each environmental aspect for the different construction activities. For each aspect, the current legislation limits were identified. (a) costs, including resources needed, acquisition costs, maintenance costs, supplier diversity, deadlines, ease of acquisition, transport and delivery, import dependence, lifetime guarantee of performance and disposal; (a) costs, including resources needed, acquisition costs, maintenance costs, supplier diversity, deadlines, ease of acquisition, transport and delivery, import dependence, lifetime guarantee of performance and disposal; (b) efficiency, including user safety, durability, productivity, observance of deadlines and integration with other subsystems, adaptability to particularities of other construction sites; and (c) ease of deployment, such as physical space, constructability, dependence on equipment, dependence on manpower, ease of mobility, and interference with time schedules. (b) efficiency, including user safety, durability, productivity, observance of deadlines and integration with other subsystems, adaptability to particularities of other construction sites; and (c) ease of deployment, such as physical space, constructability, dependence on equipment, dependence on manpower, ease of mobility, and interference with time schedules. (c) ease of deployment, such as physical space, constructability, dependence on equipment, dependence on manpower, ease of mobility, and interference with time schedules. dependence on manpower, ease of mobility, and interference with time schedules. Thomas, N. I. R.; Costa, D. B. 14 Selection and implementation of best practices The spreadsheet presented in Table 4 was developed to determine how and when to deploy each solution, the minimum conditions for Ambiente Construído, Porto Alegre, v. 17, n. 4, p. 9-24, out./dez. 2017. deployment and necessary resources. From this, it was possible to compile the Best Practice Deployment Plan according to the construction schedule, setting goals and responsibilities for implementation and indicators for monitoring and control. This plan and its policies were included into the general contractor´s quality management system and the specific control datasheets were elaborated and master sheets updated. The best practices were inserted into the operational and management training program and all of the construction site projects were adjusted accordingly. The implementation of the good practices was carried out through training workers at daily health and safety meetings and appointing the health and safety manager to supervise the implementation progress. In addition, a visual communication program showing good practices was provided. Table 3 - Example of matrix of environmental aspect and potential environmental impact for new Sinduscon-BA headquarters Environmental Aspect Consumption Emissions and Waste Interface with External Environment Material Resource Energy Efficiency Water Management Solid Waste Air Pollution Water Pollution Urban Quality Noise Visual Pollution Preliminary Works Demolition X X X X X X X X Land Cleaning X X X X X X X Temporary Installation X X X X X X X X Temporary electrical and water installation X X X X X Source: based on Araújo (2009). Table 3 - Example of matrix of environmental aspect and potential environmental impact for new Sinduscon-BA headquarters Environmental Aspect Consumption Emissions and Waste Interface with External Environment Material Resource Energy Efficiency Water Management Solid Waste Air Pollution Water Pollution Urban Quality Noise Visual Pollution Preliminary Works Demolition X X X X X X X X Land Cleaning X X X X X X X Temporary Installation X X X X X X X X Temporary electrical and water installation X X X X X Source: based on Araújo (2009). Table 4 - Evaluation of the implementation of good practices Good Practices How to implement the Good Practice Responsible Resource needed Phase for Application Cost Easy of deployment Efficiency Total Y/N Reduce materials losses during the carrying out of services, deliveries, internal transport, storage, and handling of materials on construction sites. Adoption of environmental practices on construction sites 15 Monitoring, action and learning Checklist criterion was evaluated via a 0–1 Scale (1 for compliance and 0 for noncompliance). The checklist was applied on a weekly basis for 13 weeks to ensure effective implementation of practices. From the checklist, 7 indicators were calculated, allowing for a quantitative analysis and definition of actions for continuous improvement, using previously raised indicators as a basis of comparison. Table 5 shows the indicators, the main good practices evaluated in the case study and the formula for each indicator. To monitor the implementation process, an operational checklist was developed aimed at assessing the main good practices implemented for each aspect – resource consumption, solid waste, emissions, temporary facilities, health and safety and outdoor quality. It is important to note that despite the effort to be comprehensive in the selection of the practices, due to the characteristics of the project, a limited number of practices were implemented. Table 5 - Indicators and good practices (Continues...) Indicator Good Practice Evaluated Formula Material, Water and Energy Consumption Index 1. Correct storage of materials 2. Materials identified in the warehouse 3. Dissemination of monthly control of water consumption 4. Daily shutdown of hydraulic devices 5. Dissemination of monthly control of energy bill 6. Daily power disconnection of electrical devices 𝛴 𝐶𝑜𝑛𝑠𝑢𝑚𝑝𝑡𝑖𝑜𝑛 𝑖𝑡𝑒𝑚𝑠 𝑐𝑜𝑚𝑝𝑙𝑒𝑡𝑒𝑑 𝑥 100 𝛴𝐶𝑜𝑛𝑠𝑢𝑚𝑝𝑡𝑖𝑜𝑛 𝑖𝑡𝑒𝑚𝑠 𝑒𝑣𝑎𝑙𝑢𝑎𝑡𝑒𝑑 Solid Waste Index 1. Segregation of aggregates in the correct location 2. No mixed waste in disposal drums 3. Conduits and material remains in correct boxes 4. Correct waste disposal with transport, and final destination tracking and control 5. Separation of hazardous waste and storage in confined area with protected floor 𝛴 𝑊𝑎𝑠𝑡𝑒 𝑖𝑡𝑒𝑚𝑠 𝑐𝑜𝑚𝑝𝑙𝑒𝑡𝑒𝑑 𝑥 100 𝛴 𝑊𝑎𝑠𝑡𝑒 𝑖𝑡𝑒𝑚𝑠 𝑒𝑣𝑎𝑙𝑢𝑎𝑡𝑒𝑑 Water, Soil and Air Pollution Index 1. Use of circular saw and bench with waste collection 2. Wetting of areas during production activities and when sweeping 3. Wetting of materials when cutting with circular saw 4. Use of plastic construction safety nets 5. Effluent control during concreting and other production activities 𝛴 𝑃𝑜𝑙𝑙𝑢𝑡𝑖𝑜𝑛 𝑖𝑡𝑒𝑚𝑠 𝑐𝑜𝑚𝑝𝑙𝑒𝑡𝑒𝑑 𝑥 100 𝛴 𝑃𝑜𝑙𝑙𝑢𝑡𝑖𝑜𝑛 𝑖𝑡𝑒𝑚𝑠 𝑒𝑣𝑎𝑙𝑢𝑎𝑡𝑒𝑑 Temporary Facilities Index 1. Visual communication signs in good condition and correctly located on jobsite 2. Use and respect of construction site project 3. Daily washing, sanitation and fumigation of internal areas, cloakroom and dining areas 4. Cleaning, organizing and sweeping of internal areas 5. Tools in specific boxes 6. Nuts and bolts in the boxes Σ 𝑇. Thomas, N. I. R.; Costa, D. B. 16 Selection and implementation of best practices Design compatibilities through BIM BIM Training sessions Design Coordinator Time, space, people, money Program 3 2 3 8 N Meeting for design compatibilities Design Coordinator Design 0 3 3 6 Y Use of BIM- REVIT Design Coordinator REVIT Design 3 1 3 7 N Materials identified in the warehouse Waste management plan Field Engineer Consultant Execution 1 1 3 5 Y Training sessions for implementation of waste management Field Engineer Consultant Execution 1 1 3 5 Y Supply plan Field Engineer Execution 0 2 3 5 Y mple of matrix of environmental aspect and potential environmental impact for new h d t Table 3 - Example of matrix of environmental aspect and potential environmental impact for new Sinduscon-BA headquarters Table 4 - Evaluation of the implementation of good practices Good Practices How to implement the Good Practice Responsible Resource needed Phase for Application Cost Easy of deployment Efficiency Total Y/N Reduce materials losses during the carrying out of services, deliveries, internal transport, storage, and handling of materials on construction sites. Design compatibilities through BIM BIM Training sessions Design Coordinator Time, space, people, money Program 3 2 3 8 N Meeting for design compatibilities Design Coordinator Design 0 3 3 6 Y Use of BIM- REVIT Design Coordinator REVIT Design 3 1 3 7 N Materials identified in the warehouse Waste management plan Field Engineer Consultant Execution 1 1 3 5 Y Training sessions for implementation of waste management Field Engineer Consultant Execution 1 1 3 5 Y Supply plan Field Engineer Execution 0 2 3 5 Y Table 4 - Evaluation of the implementation of good practices ce materials losses during the carrying out of services, deliveries, internal transport, storage, and ing of materials on construction sites. Adoption of environmental practices on construction sites 15 Ambiente Construído, Porto Alegre, v. 17, n. 4, p. 9-24, out./dez. 2017. Adoption of environmental practices on construction sites Monitoring, action and learning 𝐹𝑎𝑐𝑖𝑙𝑖𝑡𝑦 𝑖𝑡𝑒𝑚𝑠 𝑐𝑜𝑚𝑝𝑙𝑒𝑡𝑒𝑑 𝑥 100 Σ 𝑇. 𝐹𝑎𝑐𝑖𝑙𝑖𝑡𝑦 𝑖𝑡𝑒𝑚𝑠 𝑒𝑣𝑎𝑙𝑢𝑎𝑡𝑒𝑑 Table 5 - Indicators and good practices (Continues...) Ambiente Construído, Porto Alegre, v. 17, n. 4, p. 9-24, out./dez. 2017. Table 5 - Indicators and good practices (continuation) Indicator Good Practice Evaluated Formula Health and Safety Index 1. Use of Individual Safety Equipment: hearing protectors, helmet, footwear, glasses, safety strap 2. Use of delimitation of areas 3. Guardrails in good condition 4. Rack and pinion elevators operated by qualified professional 5. Use of trays and safety protection screens against falls 6. Suspended scaffolding correctly used Σ 𝐻. 𝑆𝑎𝑓𝑒𝑡𝑦 𝑖𝑡𝑒𝑚𝑠 𝑐𝑜𝑚𝑝𝑙𝑒𝑡𝑒𝑑 𝑥 100 Σ 𝐻. 𝑆𝑎𝑓𝑒𝑡𝑦 𝑖𝑡𝑒𝑚𝑠 𝑒𝑣𝑎𝑙𝑢𝑎𝑡𝑒𝑑 Outdoor Quality Index 1. Access and material flow management – Isolation of loading and unloading areas and Obedience of loading and unloading times 2. Maintenance and cleaning of the surroundings - routine cleaning around construction site with daily sweeping of the pavements and Use of canvas during concrete discharge 3. Good Neighbor Relationship - use of signs on jobsite facade with e-mail for complaints and Book of occurrences in the jobsite entrance 4. Noise pollution - Definition of strategic locations for noisy equipment 5. Visual pollution - The fences are well maintained and around the material storage deposits, and other obstacles belonging to jobsite Σ 𝑂. 𝑄𝑢𝑎𝑙𝑖𝑡𝑦 𝑖𝑡𝑒𝑚𝑠 𝑐𝑜𝑚𝑝𝑙𝑒𝑡𝑒𝑑 𝑥 100 Σ 𝑂. 𝑄𝑢𝑎𝑙𝑖𝑡𝑦 𝑖𝑡𝑒𝑚𝑠 𝑒𝑣𝑎𝑙𝑢𝑎𝑡𝑒𝑑 Low-impact Environmenta l Good Practice Index Overall Evaluation Σ 𝐼𝑡𝑒𝑚𝑠 𝑐𝑜𝑚𝑝𝑙𝑒𝑡𝑒𝑑 𝑥 100 Σ 𝐴𝑙𝑙 𝑖𝑡𝑒𝑚𝑠 𝑒𝑣𝑎𝑙𝑢𝑎𝑡𝑒𝑑 Table 5 - Indicators and good practices (continuation) Results and discussion Sinduscon-BA project description solutions throughout the design concept stage, construction stage and its operation and use stage. Sustainable building solutions include: solar panels, wind turbines, elevated floors, building management systems with real time energy monitoring, bioclimatic facades, Dali system for natural light optimization, LEDs activated by light sensors, green roofs, green walls, natural gas central air conditioning system, enthalpic wheel, CO2 control for indoor air quality, electric vehicle charging points, showers for cyclists and cycle racks, rainwater harvesting and greywater reuse, water and energy economizing gadgets and a centralized vacuum system. Sinduscon-BA project description The Sinduscon-BA Headquarters is located in the city of Salvador-Bahia-Brazil and was built where the former headquarters once stood. The 10-story commercial building occupies an area of 594.70 m2 with a built area of 2449.06 m2, consisting of 5 office floors, an auditorium, a flexible corporate office lounge with meeting rooms for seminars, lectures and events, a roof top with garden and 2 floors for car parking. The construction process lasted 16 months, from July 2012 to November 2013. The building was built with a reinforced and pre-stressed concrete structural system, double concrete block cavity walls and steel column foundations with reinforced concrete blocks. The external ventilated facades were composed of aluminum composites and high performance glass panels. The AQUA-HQE Certification for Sustainable Buildings, applied by the Vanzolini Foundation evaluates the environmental quality of a building in 14 categories. Performance in each category is classified as good (minimum acceptable level), superior (corresponding to best practices level) and excellent (based on the maximum performances found in Sustainable Developments). The building was certified in the execution phase achieving excellent performance in six categories, and good The Sinduscon-BA new headquarters was built under the AQUA-HQE sustainability certification criteria incorporating technologies and sustainable 17 Adoption of environmental practices on construction sites Ambiente Construído, Porto Alegre, v. 17, n. 4, p. 9-24, out./dez. 2017. in two categories. Two AQUA categories are concerned with the construction phase, Category 2 - Integrated selection of Products, Systems and Construction Processes and Category 3 - Low Environmental Impact Construction Sites. in two categories. Two AQUA categories are concerned with the construction phase, Category 2 - Integrated selection of Products, Systems and Construction Processes and Category 3 - Low Environmental Impact Construction Sites. were produced within 300km and 100% of the materials used in the building could be recycled in other applications after service life. All wood suppliers within a 300 km radius with FSC or Cerflor certification, providing full chain of custody including their environmental licenses and forest management plans were identified. Only suppliers of wood and wood products (including formwork) that were able to provide this information were used. All paints, varnishes and releasing agents for formwork were VOC free. Training of the materials procurement staff and workers on respective policies was required. Sinduscon-BA project description Directives were given to the design team in the project brief, regarding the adoption of modular coordination, pagination projects for tiles and bricks and the requirement to prioritize local materials with quality certification. Thomas, N. I. R.; Costa, D. B. 18 Implementation, monitoring and learning of environmental practice on jobsite Initially, a selective deconstruction plan in order to remove the former headquarters building and guarantee optimal valorization of materials and minimization of impacts was elaborated. All electrical and air conditioning equipment, doors and window frames, railings, removable gutters, wooden floor boards, skirting boards, lamps and lighting fixtures, switches/sockets, etc. were donated to employees and NGO'S. The remaining wood was sent to a ceramic industry for heat energy generation, plastic and glass was recycled by cooperatives. The waste masonry, slabs, mortar, blocks, floors, tiles etc. went to a recycling company for the production of aggregates. 24.4% of the construction waste aggregate was used in the basement floor of the building, replacing natural aggregates thus preventing the need to extract new resources and reducing waste disposal in landfills. Practices to reduce energy and water consumption were implanted such as using energy and water efficient equipment and appliances, insertion of procedures for switching off gadgets as well as the energy and water supply at the end of each day, and monthly monitoring of energy and water consumption. These guidelines were also passed on to the subcontractors and suppliers of equipment. Figure 2 presents water and energy consumption indicators, showing that despite the high progress of construction from June to September 2013, the relation between water and energy consumption and Man-hour maintained quite stable. These consumption practices were implemented at the beginning and therefore inserted as a policy in the contractor´s management system. From the viewpoint of consumption, a materials selection policy was adopted based on performance criteria guaranteed through appropriate quality certification, durability, life cycle concepts, distance (privileging materials produced within a 300 km radius) and ease of maintenance, which was monitored by a spreadsheet. Implementation, monitoring and learning of environmental practice on jobsite 96.58% of the materials in weight Figure 2 - Water and Energy consumption per man-hour along the project 0% 2% 4% 6% 8% 10% 12% 14% 16% 0,000 0,002 0,004 0,006 0,008 0,010 0,012 0,014 0,016 0,018jul/12sep/12nov/12jan/13mar/13may/13jul/13sep/13nov/13 Construction progress along the projectWater Consumption (m3/man hour) Water Consumption Construction Progress 0% 2% 4% 6% 8% 10% 12% 14% 16% 0,00 0,05 0,10 0,15 0,20 0,25jul/12sep/12nov/12jan/13mar/13may/13jul/13sep/13nov/13 Construction progress Energy Consumption (KVa/man hour) Energy Consumption Construction Progress Figure 2 - Water and Energy consumption per man-hour along the project Figure 2 - Water and Energy consumption per man-hour along the project our along the project 0% 2% 4% 6% 8% 10% 12% 14% 16% 0,00 0,05 0,10 0,15 0,20 0,25jul/12sep/12nov/12jan/13mar/13may/13jul/13sep/13nov/13 Construction progress Energy Consumption (KVa/man hour) Energy Consumption Construction Progress Ambiente Construído, Porto Alegre, v. 17, n. 4, p. 9-24, out./dez. 2017. A waste management plan was implemented. A spreadsheet was developed, discriminating waste type and quantity for each month allowing comparisons between previously established indicators. The first targets for waste generation were set based on the estimated volume of waste generated per construction activity and material type after having considered and implemented actions to minimize waste production. Each month, new reduced production targets were created based on the results from previous months. Costs with transportation and disposal as well as income with sales were also monitored to determine the critical waste type in terms of quantity and cost using an ABC curve. Another spreadsheet was compiled to control the waste generation and valorization. Results showed that 83.7% of all waste generated during construction was reused or valorized as raw material in the studied project. A waste management plan was implemented. A spreadsheet was developed, discriminating waste type and quantity for each month allowing comparisons between previously established indicators. The first targets for waste generation were set based on the estimated volume of waste generated per construction activity and material type after having considered and implemented actions to minimize waste production. Each month, new reduced production targets were created based on the results from previous months. environmental principals, and rational use of water, energy, waste material, waste management and risk prevention. A digital inclusion program was offered to the workers, families and the neighborhood car valets, with an Employee of the Month Program awarding one worker per month for exemplary behavior based on set criteria. Implementation, monitoring and learning of environmental practice on jobsite Groundwater levels were controlled, surface runoff and rainwater collection with adequate drainage systems were implanted with the provision of effluent treatment systems before directing it to the public network, thus avoiding soil and water pollution and minimizing risks of contamination on the construction site. During unloading of materials and concrete, plastic sheets were placed on roads under the truck to collect the remains of materials allowing for its reuse, or referral to the effluent box, avoiding obstruction of public drainage systems. Greywater was reused, such as water from the mortar mixer and from cleansing of equipment and concreting. Water from sinks was reused in the urinals. Training of workers conducted by the health and safety manager occurred on a daily basis in order to ensure the implementation of the practices. Topics included health and safety programs, Table 6 - Neighborhood feedback during the cons Neighbourhood positive feedback or claims Nov/12 Dec/12 Positive feedback 1 1 Noisy claims Particulate emission claims 1 Neighbour house damages 2 2 Dirty claims 1 1 Costs with transportation and disposal as well as income with sales were also monitored to determine the critical waste type in terms of quantity and cost using an ABC curve. Another spreadsheet was compiled to control the waste generation and valorization. Results showed that 83.7% of all waste generated during construction was reused or valorized as raw material in the studied project. Temporary facility designs were developed for each construction phase, defining strategic positions for noisy equipment and acoustic treatments thereby reducing the impacts on the neighborhood. Delivery times were agreed with neighbors banning noisy work at weekends and before 8 am and after 6 pm on week days. Sound levels were monitored during concreting as well as ensuring that equipment was in good condition, favoring equipment with lower noise and vibration emissions. The production of onsite mortars was minimized, using prefabricated and industrialized materials. Site access and pedestrian flows were managed with daily cleaning and maintenance of roads and sidewalks. A good neighborhood policy was implemented promoting transparency through signs with contact details for complaints and suggestions. Neighborhood buildings were regularly visited. Notification letters were sent out 48 hours prior the concreting and other noisy activities. Complaints were registered and monitored with corrective actions implemented. Implementation, monitoring and learning of environmental practice on jobsite Best practices aimed at controlling particle emissions and minimizing their risks were implanted such as using water during earthworks, irrigation of soils, wetting objects while sweeping and before cutting with a saw to avoid dust. Large facade safety nets were attached to each face of the construction site, to protect the neighbors from falling debris and dust. Groundwater levels were controlled, surface runoff and rainwater collection with adequate drainage systems were implanted with the provision of effluent treatment systems before directing it to the public network, thus avoiding soil and water pollution and minimizing risks of contamination on the construction site. During unloading of materials and concrete, plastic sheets were placed on roads under the truck to collect the remains of materials allowing for its reuse, or referral to the effluent box, avoiding obstruction of public drainage systems. Greywater was reused, such as water from the mortar mixer and from cleansing of equipment and concreting. Water from sinks was reused in the urinals. Table 6 shows that the critical period of the project concerned to claims was in November and December 2012, during superstructure works with a total of 7 claims related to neighborhood house damages, cleanness and particulate matter emissions. Table 7 shows the findings for the monitoring of low impact environmental good practices from July to October 2013, according to the indicators, good practices and criteria aforementioned at Table 2. Training of workers conducted by the health and safety manager occurred on a daily basis in order to ensure the implementation of the practices. Implementation, monitoring and learning of environmental practice on jobsite Sound levels were monitored during concreting as well as ensuring that equipment was in good condition, favoring equipment with lower noise and vibration emissions. The production of onsite mortars was minimized, using prefabricated and industrialized materials. Site access and pedestrian flows were managed with daily cleaning and maintenance of roads and sidewalks. A good neighborhood policy was implemented promoting transparency through signs with contact details for complaints and suggestions. Neighborhood buildings were regularly visited. Notification letters were sent out 48 hours prior the concreting and other noisy activities. Complaints were registered and monitored with corrective actions implemented. Table 6 shows that the critical period of the project concerned to claims was in November and December 2012, during superstructure works with a total of 7 claims related to neighborhood house damages, cleanness and particulate matter emissions. Table 7 shows the findings for the monitoring of low impact environmental good practices from July to October 2013, according to the indicators, good practices and criteria aforementioned at Table 2. Table 6 - Neighborhood feedback during the construction Neighbourhood positive feedback or claims Nov/12 Dec/12 Jan/13 Feb/13 Mar/13 Apr/13 May/13 Total Positive feedback 1 1 1 3 Noisy claims 1 1 Particulate emission claims 1 1 Neighbour house damages 2 2 1 1 6 Dirty claims 1 1 1 3 type after having considered and implemented actions to minimize waste production. Each month, new reduced production targets were created based on the results from previous months. Costs with transportation and disposal as well as income with sales were also monitored to determine the critical waste type in terms of quantity and cost using an ABC curve. Another spreadsheet was compiled to control the waste generation and valorization. Results showed that 83.7% of all waste generated during construction was reused or valorized as raw material in the studied project. Best practices aimed at controlling particle emissions and minimizing their risks were implanted such as using water during earthworks, irrigation of soils, wetting objects while sweeping and before cutting with a saw to avoid dust. Large facade safety nets were attached to each face of the construction site, to protect the neighbors from falling debris and dust. Adoption of environmental practices on construction sites 19 Implementation, monitoring and learning of environmental practice on jobsite Regular jobsite inspections were performed by the health and safety manager to ensure effective implementation of good practices. Integration trainings were carried out with new teams and subcontractors. Adoption of environmental practices on construction sites 19 type after having considered and implemented actions to minimize waste production. Each month, new reduced production targets were created based on the results from previous months. Costs with transportation and disposal as well as income with sales were also monitored to determine the critical waste type in terms of quantity and cost using an ABC curve. Another spreadsheet was compiled to control the waste generation and valorization. Results showed that 83.7% of all waste generated during construction was reused or valorized as raw material in the studied project. Best practices aimed at controlling particle emissions and minimizing their risks were implanted such as using water during earthworks, irrigation of soils, wetting objects while sweeping and before cutting with a saw to avoid dust. Large facade safety nets were attached to each face of the construction site, to protect the neighbors from falling debris and dust. Groundwater levels were controlled, surface runoff and rainwater collection with adequate drainage systems were implanted with the provision of effluent treatment systems before directing it to the public network, thus avoiding soil and water pollution and minimizing risks of contamination on the construction site. During unloading of materials and concrete, plastic sheets were placed on roads under the truck to collect the remains of materials allowing for its reuse, or referral to the effluent box, avoiding obstruction of public drainage systems. Greywater was reused, such as water from the mortar mixer and from cleansing of equipment and concreting. Water from sinks was reused in the urinals. Training of workers conducted by the health and safety manager occurred on a daily basis in order to ensure the implementation of the practices. Topics included health and safety programs, Regular jobsite inspections were performed by the health and safety manager to ensure effective implementation of good practices. Integration trainings were carried out with new teams and subcontractors. Temporary facility designs were developed for each construction phase, defining strategic positions for noisy equipment and acoustic treatments thereby reducing the impacts on the neighborhood. Delivery times were agreed with neighbors banning noisy work at weekends and before 8 am and after 6 pm on week days. Implementation, monitoring and learning of environmental practice on jobsite In addition, the production team did not always wet areas during the production activities and when sweeping. construction safety nets attached to the facade. The wind destroyed these quickly and so, there were often periods completely without them. In addition, the production team did not always wet areas during the production activities and when sweeping. Despite the intense and greater concern with health and safety training enforced by the Brazilian Regulatory NR 18 standards (BRASIL, 2015), this indicator showed a fall in September 2013 for different reasons, such as not using individual safety equipment (Hearing Protectors, Footwear, Glasses) and also equipment for delimitation of areas and guardrails in bad conditions. In terms of the Low- impact Environmental Good Practice Index (LIEGPI), performance increase was noticed, despite a drop in October 2013, which might be attributed to the amount of work in progress in the last two months of the construction and the focus of the project team on meeting the construction schedule rather than the environmental issues. Due to a structured waste program under an external consultancy, almost 80% of the practices were systematically implemented during the study. One of the difficulties was concerned with plaster waste, as despite the legal requirement set out by the CONAMA Resolution N. 307 (BRASIL, 2002), there is to date, no industry to receive plaster waste in Salvador, so it was sent to a dangerous waste landfill. Another important consideration is that waste management requires continual monitoring and training in order to ensure correct segregation. Recommendations for the adoption of environmental practice on construction site Often waste was found mixed together therefore requiring more bins located in strategic work areas to promote reuse within the jobsite. At times, subcontractors showed a resistance in adapting to the contractors’ sustainable construction policies. Even with daily training on waste management and other practices emphasizing importance, the construction site had some difficulties in making the workers participate, due to the high employee turnover and the fact that the subcontractor employees would only be working on the site for a predetermined time. Meetings were held with the subcontractor managers to enforce employee commitment, thus confirming the need to determine measures and include previously agreed contractual terms guaranteeing adherence to best practices and project policies when hiring subcontractors. The construction site is a dynamic space in which several changes are made during the execution phase. Therefore, solutions and potential barriers should be forecasted before the construction phase or major activities start. The implementation of low environmental impact construction sites requires a set of guidelines, as discussed below. Thomas, N. I. R.; Costa, D. B. 20 Implementation, monitoring and learning of environmental practice on jobsite Topics included health and safety programs, Adoption of environmental practices on construction sites 19 Table 6 - Neighborhood feedback during the construction Neighbourhood positive feedback or claims Nov/12 Dec/12 Jan/13 Feb/13 Mar/13 Apr/13 May/13 Total Positive feedback 1 1 1 3 Noisy claims 1 1 Particulate emission claims 1 1 Neighbour house damages 2 2 1 1 6 Dirty claims 1 1 1 3 Adoption of environmental practices on construction sites 19 Table 6 - Neighborhood feedback during the construction Neighbourhood positive feedback or claims Nov/12 Dec/12 Jan/13 Feb/13 Mar/13 Apr/13 May/13 Total Positive feedback 1 1 1 3 Noisy claims 1 1 Particulate emission claims 1 1 Neighbour house damages 2 2 1 1 6 Dirty claims 1 1 1 3 Table 6 - Neighborhood feedback during the construction Neighbourhood positive feedback or claims Nov/12 Dec/12 Jan/13 Feb/13 Mar/13 Apr/13 May/13 Total Positive feedback 1 1 1 3 Noisy claims 1 1 Particulate emission claims 1 1 Neighbour house damages 2 2 1 1 6 Dirty claims 1 1 1 3 Table 6 - Neighborhood feedback during the construction Adoption of environmental practices on construction sites 19 Ambiente Construído, Porto Alegre, v. 17, n. 4, p. 9-24, out./dez. 2017. Table 7 - Monitoring of environmental good practice indicators per month Indicators Jul/13 Aug/13 Sep/13 Oct/13 Average Material, Water and Energy Consumption Index 71% 54% 89% 50% 66% Solid Waste Index 80% 80% 73% 80% 78% Water, Soil and Air Pollution Index 80% 85% 93% 60% 80% Temporary Facilities Index 73% 86% 67% 91% 79% Health and Safety Index 75% 86% 71% 79% 78% Outdoor Quality Index 78% 84% 89% 89% 85% Low-impact Environmental Good Practice Index 76% 79% 80% 75% 78% Table 7 - Monitoring of environmental good practice indicators per month Difficulty in meeting the consumption items criteria was found. Despite the training of workers on the rational resource use, the dissemination of these onto workers was not a routine. Often the storage of materials was also not appropriate. From the material purchase viewpoint, the project team faced difficulties in finding material manufacturers near to the city of Salvador, such as high flag ash Portland Cement, ceramic tiles, aluminum composites and glass. It is therefore necessary to incentivize local industries. construction safety nets attached to the facade. The wind destroyed these quickly and so, there were often periods completely without them. Hiring of subcontractors The responsibility of adhering to the construction site environmental management plan, policies and procedures must also be passed on to the subcontractors and inserted during negotiations and formalization of contracts. Existing management systems should be extrapolated and extended to subcontractor management systems, defining parameters for the formulation of contracts, stipulation of goals and rules regarding the reduction of losses, etc. Monitoring and control To evaluate the effective implementation of best practices, a deployment checklist can be applied weekly generating indicators, which can also be used to motivate site workers. Spreadsheets should be developed to control and monitor all data. Weekly meetings also help enforce the effective implementation of the best practices; they should be held with the presence of the production engineer, the project manager, and health and safety manager with minutes distributed via email after the meeting for all participants. Attention should be given for producing and dissemination metrics that also measure the successful realization of design intent in order to recognize the construction phase as an important component of sustainable buildings (HOLLOWAY; PARRISH, 2015). Implementing practices The implementation of low impact construction sites should start with the training of workers and assigning a responsible for the work to guarantee effective implementation and monitoring of the low environmental impact construction site. This responsibility can be attributed to health and safety managers or an environmental manager. Provision of a visual communication program will assist implementation as well as training of workers in short daily meeting and specific lectures, with the aid of good practice photos and posters. Considering site sustainability in the design phase During the project briefing stage, all designers should be given the necessary recommendations regarding the sustainability attributes to be adopted also considering the construction phase, such as the materials policy and construction methods with the aim of minimizing waste production and promoting rational resource use. On the other hand, contractors need to share their knowledge of construction cost and quality to promote cost- Concerning pollution, there was an evolution in the implementation of good practices until September 2013 after which there was a drop. The recurrent problems identified were the lack of maintenance and often complete removal of the Ambiente Construído, Porto Alegre, v. 17, n. 4, p. 9-24, out./dez. 2017. effective sustainability (HOLLOWAY; PARRISH, 2015). These two authors state that early contractor involvement in the design phase will allow enhanced cost and sustainability feedback regarding design option. (a) training on reducing loss of material in the assignment, delivery, internal transport, storage and handling of materials on the construction site; (b) training on procurement of materials and products, including training on criteria and purchasing policies, reverse logistics, and supplier evaluation; Planning the construction site Before starting any construction work, it is necessary to characterize and analyze the construction site, the surrounding neighborhood and the legal requirements. Then, the environmental aspects and impacts relevant to each construction activity should be identified and prioritized. The selection of the practices should consider cost, efficiency and ease of employment criteria. The selected practices should put into a Construction Site Environmental Management Plan containing a detailed plan on how and when each solution should be implemented, what resources are required, who will be responsible and how it will be monitored. The Main Contractors’ Quality Management system can be adjusted including the data control sheets, and relevant training material. (c) training on the implementation of waste management plans, ensuring separation of waste at the source to maintain the quality of waste material and its potential to be reused or valorized, and allocation of storage areas on each floor of the construction site; (d) training on monitoring and minimizing the emission of particulates and integration of these practices as operational procedures; and (e) training and awareness about sound pollution, methods to characterize activities that emit noise and mitigation plans. Conclusion and recommendations The main theoretical contribution of this paper is the establishment of conditions, criteria, and metrics during the construction phase to evaluate the implementation process of management practices for low environmental impact construction sites, also presenting the effects of a comprehensive on-site sustainable practice program. At a practical level, the paper provides tools and recommendations for the implementation and assessment of environmental good practices on construction sites, allowing the development of a methodology elaborated based on a real case considering important requirements. Operational training BERGE, B. The Ecology of Building Materials. 2nd. ed. Oxford: Architectural Press, 2009. Furthermore, incentives are required for the development of industrialized materials and technologies decentralizing factories as a whole in Brazil. Subsidies should be created in order to promote innovative technologies that are energy efficient, use resources rationally and are economically feasible. It is important to develop certification systems and other methods to ensure compliance and quality of materials, products and systems. BUILDING RESEARCH ESTABLISHMENT ENVIRONMENTAL ASSESSMENT METHODOLOGY. Europe Commercial Assessor Manual. London: BRE Global, 2009. BUILDING RESEARCH ESTABLISHMENT ENVIRONMENTAL ASSESSMENT COUNCILS LONDON. Guidance Note Construction Sites. London: Local Government: Air Quality Toolkit, 2010. DEGANI, C. M. Sistemas de Gestão Ambiental em Empresas Construtoras de Edifícios. São Paulo, 2003. Dissertação (Mestrado em Engenharia Civil) – Escola Politécnica, Universidade de São Paulo, 2003. From this study, a methodology for adoption of low environmental impact construction sites was developed. However, further studies should be carried out for its validation. A best practices manual is under development in alignment with national and international protocols on reducing environmental impacts on construction sites. DING, G. K. C. Sustainable Construction: the role of environmental assessment tools. Journal of Environmental Management, v. 86, n.3, p. 451– 464, 2008. DING, G. K. C. Sustainable Construction: the role of environmental assessment tools. Journal of DING, G. K. C. Sustainable Construction: the role of environmental assessment tools. Journal of Environmental Management, v. 86, n.3, p. 451– 464, 2008. Environmental Management, v. 86, n.3, p. 451– 464, 2008. Operational training To ensure the effective implementation of good practices in the construction site, it is necessary to develop operational training programs and encourage the integration of subcontractors and labor to enhance their commitment, such as: 21 Adoption of environmental practices on construction sites Ambiente Construído, Porto Alegre, v. 17, n. 4, p. 9-24, out./dez. 2017. ARAÚJO, I. P. S.; COSTA, D. B., MORAES, R. J. B. Identification and Characterization of Particulate Matter Concentrations at Construction Jobsites. Sustainability, v.6, n.11, p. 7666-7688, 2014. Although this case study was based on a single construction project, the study was long and intense, involving the participation of various entities from the industry and the academia, which required a strong commitment and input from professionals from all hierarchical levels. Despite these limitations, this methodology can be used to evaluate the implementation of different best practices and provides guidance on the various levels of training and incentives for the adoption of these practices. ARAUJO, V. M. Práticas Recomendadas Para a Gestão Mais Sustentável de Canteiro de Obras. São Paulo, 2009. 228 f. Dissertação (Mestrado em Engenharia Civil) – Escola Politécnica, Universidade de São Paulo, São Paulo, 2009. BRASIL. Ministério do Meio Ambiente. Conselho Nacional do Meio Ambiente. Resolução n. 307, julho 2002. Disponível em: <hp://www.mma.gov.br/port/conama/res/ res02/res30702.html>. Acesso em: 12 nov. 2016. Through this case study, it was observed that, in order to assist in achieving sustainability on construction sites, construction site design plans should be developed, redeeming design solutions and management practices adopted for permanent buildings and exploiting them into the design of temporary facilities, with assigned technical responsibility. BRASIL. Ministério do Trabalho e Emprego. NR- 18: condições e meio ambiente de trabalho na indústria da construção. 2015. Disponível em: <hp://trabalho.gov.br/seguranca-e-saude-no- trabalho/normazacao/ normas- regulamentadoras/norma-regulamentadora-n-18- condicoes-e-meio-ambiente- de-trabalho-na- industria-da-construcao>. Acesso em: 15 nov. 2015. In addition, the implantation of sustainable construction sites also depends on an entire supply chain that must also be engaged in achieving sustainable development, which requires a commitment from all parties involved. It is necessary to encourage enterprises to develop training programs and use local labor, training engineers and architects and creating new public policy agendas, promoting forums aimed at strengthening the link between the construction industry, academia and government. BALLESTEROS, M. J. et al. Noise Emission Evolution on Construction Sites: measurement for controlling and assessing its impact on the people and on the environment. Building and Environment, v. 45, n. 3, p. 711–717, 2010. Thomas, N. I. R.; Costa, D. B. 22 References FUNDAÇÃO VANZOLINI. Referencial Técnico de Certificação: edifícios habitacionais, Processo AQUA. São Paulo: Fundação Vanzolini, 2010. ABDUL-RAHMAN, H. et al. Integrating and Ranking Sustainability Criteria for Housing. Engineering Sustainability, v. 169, n.1, p. 1-30, 2016. Ambiente Construído, Porto Alegre, v. 17, n. 4, p. 9-24, out./dez. 2017. KHASREEN, M. M.; BANFILL, P. F. G.; MENZIES, G. F. Life-Cycle Assessment and the Environmental Impact of Buildings: a review. Sustainability, v. 1, n. 3, p. 674-701, 2009. GANGOLELLS, M, et al. A Methodology for Predicting the Severity of Environmental impaCts Related to the Construction Process of Residential Buildings. Building and Environment, v.44, n.3, p 558-571, 2009. KILBERT, C. J. Sustainable Construction: green building design and deliver. London: John Wiley and Sons, 2005. GANGOLELLS, M. et al. Model for Enhancing Integrated Identification, Assessment and Operational Control of On-Site Environmental Impacts and Health and Safety Risks in Construction Firms. Journal of Construction Engineering and Management, v. 139, n. 2, p 138–47, 2013. KIM, S. S. et al. Development of a Housing Performance Evaluation Model for Multi-Family Residential Buildings in Korea. Building and Environment, v. 40, n. 8, p. 1103–1116, 2005. Construction Firms. Journal of Construction Engineering and Management, v. 139, n. 2, p 138–47, 2013. KUKADIA, V.; UPTON, S.; HALL, D. Control of Dust From Construction and Demolition Activities. London: BRE, DTI, UK, 2003. GANGOLELLS, M. et al. Analysis of the Implementation of Effective Waste Management Practices in Construction Projects and Sites. NETREGS ENVIRONMENT AGENCY UK. Presents a Good Practice Guide and How to Abide to Environmental Laws. 2012. Disponível em: <http://www.netregs.gov.uk>. Acceso em: 15 nov. 2012. Resources, Conservation and Recycling, v. 93, n. 12, pp 99–111, 2014. GANGOLELLS, M. et al. Assessing Concerns of Interested Parties When Predicting the Significance of Environmental Impacts Related to the Construction Process of Residential Buildings. GANGOLELLS, M. et al. Assessing Concerns of Interested Parties When Predicting the Significance of Environmental Impacts Related to the Construction Process of Residential Buildings. Building and Environment, v. 46, n. 5, p. 1023- 1037, 2011. PROBERT, A. J. et al. 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Acknowledgements We are grateful to the Cantechis Project (Technologies for Sustainable Construction on Social Housing Jobsites), the Ministry of Science, Technology and Innovation, through FINEP Natasha Ilse Rothbucher Thomas Faculty of Health and Life Sciences, School of Pharmacy | De Montfort University | The Gateway, Leicester LE1 9BH, Leicester, United Kingdom | Tel.: +44 (0) 116 230 6443 | E-mail: nashatasha@hotmail.com Dayana Bastos Costa Departamento de Construção e Estruturas, Escola Politécnica | Universidade Federal da Bahia | Av. Aristides Novis, 2, Federação | Salvador - BA – Brasil | CEP 40210-630 | Tel.: (71) 3283-9731 | E-mail: dayanabcosta@ufba.br Dayana Bastos Costa Departamento de Construção e Estruturas, Escola Politécnica | Universidade Federal da Bahia | Av. Aristides Novis, 2, Federação | Salvador - BA – Brasil | CEP 40210-630 | Tel.: (71) 3283-9731 | E-mail: dayanabcosta@ufba.br Revista Ambiente Construído Associação Nacional de Tecnologia do Ambiente Construído Av. Osvaldo Aranha, 99 - 3º andar, Centro Porto Alegre – RS - Brasil CEP 90035-190 Telefone: +55 (51) 3308-4084 Fax: +55 (51) 3308-4054 www.seer.ufrgs.br/ambienteconstruido E-mail: ambienteconstruido@ufrgs.br
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Keywords Keywords Nationalism, theories, ethnic diversity Article Ethnicities 2022, Vol. 22(4) 573–588 © The Author(s) 2022 Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/14687968221085260 journals.sagepub.com/home/etn Contextualising nationalism Anna Triandafyllidou Ryerson University, Faculty of Arts, Department of Sociology, Toronto, ON, Canada. Corresponding author: Anna Triandafyllidou, Ryerson University, Faculty of Arts, Department of Sociology, 350 Victoriastreet, Toronto ON M5B 2K3, Canada. Email: anna.triandafyllidou@ryerson.ca Abstract Th This paper seeks to relate the scholarly analysis of nationalism – and of the ways in which nation-states relate to minorities and migrants – with the actual socio-political context within which such analysis takes place. Looking back into the theories of nationalism as they have developed since World War II, the focus of nationalism theorists has shifted from the effort to explain why nations emerged and when they emerged, looking at the wider processes of industrialisation and print capitalism; to the effort to analyse na- tionalism from the ground up through the lived experiences of citizens; to, more recently, the effort to explain why and how nationalism persists and whether and how it evolves in more plural or more exclusionary ways. I am arguing in this paper that it is important to adopt a self-reflexive approach so as not only to link our understanding of nations and nationalism in their contemporary socio-economic and political context, but that we also need to adopt such self-reflexivity in relation to our own work and ask: why do we focus on a particular perspective or evolution? How does this relate to our wider context and positionality as scholars? In this paper, I am proposing a periodisation of nationalism studies from the post-WW II period to this day, arguing that the focus of nationalism theories was guided interactively by the wider socio-economic developments of each period. I conclude with a critical reflection on nationalism in a (post-)pandemic world. Keywords Nationalism, theories, ethnic diversity Introduction This paper seeks to relate the scholarly analysis of nationalism – and of the ways in which nation-states relate to minorities and migrants – with the actual socio-political context within which such analysis takes place. Looking back into the theories of nationalism as 574 Ethnicities 22(4) Ethnicities 22(4) they have developed since World War II, the focus of nationalism theorists has shifted from the effort to explain why nations emerged and when they emerged (connecting those with the wider processes of industrialisation and print capitalism), to the effort to analyse nationalism from the ground up through the lived experiences of citizens (notably the so- called banal or everyday nationalism), to, more recently, the effort to explain why and how nationalism persists and whether and how it evolves in more plural or more exclusionary ways (multicultural, transnational or plural nationalism). One might wonder whether these theories emerged out of the intellectual curiosity of the scholars or their preoc- cupation with a particular case study or whether they relate in a more self-reflexive way to the positionality of the scholars and their situatedness in a given period with its specific social, economic and geopolitical features. While one may argue that this is the nature of the scholarly inquiry, it is important and necessary to ask ourselves not only why and how we select our research topics, but also our development of specific theoretical concepts and methodologies are indirectly guided by the socio-political transformations that happen around us. The interactive link between nationalism studies and the broader socio- political context is taken for granted or simply rendered invisible but our future research can benefit from a critical account of how nationalism studies have evolved. My critical perspective here is not only scholarly and theoretical – bringing different strands of literature in dialogue with one another – but also sociological and political in the wider sense: it seeks to help us understand better ourselves and our societies and a certain level of polarisation that we are witnessing in Europe today between, on the one hand, the re-emergence of ethno-cultural nationalism currents and exclusionary discourses (par- ticularly against Muslims and significantly vocal in the immediate post-2015 period) and, on the other hand, a notable mobilisation against racism and discrimination such as that we have witnessed in 2020–2021 through the Black Lives Matter movement not only in North America but also in Europe. Introduction It is in this effort to relate the scholarly work to its socio-political context that I discuss some of the most important works on nationalism and their distinctive analytical con- tributions to understanding this phenomenon. For this reason, I have opted for organising this chapter not in relation to clusters of theories (e.g. modernists vs perennialists, constructivists vs ethno-symbolists and multiculturalists vs nativists) nor in relation to themes (e.g. nationalism and the state, nations and ethnic groups and national heritage) but rather into chronological periods marked by relevant socio-economic and political changes that have influenced not only nations and nationalism but also the analytical perspectives and methodological choices of scholars studying nationalism. Imperfect though my periodisation may be, I propose to divide the last 40 years, notably the period running from 1980s to today (2021) in four periods which are marked by important social and political transformations. I chose to start with the 1980s with a view to including the period prior to 1989 and the dramatic changes that 1989 brought to Europe (and the world) but also with a view to emancipating my review from the post- World War II considerations and placing it more firmly in a context of turn of the century, start of 21st century. While I am going to discuss in more detail in the following sections the context of each of these periods, I would like to briefly justify here my periodisation and how it is linked to important developments in nationalism research. 575 Triandafyllidou The first period starts in the early 1980s until the early 1990s notably in the last period of the Cold War when nation-states still dominated international politics while European integration remained still rather weak and also predominantly economic in its outlook. During this period, nationalism research concentrated on explaining when and how nations have developed and why and how they have turned into the primary and dominant form of political community in the modern period. Thus, theoretical debates between the so-called modernists that argue that the nation is an outcome of the modern period (Anderson, 1981; Gellner, 1983; Hobsbawm, 1990), responding to the new forms of production and socio-political organisation that emerge after the industrial revolution, and the perennialists (Armstrong 1982; Hastings 1997) who search for the origins of nations in earlier ethnic identities and communities, remained dominant in this period. Introduction It is in this period that John (Breuilly, 1994) discussed the links – or rather the tensions – between nationalism and the state in the modern period while Walker (Connor, 1994) critically investigated ethnonationalism and its political implications. The common thread that can be seen as running through these important scholarly works on nationalism is the desire for an explanation: where do nations come from, and how can we explain that they have become the most important form of political organisation in the contemporary world. The second period that I identify starts in the early/mid-1990s and lasts till the mid- 2000s. This period reflects the aftermath of the sweeping geopolitical changes that took place in 1989 and the following years as Communist regimes in Central and Eastern Europe imploded. This political and economic reconnection of Europe has been ac- companied by the re-emergence of the ‘national question’ in Central Eastern Europe and the revival of both national majorities and national or ethnic minority identities. At the same time, this period saw important challenges in terms of diversity and Europe’s Muslims. Last but not least this period was marked by important steps in European integration – not least the creation of a European citizenship and the big bang Eastern Enlargement of 2004 (and 2007). Thus this period raised important questions as to what is the role of individuals and groups in nations, how much nations should be taken for granted or can be superseded by alternative forms of political organisation, how can we understand nationalism in a changing world order and does nationalism need to end up in war. It is no coincidence that there is a new interest in this period in understanding nationalism from a social psy- chological perspective, looking at the common elements within nationalism dynamics beyond (to the extent possible) historical specificities. It is in this period that Michael Billig wrote his by now-famous Banal Nationalism (1995) looking at how our everyday lives are imbued by tacit understandings of us and them, here and there and how such understandings are reinforced by everyday routines. (Reicher and Hopkins, 2001) also wrote their more systematic analysis of nationhood, national identification and nationalist mobilisation from a social psychological perspective. Introduction There was an increasing interest as to the role of minority/majority groups in Central Eastern Europe and the analytical use of the term ‘identity’ (Brubakers and Cooper, 2000 but also Brubaker, 2006; Brubaker et al., 2007, Brubaker et al. 2007). While the early 2000s are of course marked by 9/11 and also the Madrid and London bombings of 2004 and 2005, these events do not directly bear on the study of nationalism 576 Ethnicities 22(4) Ethnicities 22(4) or at least not in the immediately subsequent years. While the rise of international jihadist terrorism triggers both a political and scholarly interest in questions of socio-economic and cultural integration of migrants and second generations, the relative success or failure of different integration models, and of course the roots of violent radicalisation and terrorism, they are not immediately reflected in the focus of nationalism studies. Indeed, the reflections on what international terrorism and violent radicalisation means for nations come into the study of nationalism only in the 2010s, after a new wave of international terrorism and the related rise of Islamophobia. The question of diversity and integration develops separately as an issue of social cohesion and migration, while the role of di- versity within nationalism is only addressed most recently in the new currents also of multicultural nationalism as discussed by the ‘Bristol school of multiculturalism’ (Levey, 2019; Modood, 2019), transnational nationalism (Kastoryano 2018) and my own work on plural or neo-tribal nationalism (Triandafyllidou 2013; 2022). Indeed, this third period expands from the second half of the 2000s through until the late 2010s. This period is dominated by two concerns: whether nations will survive and whether they are fit for purpose in an increasingly globalised and transnationalised world where major challenges (including climate change, security or indeed trade and work and the economy) transcend state boundaries (see for instance Calhoun 2007). At the time of writing we are going through an unprecedented global pandemic that has initiated a complex discussion between the so-called ‘vaccine nationalism’ and the wish of national states to close their borders so as to protect their populations from the spreading of the virus. Introduction While most states have restricted international travel to extents that we would have never imagined, it became clear that sealing the borders was not feasible, that the virus travelled nonetheless and that, at the end of the day, we are very inter- dependent for a number of reasons (exchange of goods and services, mobility of people for work or leisure). The question that emerges is how this new situation and the new challenges and reflections that it brings will affect notions of the nation – the ingroup, national membership and the role of borders. The paper concludes with a reflection on what we have learnt from the pandemic and how nationalism studies are likely to evolve in the coming years. This paper discusses the three main periods identified notably the 1980s till the mid- 1990s, the late 1990s till mid-2000s, the late 2000s till late 2010s and the tentative new period opening up in their chronological sequence. Unavoidably my analysis is selective; I do not cover the entire bibliography but I do discuss the important and influential works and currents of thought in nationalism studies as they have developed in the scholarly literature. Providing a full annotated bibliography is certainly not the aim of this paper. Rather I am seeking to highlight how living and studying nationalism and diversity are inextricably intertwined and that we need more self-reflexive perspectives in our work. Nationalism studies in the pre- and post-1989 period: preoccupied with grand theoretical explanations Looking back, the 1980s were a pivotal decade. While there were important socio- political mobilisations in Europe, notably in Poland with Solidarnosz and the quest for 577 Triandafyllidou democratisation, and the rise of socialist governments across several European countries like France, Germany or Spain, there was no clear sign that the Cold War was about to end and that the Soviet Union and the Warsaw Pact were about to implode. However, during the 1980s one might argue that while the new was about to be born, the old had not yet died in Europe and none seemed to imagine the dramatic socio-political changes that were forthcoming. It may thus come as no surprise that during this period, nationalism studies were still dominated by the classical theoretical questions of whether nations are a product of modernity or have always existed in some form. It is in those years that Ernest Gellner (1983), Benedict Anderson, 1981, Anthony D. Smith (1986, 1991) and Eric (Hobsbawm, 1990) wrote their foundational works on nations and national identity linking up the rise of nations and nationalism with wider processes of socio-economic transformation in modern times. These thinkers were preoccupied with explaining why and how the nation had become a dominant form of political organisation in the last two centuries, commanding the primary loyalty of the citizens. Primordialists like Clifford Geertz (1963) emphasised the importance of ethnic bonds as the ‘givens’ of social existence (Geertz 1963: 109) and as the main boundary markers (Barth 1969) that organise society as well as economic exchange. John Armstrong (1982) in his influential book on Nations before Nationalism pointed to the fact that ethnic bonds have important political consequences as they form the basis of structures of power and legitimise them. Armstrong was rather radical in his view to the extent that he saw the modern post-eighteenth century nations as a novel type of ethnic groups that conformed to the new socio-economic conditions, rather than as a signifi- cantly different social formation. Adrien Hastings (1997) argued that ethnic polities in history provided the building blocks of modern nations, but agreed that these last were qualitatively different from ethnic groups because of the processes that were set in motion in Europe in the 18th century notably industrialisation, Englightenment and bureaucratisation. Nationalism studies in the pre- and post-1989 period: preoccupied with grand theoretical explanations Modernist thinkers like Benedict Anderson (1981) and Nairn (1997), but also Ernest Gellner (1983) and Eric (Hobsbawm, 1990), disagreed with both primordialists and perennialists and argued that the emergence of modern nations and of nationalism as a modern social movement have much more to do with the process of print capitalism (as Anderson put it) and industrialisation than with the cultural fabric of pre-existing ethnic groups. Anderson (1981) explained this process of modernisation and emergence of modern nations in relation to the spread of print capitalism. Of course print capitalism spreads along with significant changes in the system of production, industrialisation, improvement in transport and communication, the emergence of urban middle-classes and the spread of literacy. Thus, printed vernacular languages emerge and spread connecting people who spoke different, local dialects. This whole process made it possible for people to imagine a (national) community beyond their locality and to connect with it. In this perspective, ethnic cultures and bonds form a malleable ground material on which capitalism and industrialisation build the modern nation. For modernists, the malleability of ethnic cultures and the fact that some survived and transformed into modern nations while others did not, is a proof of their limited importance in the formation of modern nations. Modernists paid also special attention to the role of the state which emerges as a 578 Ethnicities 22(4) Ethnicities 22(4) powerful political actor through industrialisation and bureaucratisation reaching out to the simple people. Indeed the nation functions as the key vehicle for the legitimisation of the nation and its control over the peasants converted into nationals and citizens. The 1980s and early 1990s are also marked by the writings of Anthony Smith (1986, 1991, refined in 2009) who argued for a middle ground – an ethnosymbolist approach that brings together some of the insights of the perennialists while it also acknowledges the qualitatively different features of modern nations. Thus, Smith (2009) accepted the importance of the scientific and economic developments that took place in the 17th and 18th century making the nation functional to this historical period, but disagreed with modernists as to the relative importance of those developments in the longue duree. He pointed to the fact that while nations cannot always be predicted on the basis of the existence of an ethnic group, there can be no nation that is totally socially and culturally engineered, without a pre-existing ethno-cultural fabric. Nationalism studies in the pre- and post-1989 period: preoccupied with grand theoretical explanations g , p g The scholarly inquiry onto the circumstances and factors driving the emergence of modern nations and nationalism in Europe in the post 18th century has shaped the classification of nations into ethnic versus civic. Ethnic nations are those whose definition is mainly based on ethno-cultural elements such as a belief in common descent, shared historical memories, a link with historical territory that is seen as the homeland of the nation and which may not coincide with its current territory, a common language and sometimes also a common religion. Ethnic nations are ascriptive. Civic nations are those whose definition privileges territorial and civic elements, notably a mass public culture, a territory and a single economy, a set of common rights and duties among its members. Naturally language is often a pre-requisite – if anything a functional one – for civic nations too. Civic nations are voluntaristic in their nature: a person who lives in the national territory, abides by its laws, contributes to society and the economy for a given period of time, can become a national. Civic nationalism is not devoid of emotive power as it is not a mere civic association but rather involves ethical obligations to fellow nationals (Greenfeld 1992). Naturally these are ideal types and most nations fall somewhere in between. In other words the ethnic versus civic definition of nations ought better be understood as a continuum rather than as a black and white distinction. However, the ethnic versus civic distinction in analysing the emergence of nations and nationalism in Europe has not simply been a theoretical or analytical one; it also emerged out of the necessity to make sense of two distinct paths to nation state formation in Europe. Thus, ethnic nationalism largely has been considered to correspond to the historical experience of Central, Eastern and Southeastern Europe, where national identities were forged in the absence of corresponding state structures and generally in opposition to Empires. Civic nationalism, by contrast, has been identified with western European pathways to nation state formation, in countries where state structures had formed before the potent onset of modern nationalism. The distinction between a good civic and a bad ethnic nationalism can be found in the work of Guibernau (1996) and Nairn (1997), while Miller (1995) tends to warn against such a false dichotomy. Nationalism studies in the pre- and post-1989 period: preoccupied with grand theoretical explanations Yael Tamir (1993) has also argued that the dividing line between ethnic and civic nationalism is not so hard, as even in an exclusive ethnic type of nationalism, there are several inroads to assimilation and 579 Triandafyllidou belonging including adopting fully the new culture and language, marrying in, aban- doning one’s former culture and where appropriate also converting. belonging including adopting fully the new culture and language, marrying in, aban- doning one’s former culture and where appropriate also converting. This political and scholarly question of distinct types of nations and nationalism has marked also the work of important political theorists seeking to make sense of nationalism from a normative perspective. This led to the emergence of two schools of thought notably the so-called liberal nationalists – like Tamir (1993), Miller (1995) and (Kymlicka, 1995) – and the so-called conservative nationalists like Scruton (1990), and also theorists like Bhikhu (Parekh, 1994, 1995); see also Uberoi 2018) that took issue with the relationship between cultural and religious diversity and national identity, advocating for a broadening and deepening of national identity that would allow for diversity to be incorporated within it. While starting off from political theory, these scholars were preoccupied with justifying why the individual could give its primary loyalty to the nation and what forms of na- tionalism were morally appropriate, or what kind of accommodation should be provided to minorities – whether considered as groups or as individuals. The historical sociological perspective and the quest for making sense of the distinct paths to nationhood in Europe, and their implications for contemporary, post-war nations dominated nationalism studies till the late 1980s but did not survive much further. While the disagreement between the perennialists and the modernists has never been settled, it is clear that this discussion and the analytical distinction between ethnic and civic na- tionalism has lost its steam after the early 1990s. Smith (2009) refined his work and answered to critics of his work. In the meantime, though, the question of which nation is civic or ethnic and whether nations are a product of modernity or have always existed, has ceased to preoccupy sociologists and political scientists or cultural anthropologists. By contrast the question of how minorities and individual minority members relate to the nation, how nationalism can be combined with liberalism and how it can reconcile diversity within the nation, remained and is re-emerging today with renewed urgency. Nationalism studies in the pre- and post-1989 period: preoccupied with grand theoretical explanations It is my contention that the sweeping geopolitical and socio-economic changes that followed 1989 in Europe made those questions rather redundant or insignificant. The reconnection of Europe and the revival of nationalism and religiosity in the former Communist countries with the parallel development of the European Union as a transnational political formation with a transnational citizenship (as of 1992) and the quest for a European identity (or identities in the plural, Triandafyllidou and Gropas 2015), shifted the focus to explaining how people and institutions navigate these turbulent times. The discursive turn in the late 1990s and the reconnection of Europe The mid-1990s in Europe were characterised by opposed developments. On one hand, Central Eastern European countries were rediscovering their national and religious identities and so did the minorities that lived within them, and, on the other hand, the European Communities transformed into the European Union opted for a big-bang enlargement to the East, incorporating these countries into a united Europe (Spohn and Triandafyllidou 2003). There was significant celebration about the reconnection of Europe and the belief that the national was no longer important. The sociologists of 580 Ethnicities 22(4) Ethnicities 22(4) globalisation (e.g. Beck and Willms 2003) celebrated the arrival of ‘banal cosmopoli- tanism’ even though scholars like Calhoun (2007) argue for caution as the cosmopolitan imaginary still needed a form of belonging to be meaningful and provide anchoring. Despite the important economic and political challenges posed by the implosion of the Communist regimes in Central Eastern Europe, the 1990s were characterised by a certain ideological enthusiasm that the reconnection of the continent ended a past of wars and division, and that the European Union would offer a platform for both economic and geopolitical integration while European identity would become intertwined with national identities enriching and not replacing them. Not only were the 1990s a decade of Eu- ropean enthusiasm and drive for unification, they were also characterised by increased attention to cultural and religious diversity in Europe. Multiculturalism was celebrated in many countries (e.g. the UK, the Netherlands and Sweden) as the most appropriate way for accommodating cultural and religious diversity and building an inclusive citizenship. This pro-diversity policy extended also to the then newly independent states in Central and Eastern Europe which were strongly encouraged to recognise their national minorities and provide appropriate guarantees for their rights as foreseen in relevant European and international legal instruments (Triandafyllidou and Ulasiuk 2014). Indeed, the end of the Cold War and the implosion of the Communist Other were celebrated in the 1990s as a liberation from Europe’s past tragedies as well as the start of a new era globally. During this period, nationalism studies shifted their focus to the everyday experiences of nationalism. The discursive turn in the late 1990s and the reconnection of Europe Rather than seeking broader explanations of why nations emerge or how they form and evolve, faced with significant geopolitical and socio-economic changes and with related massive migration from eastern to western and southern Europe, the focus turned to exploring what people ‘do’ with the nation, how they speak about it and how they seek also to redefine it (Fox and Miller Idriss 2008). (Billig, 1995) along with Calhoun (1997) were the precursors of the discursive turn, pointing to the ways in which people experience nationalism and actually arguing that nationalism while pervasive and persisting is largely invisible because banal and embedded in the everyday routines (Billig, 1995). Indeed Calhoun was writing in 1997 that ‘nations are constituted [...] by the way of talking and thinking and acting that relies on these sorts of claims to produce collective identity, to mobilize people for collective projects, and to evaluate peoples and practices’ (1997: 5). p ( ) Billig’s work was particularly influential but also was subjected to several criticisms including the fact that Billig assumed a uniform national audience and disregarded the fact that western nations become increasingly diverse as a result of international migration (Skey, 2009a, 2009b). During the same period indeed, Basch et al. (1996) were arguing that the increasing pace and intensity of global flows impact group and national identities, while Appadurai (2003) argued that people’s inter-connectivity offered them new ways of imagining the world. Most importantly the critiques of Billig like Edensor (2002) pointed to the fact that globalisation and national identity should not be conceived as a zero sum game as Beck and Willms may seem to have argued, but rather as interacting phenomena that can lead to the reinforcement or transformation of national identities or to their diminished importance. 581 Triandafyllidou Focussing on the specific realities of Central Eastern Europe, for instance, Brubaker (2006) emphasised that the importance of the nation is contextual and becomes visible under specific circumstances. Indeed as Fox and Miller-Idriss (2008) have further il- lustrated that people may respond to, engage with or overlook particular symbols of the nation in specific moments and situations. Indeed the work of scholars like Edensor, 2002; Skey, 2009a, 2009b, 2011 have explored the agency of lay people, simple citizens, in forming and transforming the nation in their everyday encounters or in specific cir- cumstances. The discursive turn in the late 1990s and the reconnection of Europe The work of these scholars pointed to the ways in which ordinary people may ignore the rhetoric of politicians and instead may construct their national identity in their own terms (Brubaker 2006; Fox and Idriss 2008). ( ) It was 9/11 and the international terrorism events that took place in Madrid in 2004 and in London in 2005 that shifted the compass again, pointing to the presumed failures of national states to integrate their immigrant populations and ethnic minorities. From the mid-2000s onwards we note a significant shift in nationalism studies towards making sense of how the nation and migration interact. Nationalism and the other: mid-2000s and beyond The new millennium started with a profound geopolitical, cultural and existential crisis for Europe and the West. The terrorist attacks of 9/11 signalled the end of the post-1989 euphoria. While the US government was proclaiming the War on Terror and attacking Afghanistan and later again Iraq, Europe was facing important internal and external challenges. Urban violence erupted in northern English cities in the summer of 2001, while the French cities followed suit in 2005. National grievances of second generation children that were failing both in school and the labour market were then coupled by global cultural crises like that surrounding the Danish cartoons of Prophet Mohammad in 2006. While jihadist, extremist forms of Islam were emerging as a global terrorist threat, more moderate versions of Islam and European Muslims started being portrayed by conservative parties as unfit for European liberal and secular societies. The Madrid (2004) and London (2005) bombings did nothing but reinforce this view. Far-right and even simply conservative politicians argued that there is something ‘fundamentally wrong’ with Islam as a religion that makes it inappropriate for European democratic societies and impossible to accommodate in a secular system. Islam emerged forcefully as an important dividing ‘civilizational’ line within Europe during the same period in which the post-1989 European re-unification enthusiasm started declining. Indeed, the magnitude of the economic and political challenges of the transition of Central Eastern European countries from Communism to free market Capitalism and liberal democracy became increasingly felt in Europe in the late 1990s when several of the former Communist countries experienced a second round of economic and political decline. Discussions about their integration into the European Union seemed to come to a dead end when the Helsinki summit of 1999 reaffirmed the political will of the EU15 to integrate the new countries possibly in one big enlargement wave by 2004. Thus, economic objectives were subsumed to the overarching political goal of re-uniting Europe, provided the new member states would be full-fledged democracies and 582 Ethnicities 22(4) would subscribe to the European values which included the accommodation of national minorities and the abandonment of irredentist claims or border disputes. In a way it was the very success of Eastern Enlargement, alongside with the emergence of international jihadist terrorism, urban tensions among post-migration minorities and native majorities that paved the way for Islam to become the necessary European Other. Nationalism and the other: mid-2000s and beyond Not only had Communism collapsed and with it the overall Cold War geopolitical and symbolic framework, but the Central Eastern European countries were fully subscribing to the by then hegemonic western European model. The Communists had been suc- cessfully ‘reformed’ – there was a need for a new Other at the European and global level against whom a united Europe and the Western/European values would be reaffirmed. Migrants and particularly Muslims emerged as a convenient Other, both internally and externally – they were accused of creating ‘parallel societies’ within European countries, while they also posed a threat to European security through terrorism. Indeed, a number of thinkers and politicians were advancing the claim that Muslims were impossible to accommodate in European countries because their cultural traditions and religious faith were incompatible with secular democratic governance, while others distinguished be- tween those who were ‘good’ and ‘bad’ and ‘compatible’ and ‘incompatible’. In a similar vein, the refugee emergency of 2015 has been represented as an ‘invasion’ of Muslims that put under strain the already scarce welfare resources while also threatening the European secular way of life (see Wodak, 2016). Turning to how nationalism studies have developed during this period since the mid- 2000s to the late 2010s, there is a new focus on the role of the Other in defining the nation. I have argued in my own work (2013, 2020) for the need to put the Other centre-stage when seeking to understand the evolution of nationalism today, whether in open and pluralistic trajectories (plural nationalism) or in nativist and exclusionary ways (neo-tribal nationalism). My preoccupation with the Other is shared also by a number of scholars working in the wider field of nationalism and citizenship and particularly by scholars in the ‘Bristol school of multiculturalism’. Tariq Modood (2019) has recently argued for a multicultural understanding of na- tionalism. Modood’s notion builds on his earlier writings, arguing that British national identity should accommodate post-migration ethnic minorities who ask for recognition and inclusion within the national self-concept (Modood 2003). Within his more refined and more recent concept, however, Modood gives more space to the national majority. Nationalism and the other: mid-2000s and beyond He avoids the pitfall of Quebec-style interculturalism or liberal nationalism which argue for the primacy of the national majority culture, and rather specifies that multicultural na- tionalism implies that ‘the predominance that the cultural majority enjoys in shaping the national culture, symbols and institutions should not be exercised in a non-minority accommodating way’ (Modood 2019: 235). Thus, argues Modood (2019: 236–237), the majority and the minorities should stand in a dialogical relationship which should recognise that both identities are ever evolving, that neither side has the right to impose itself on the others, in ways that do not allow these others to coexist. Tahseen Shams (2020) has situated the migrant’s national identity formation in a triangular relational framework of the here, there and the elsewhere (country of origin, new homeland, and other significant ‘places’). Shams (2020) is looking at US citizens 583 Triandafyllidou who are Muslims of different origins and who are obliged to make sense of their identity through reference not only of their own country of origin or the country of destination but by reference to Muslim majority countries or an imagined Islam to which they are as- sociated in media or political discourses or even in their everyday encounters with fellow citizens. Concerned with the rising tendency of exclusionary and nativist disourses not only the part of majorities but also by minorities, Riva Kastoryano (2018) speaks of transnational nationalism. Kastoryano reflects on the phenomenon of nationalist exclusion through transnational exposure and openness. She notes that in a world of increased migration and inter-dependence, we witness the re-territorialisation of global identities through the backdoor. Communities and states, argues Kastoryano (2018: 7), strive to create new configurations of nations and nationalism that are relevant in a globalised context. One strategy for achieving this is to argue for transnational solidarity. Such transnational solidarity can be that of a global ‘nation’ –a global diaspora, such as, argues Kastoryano, a transnational European Muslim community. Recent work on nationalism has also turned to old concepts like nativism. Nativism emerged as a political movement in the early 19th century in the United States in response to mass immigration from Europe (Betz 2017). Literature on nativism is scant for a good part of the 20th century until the concept resurfaced to express anti-immigrant sentiment, during the last 20 years (Guia, 2016). Nationalism and the other: mid-2000s and beyond Such work has put centre-stage the role of immigrants as both socio-economic and moral threats to the nation, giving rise to exclusionary discourses in Europe and North America (ibid). While concerns with exclusionary nationalism and with the overall interaction between the national majority and ethnic or religious minorities rightly occupied the centre-stage of nationalism studies, we have experienced in 2020 a new challenge, a global pandemic, that has further put to the test both our feelings of community, belonging and exclusion as well as our scholarly efforts to understand these phenomena. Concluding remarks I have argued in this paper that not only nationalism and the ways in which we define and negotiate our minority and majority identities needs to be seen in its specific socio- political context, but that also nationalism studies require the same contextual reading. Reviewing some of the main theories of nationalism I have tried to make sense of why nationalism theorists have focused on specific theoretical and empirical questions or have adopted methodologies of broader political sociology or more of cultural anthropology and qualitative sociology in understanding the evolution of nations and nationalism. I have defined three periods in the last 40 years within which I identify the main research questions that dominate nationalism studies. In the first period that extends from the early 1980s till the mid-1990s, nationalism studies are dominated by a focus on grand the- oretical questions and the methods of political sociology. The most important nationalism theorists seek to explain whether nations are perennial or modern and whether nation- states and nationalism as a political movement is mainly ethnic or civic in its orientation. Political theorists during the same period also seek to define the contours of ‘good’ liberal 584 Ethnicities 22(4) Ethnicities 22(4) nationalism investigating the tensions between liberalism and nationalism. The main nationalism theorists of this period disagree on their explanations but agree on the focus of their research questions. This focus persists though until shortly after 1989 and the landslide changes that happened in Europe and the world after that date. Looking at the contrasting developments of the 1990s – the focus on national minority and religious identities and their revival in Central Eastern Europe on one hand, and the process of European integration and reconnection of Europe under a common Union – it comes as no surprise that nationalism theorists focus on what people ‘do’ with their identities and how they re-elaborate political and media discourses in their everyday encounters. Even though perspectives in this period too may differ between those that see in the new socio-economic and political realities the emergence of a cosmopolitan and post-national world and those that emphasise the persistence of the nation, the focus in both perspectives is on the everyday nationhood. The methodology is constructivist and discursive rather than focussing on institutions. Concluding remarks A new turn is registered though in the mid-2000s and the emergence of international terrorism and increased tensions between national majorities and ethnic minorities, particularly Muslims. The discursive and constructivist turn persists as a methodology, but the focus now is on the interaction between the nation and the Other and the ways in which diversity and even transnational influences may shape nations and nationalism. This preoccupation with the interaction between the nation and the Other reflects also the relevant politics in Europe where several far-Right and generally conservative political parties seek to capitalise on anti-immigrant and anti-Muslim sentiment. Taking note of how our research questions are interactively and perhaps at times inadvertently shaped by the socio-political developments in the context in which we work suggests the need for more self-reflexive work in nationalism studies. It is important to acknowledge that our perspective, research questions and even methodologies are in- directly guided by the important socio-political developments of the society in which we live, rather than simply by our intellectual curiosity. This invites for a more self-reflexive approach to our own work which resonates particularly with the current pandemic context and the renewed anti-racism mobilisations of the last 2 years (2020–2021). Looking at the challenges that emerge today for the coming decades, three issues arise: the pandemic and the upheaval as well as the innovation that it has brought; the persistent rise of populist tendencies that are closely intertwined with nationalism; and the powerful emergence of anti-racism and decolonisation as a potential new political narrative. Scholars in nationalism studies need to consider what the pandemic re-bordering and ‘vaccine nationalism’ tell us about hierarchies of membership and transnational inter- dependence. During the pandemic lockdowns we have witnessed innovative approaches to membership that have valued ‘effective residence’ over legal status of a migrant (as temporary or permanent) (Triandafyllidou 2022). We have seen a reconsideration of temporary migrants or asylum seekers as ‘essential workers’ that should be given a preferential path to permanent residency or citizenship on the basis of their contribution to the community (by ensuring the food supply chain or the care services do not break down). Concluding remarks Fundamental rights such as the right to health and life took also precedence over status considerations during the pandemic leading thus to universal access to public health 585 Triandafyllidou services in several European countries and in Canada, and for instance to provisional automatic ‘regularisations’ for people with precarious status to avoid them being exposed to further challenges during the pandemic (Triandafyllidou 2022). At the same time the pandemic has also given rise to calls for less transnational dependency and more self- sufficiency for some goods that could be crucial for the safety of the nation (e.g. in relation to the production of protective equipment or of vaccines). These remain open questions and we are still to see whether the pro-immigrant and pro-solidarity dimensions of the pandemic emergency will persist or whether vaccine nationalism will win the day. A second set of phenomena that has arisen in the second part of the 2010s and persists to this day is the rise of populism and the powerful combination of populism and na- tionalism among Far Right parties and movements but also among those on the left side of the political spectrum (obvious examples being Podemos in Spain and SYRIZA in Greece). The most strident forms of populist and exclusionary nationalism have arisen in countries that were considered to be the cradles of civic nationalism notably the US (Trump) and the UK (Brexit). What is interesting in these developments is the combi- nation of political ideologies with national pride. Thus in Trump and in Brexit discourses we find an exclusionary statist nationalism (Joppke 2021) which builds walls to protect the nation (and particularly those ‘left behind’) and compensate for the threats of globalisation and the neoliberal order while at the same time it argues that citizens are responsible for looking after themselves and should not burden the welfare state. In a symmetrical but radically different dynamic, left wing forces like the Greek party SYRIZA or the Spanish Podemos (Custodi 2021) developed a populist discourse where national identity became a vehicle for constructing a moral, political community arguing for an inclusive welfare state, an egalitarian society and a nation that is open to cultural and religious diversity. Such discourses are counter-hegemonic both from a nationalist and a political ideological perspective but can also become divisive as they are based on an anti-elite rhetoric. Concluding remarks A third and very important phenomenon that we have witnessed during the last few years is the calls for decolonisation (Tuck and Yang 2012) and for more effective anti- black racism policies that have crystallised in the black and indigenous lives matter movements (Habtom and Scribe 2020) and for a renewed call for decolonisation not just in settler colonial states like the US, Canada or Australia but also as a broader concept and a policy pertinent to Europe and North America alike. What remains though unclear is the extent to which these calls align with those fighting Islamophobia and whether these counter-hegemonic discourses can develop into a new form of nationalism or within each country as a new national narrative. Scholars preoccupied with nationalism need to remain vigilant and self-reflexive as to what are the research questions we should be asking in the coming years. The pandemic may actually be seen as catalyst that has both raised new questions about membership, catalysed ongoing trends of populist nationalism (both affirming the importance of science but also fuelling the ranks of anti-elite movements among the anti-vaxxers) and made room for anti-racism currents to emerge with a stronger voice. 586 Ethnicities 22(4) Anna Triandafyllidou https://orcid.org/0000-0001-6760-0033 Anna Triandafyllidou https://orcid.org/0000-0001-6760-0033 Anna Triandafyllidou  Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research has benefited from the Canada Excellence Research Chair in Migration and Integration program’s funding support generously provided by the Social Sciences and Humanities Research Council of Canada for the period 2019-2026. 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https://openalex.org/W4391757824
https://hrmars.com/papers_submitted/20550/the-association-of-fanship-in-psychosocial-outcomes-among-k-pop-fans.pdf
English
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The Association of Fanship in Psychosocial Outcomes among K-Pop Fans
International journal of academic research in progressive education and development
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cc-by
6,448
Abstract Korean pop culture (K-Pop) has reached a global fan base outside of Korea. K-pop fanship is still thought to be a passing fad in society, neglecting the influence it has on its fans. Thus, the purpose of this quantitative study is to look into K-pop fanship from a psychological standpoint. Social Identity Theory and Self-Categorization Theory was applied as a theoretical framework. The study used purposive sampling to determine participants. In total, 203 K-pop fans ranging from 18 to 35 years old from Malaysia fully completed an online survey with three different instruments measuring fanship, and psychosocial outcomes (happiness and self-esteem). Descriptive analysis, T-Test, One-Way ANOVA and simple linear regression were utilized to analyze the data. Results of this study indicated that K-Pop fanship was significant predictors of happiness and self-esteem. Implications of the findings, limitations, recommendations for future studies were also presented. Keywords: K-pop, Fanship, Psychosocial Outcomes, Happiness, Self-Esteem Published Online: 25 January 2024 Published Online: 25 January 2024 To Link this Article: http://dx.doi.org/10.6007/IJARPED/v13-i1/20550 DOI:10.6007/IJARPED/v13-i1/20550 Published Online: 25 January 2024 //dx.doi.org/10.6007/IJARPED/v13-i1/20550 DOI:10.6007/IJARPED/v13-i1/20550 Vol 13, Issue 1, (2024) E-ISSN: 2226-6348 Nevhishna Nisha Sivanesan & Daniella Mokhtar Centre for Research in Psychology and Human Well-being, Faculty of Social Science and Humanities, Universiti Kebangsaan Malaysia Corresponding Author Email: a183084@siswa.ukm.edu.my Nevhishna Nisha Sivanesan & Daniella Mokhtar Centre for Research in Psychology and Human Well-being, Faculty of Social Science and Humanities, Universiti Kebangsaan Malaysia Corresponding Author Email: a183084@siswa.ukm.edu.my To Link this Article: http://dx.doi.org/10.6007/IJARPED/v13-i1/20550 DOI:10.6007/IJARPED/v13-i1/20550 Published Online: 25 January 2024 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 South Korea for protection. As a result, Western music gained popularity after the American Forces Korea Network radio began broadcasting in 1957. The end of the 1960s and beginning of the 1970s saw another revolution in Korean pop music. Ballad singers began to gain popularity in the 1980s after the release of an album in 1985. Early K-pop artists adopted American popular music genres like rap, rock, and techno into their music during the 1990s (Hartong & Laurens, 2016). The exposure to American music trends aided in the establishment of entertainment companies. Talent agencies started marketing K-pop stars in the late 1990s by adopting the idol business model from J-pop (Gingold, 2019), in which talents are chosen and taught to attract a worldwide audience through formal courses or through residency programmes (Oh, 2013). Potential talents are selected through a lengthy and rigorous process that includes physical and language training as well as height selection. Regardless of whether they are male or female singers, K-pop prioritizes slim, tall, and feminine appearances with teenage or occasionally very cute facial expressions, according to sociology professor Ingyu Oh (2013). These initiatives increase the marketability of K-pop while simultaneously strengthening South Korea's soft power, which has grown to be a significant component of the official policy (Walsh, 2014). Further with the emergence of illegal underground music clubs and punk rock bands in the 1990s, there was a reactionary movement against mainstream popular culture (K-Pop: A New Force in Pop Music, 2011, pp. 63–66). In addition to encouraging South Korean entertainers to seek out new markets, the 1997 Asian financial crisis also encouraged the national leaders to concentrate on enhancing the country's cultural influence through music (Hong, 2014). K-pop's rising recognition in the twenty-first century is a component of Hallyu, also known as the Korean Wave, which refers to the spread of South Korean culture abroad (Ryoo, 2008). The growth of social media websites has been an essential instrument for the Korean music industry in expanding its audience. The South Korean government has embraced K-pop as a component of the Korean Wave and as a vehicle for projecting South Korea's soft power abroad, particularly towards international youth (Linda, 2012). Introduction K-pop, or Korean popular music, is a genre of popular music that has its roots in South Korea and is a component of that nation's culture. On top of its traditional Korean music roots, it incorporates styles and genres from around the world, including pop, hip hop, R&B, rock, jazz, gospel, reggae, electronic dance, folk, country, disco, and classical music (Laurie, 2016). Korean pop music has been gaining popularity in Asian countries for over a decade. The K- pop Wave referred to as Hallyu is known as the rise of the Korean pop music phenomenon which successfully penetrated the overall music industry all over the world (Boman, 2019). PSY, BTS, and Blackpink are internationally recognized K-pop artists who have demonstrated the extent of K-pop culture's global popularity in recent years through the use of various digital media technologies. The origins of Korean popular music first emerged back in 1885, when Henry Appenzeller, an American missionary, began teaching American and British folk songs at a school (K-POP: A New Force in Pop Music, 2011, pp.47-48). They frequently featured Korean lyrics sung to a well-known Western melody. As the Korean Peninsula was partitioned into North and South after being freed from Japanese control in 1945, Western culture was gradually imported into South Korea between the 1940s and 1960s. After the Korean War, which lasted from 1950 to 1953, American troops stayed in 943 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 extraversion personality qualities are more likely to be found in a subculture like fandom. Extraverts are individuals who are friendly, talkative, and assertive. According to one study, extraversion is highly associated with self-esteem, life satisfaction, and general well-being (Kadir et al., 2011). Despite the influence on lifestyle and social relationships, all of this has surely had a large impact on the culture of consumerism toward K-pop products too. According to the findings of a study, capitalist elements such as individual factors, environmental factors, and mass media factors have an impact on the consumption habits of K-pop fans (Ayob & Abdullah, 2021). extraversion personality qualities are more likely to be found in a subculture like fandom. Extraverts are individuals who are friendly, talkative, and assertive. According to one study, extraversion is highly associated with self-esteem, life satisfaction, and general well-being (Kadir et al., 2011). Despite the influence on lifestyle and social relationships, all of this has surely had a large impact on the culture of consumerism toward K-pop products too. According to the findings of a study, capitalist elements such as individual factors, environmental factors, and mass media factors have an impact on the consumption habits of K-pop fans (Ayob & Abdullah, 2021). There are fewer studies that focus on the psychological aspect of K-pop fanship. Kpop fanship is still assumed to be a random youth phase trend in society, ignoring the influences it has on its fans' lives. Undoubtedly, music is a brilliant tool for improving mental health. However, the presence of stigma towards K-pop fans can still be seen & it was mentioned in few studies (Ezani, 2019; Othman & Aini, 2021) where K-pop fans were perceived as being obsessive towards their idols. Apart from being discriminated against as obsessive, the fans from different countries or races are called “Korean Wannabe”. Another stigma associated with K-pop fans is that Kpop fanboys experience more hardship than girls as a result of the stereotype known as toxic masculinity. The fact that music is gender neutral and universal appears to be forgotten by society. When fanboys support idols, particularly male idols, and show interest in the K-pop genre, they are ridiculed as homosexuals. Therefore, this study is intended to explore the insight of K-pop fanship from a psychological aspect and dispel these stereotypes and demonstrate to the community the good it has been doing. INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 The findings of this study would reveal what is the influence of K-pop fanship on happiness and self-esteem among the fans. This study would benefit many parties since it has a lot of advantages and would yield more insights about this topic as well. Some parties that will be benefitted are the K-pop fans themselves, the Malaysian community, parents, future researchers and the Malaysian and South Korean Government. In a society that increasingly values online connections and global communities, this research offers a unique perspective on the power of music and fandom in shaping individuals' psychosocial well-being. This study not only explores the profound motivation that drives individuals to become fervent K-Pop fans but also investigates how their strong sense of belonging to a fandom can have far-reaching effects on their mental and emotional health. By delving into the intricate web of fan culture, it unravels the enigma of why K-Pop fans are often so dedicated, shedding light on the immense joy and fulfilment they derive from their fandom. Furthermore, the research illuminates the contribution of these passionate fans to the broader cultural landscape. As K-Pop continues its global ascendancy, understanding the psychosocial dynamics of fandom becomes increasingly relevant. This study is poised to uncover valuable insights that can not only benefit the fans themselves but also inform the entertainment industry, academia, and society at large about the transformative power of music and fandom on individuals' lives. INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 The K-pop market had collapsed at the start of the twenty-first century, and any of the first-generation idol groups that had gained popularity in the middle of the 1990s had either disbanded or stopped performing together (K-Pop: A New Force in Pop Music, 2011, p. 66). However, the popularity of a new boy band upon its debut in 2003 marked the return of idol groups to Korean entertainment and the expansion of K-pop as a component of Hallyu. After the successful debuts, second-generation K-pop began to emerge. K-pop has dominated a significant share of the East Asian music market since the mid-2000s. Also, Hallyu began to have a big impact on K-pop fans' lifestyles all over the world. For instance, the application of Korean culture can be seen more clearly nowadays among the fans in Malaysia just like how Western culture has been. In order to learn Korean, some Malaysians are even enrolling in language classes. It's amazing how well-versed most K-pop fans are in the lyrics. In addition, K-pop fashion is a significant factor in fan impact. Several Malaysians can be spotted adopting the fashion trends of their favorite K-pop idols. Besides, as an increasing number of people learn to understand and enjoy Korean cuisine, Korean eateries are proliferating throughout Malaysia. Korean noodles and "Kimchi" are gaining popularity. K-pop has undoubtedly had a fascinating cultural impact on Malaysian living. Hallyu leads social interactions in their daily lives as well. In order to join fandoms without disclosing their true identity, some fans create their own online persona. This allows the fans to form social bonds based only on shared interests (Maros & Basek, 2022). Individuals with 944 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 Branscombe in 2010 (Laffan, 2020). The K-pop fan identity may be examined within the framework of an empirically supported psychological construct, which is highlighted by the way that group memberships and affiliations affect one's self-concept. Branscombe in 2010 (Laffan, 2020). The K-pop fan identity may be examined within the framework of an empirically supported psychological construct, which is highlighted by the way that group memberships and affiliations affect one's self-concept. According to Henri Tajfel and John Turner-developed Social Identity Theory (SIT) in 1979, individuals will construct their self-concept or define their identities when they are surrounded with specific social groups. According to Tajfel (1987), a social identity is "that part of an individual's self-concept which derives from his knowledge of his membership of a social group together with the value and emotional significance attached to that membership" (p. 63). According to this concept, individuals will display proper behavior following the norms owned by their specific organization. For example, if the organization has a research culture that encourages academic excellence, the individual will be involved in research as well. This individual's self-esteem is also linked to organizational membership, and he will compare his organization to others (Nor et al., 2017). Moreover, the self- categorization theory by Turner and his colleagues in 1987 explains that individuals tend to characterize few identities in different settings when they are alone or ingroup entities (Levine & Hogg, 2010). And, through the process of self-categorization, people form their own social identity. As a result of self-categorization, individuals also report several beneficial psychosocial outcomes, including a positive perception of fellow group members and changes in self-esteem and self-enhancement (Tajfel & Turner, 1979). SIT might provide some useful insights regarding how the social identity of K-pop fans has evolved. From this vantage point, K-pop fans might think about their social and personal selves in relation to their K-pop fandom. K-pop enthusiasts have been known to organize themselves into different online fan organizations by starting, joining, and managing them (Kim & Kim, 2017). Yet, there hasn't been a lot of work done to investigate the psychosocial scope of K-pop fans' self-categorization behaviors. In contrast to the assumptions of the theory, qualitative research on K-pop fan identity has shown the existence of a pathological fan type termed Sasaengpaen (Williams & Ho, 2014). Sasaengpaens have been known to display stalker like behavior and an extreme level of fanship. Literature Review There are two theories that can be used to explain fan behavior and cognition, the Social Identity Theory (SIT) and Self Categorization Theory (Reysen & Branscombe, 2010). It appears that K-pop followers have a social identity based on their compassion to the genre. As a matter of fact, the psychological fanship depth of the K-pop fan identity is still mostly unknown and is a crucial part of the fan social identity conception put forward by Reysen and 945 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 b. Fanship Scale The Fanship Scale was used to assess K-pop fanship (Reysen & Branscombe, 2010). The Fanship Scale is an 11-item, unidimensional self-report instrument that measures psychological fanship and was created to accommodate all types of fan interest. Using a likert scale that ranges from one to nine, each item is self-reported. The Fanship Scale's scoring process entails adding up the item scores, with one item being scored in reverse, and interpreting the total result as the fanship level. Greater scores are correlated with higher fanship levels.Sample questions of this questionnaire are “I am emotionally connected to my interest” and “My interest is part of me”. This Fanship scale adapted for K-pop fanship is highly reliable (11-items, α = 0.88). INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 But this kind of fan is not typical of K-pop fans in general (Williams, 2016). The study conducted by Reysen and Branscombe in 2010 was intended to examine the similarities and differences between sports fans and non-sports fans with fanship and fandom on entitativity, and collective happiness. The findings of the study produced a positive correlation between fanship and collective happiness among sports fans (Reysen & Branscombe, 2010). Another study has been conducted to find the differences in happiness between otaku and non-otaku college students (Jang & Park, 2021). Otaku is a Japanese term for those who have an intense interest towards something, especially in anime, manga and video games. The study results proposed that otaku students had a significantly higher happiness score compared to non otaku students. Laffan (2020) investigated the relationship between K-pop fanship and psychosocial outcomes which included happiness and self- esteem too, along with another domain among K-pop fans. The findings of this study in the end demonstrated K-Pop fanship as a significant predictor of increased happiness, self- esteem, and social connectedness. The attitude of fans has been demonstrated in another study to play a mediating function in the relationship between psychological well-being and self-esteem (Ismi & Badayai, 2023). 946 Research Method and Design This study used a quantitative approach to study the association of fanship on psychosocial outcomes (happiness and self-esteem) among K-pop fans. Research design that was used in this study is regression analysis. Regression design was used to solve the research questions included in this study. Since the objective of simple linear regression is to predict the value of an output variable based on the value of a predictor variable, it is suitable for these research questions. Moreover, it simplifies the calculation technique and, more significantly, these linear equations have a simple interpretation. a. K-pop based demographic questionnaire a. K-pop based demographic questionnaire Participants were queried about their demographic details regarding age, gender, nationality, favorite K-pop artist or band, how long they have been fans of that artist or band, and how frequently they listen to K-pop music. Sample The target population of this study were Malaysian K-pop fans. Participants in this study were required to meet the eligibility requirements, which include being between the ages of 18 and 35 and having listened to K-pop for at least a year. Purposive sampling approach was used to determine participants for this study. The Cochran Formula was used to determine the required sample size which was 384. However, only 203 responses were collected. The setting of this study was based in Malaysia only. c. Subjective Happiness Scale (SHS) A 4-item scale of global subjective happiness is called the SHS. The Subjective Happiness Scale was developed by Sonya Lyubomirsky and Heidi Lepper. Two items ask respondents to rate themselves both in absolute terms and relative to peers. The other two items provide brief descriptions of happy and unhappy people and ask respondents to what extent each categorization describes them. A range of 1 to 7 is produced by averaging responses to the four items. A higher score means that the person is happier. "In general, I consider myself not a very happy person (1) or a really happy person (7)" is one of the example items. INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 in reverse. An example question is “At times I think I am no good at all”. Higher total scores indicate higher levels of self-esteem. The SES is satisfactorily reliable according to McDonald’s Omega reliability computation (10-items, α = 0.79). in reverse. An example question is “At times I think I am no good at all”. Higher total scores indicate higher levels of self-esteem. The SES is satisfactorily reliable according to McDonald’s Omega reliability computation (10-items, α = 0.79). Data Analysis The SPSS statistical analysis software was used in this study. Data cleansing was done first to find any potential inaccuracies or errors in the database's data. This is accomplished by eliminating erroneous, incomplete, and improperly formatted data (Warudkar, 2022). The data was then validated and evaluated for reliability. The data was next subjected to a normality test to confirm that it is distributed normally. As the data was trustworthy, legitimate, and normal, the research was continued with the data analysis. Simple linear regression analysis was utilized to examine the relationship between two continuous variables. Since the focus is to find out if psychosocial outcomes can be predicted by K-pop fanship, simple regression analysis was used to determine how much these as a whole affect psychosocial outcomes. d. Rosenberg Self-Esteem Scale Rosenberg developed the Rosenberg SES in 1965. The scale is assessed by adding the totals of the 10 items' scores on a four-point Likert scale, with half of the items' scores being given 947 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 RNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT 3, No. 1, 2024, E-ISSN: 2226-6348 © 2024 Female 134 66 Frequency of Listening to K-Pop Music Very rarely 0 Rarely 0 Occasionally 64 31.5 Frequently 88 43.3 Very frequently 51 25.1 Note: Sd. = Standard deviation, Min.= Minimum, Max. = Maximum. Note: Sd. = Standard deviation, Min.= Minimum, Max. = Maximum. Descriptive Analysis Means, Standard deviation, Minimum and Maximum of three variables are shown in Table 1, including age, gender and the frequency of the fans listening to K-pop music. A total of 203 K- pop fans have participated in this research. A majority of the fans were female (66%) and 34% were male. Participants ranged in age from 18 to 35 divided into three groups, with a mean age group of 1.4, standard deviation of 0.69, and lastly, a minimum age of 18 years old and a maximum age of 26 years old. According to the data collected, 51 (25.1%) out of 203 fans listen to K-pop music excessively. Then, another 43.3% listen to this genre frequently and 31.5% of them listen to it occasionally. Table 1 Means, Standard deviation, Minimum and Maximum for K-pop Fan’s characteristics (n=203) Variable N % Age (Years) 18 – 23 144 70.9 24 – 29 36 17.7 30 – 35 23 11.3 Mean 1.40 Sd. 0.69 Min. 18 Max. 35 Gender Male 69 34 Standard deviation, Minimum and Maximum for K-pop Fan’s characteristics (n=203) le N % Means, Standard deviation, Minimum and Maximum for K-pop Fan’s characteristics (n=2 Variable N % 69 948 Results Simple linear regression was carried out to examine if K-pop fanship predicts happiness. The model was statistically significant (F (1,201)= 178.93, p < 0.001). The adjusted R2 indicated that 46.8% of the variance in happiness can be explained by the variance in fanship. Fanship significantly predicted happiness among K-pop fans. Thus, the alternative hypothesis is accepted. The regression equation for this model was: Happiness = 9.56 + (0.13 x Fanship). Table 2 Simple Regression Analysis for Happiness. Variable B SEB β p Fanship 0.125 0.009 0.686* 0.000 (Constant) 9.557 0.671 0.000 Note: *p<0.05, **p<0.01, ***p<0.001 Simple linear regression was carried out to examine if K-pop fanship predicts self-esteem. The model was statistically significant (F (1,201)= 231.43, p < 0.001). The adjusted R2 indicated that 53.3% of the variance in self-esteem can be explained by the variance in fanship. Fanship significantly predicted self-esteem among K-pop fans. Thus, the alternative hypothesis is accepted. The regression equation for this model was: Self-esteem = 29.59 + (-0.08 x Fanship). Simple linear regression was carried out to examine if K-pop fanship predicts self-esteem. The model was statistically significant (F (1,201)= 231.43, p < 0.001). The adjusted R2 indicated that 53.3% of the variance in self-esteem can be explained by the variance in fanship. Fanship significantly predicted self-esteem among K-pop fans. Thus, the alternative hypothesis is accepted. The regression equation for this model was: Self-esteem = 29.59 + (-0.08 x Fanship). Simple linear regression was carried out to examine if K-pop fanship predicts self-esteem. The model was statistically significant (F (1,201)= 231.43, p < 0.001). The adjusted R2 indicated that 53.3% of the variance in self-esteem can be explained by the variance in fanship. Fanship significantly predicted self-esteem among K-pop fans. Thus, the alternative hypothesis is accepted. The regression equation for this model was: Self-esteem = 29.59 + (-0.08 x Fanship). Table 3 Simple Regression Analysis for Self-Esteem. Variable B SEB β p Fanship -0.076 0.005 0.732* 0.000 (Constant) 29.587 0.357 0.000 Note: *p<0.05, **p<0.01, ***p<0.001 949 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 b. Fanship as A Predictor of Self-Esteem This study aims to examine if K-pop fanship predicts self-esteem. The hypothesis stating that K-pop fanship predicts self-esteem is accepted. Results found that indeed fanship is a predictor of self-esteem among K-pop fans. Thus, fanship can boost self-esteem. Previous studies have shown that being associated with successful organizations such as K-pop bands or football clubs can raise an individual's self-esteem and self-appraisal. Feeling connected socially can have a positive effect on self-esteem and wellbeing. O'Donovan (2016) found that participating in fandom activities tended to lead to the development of the individual’s skills and the boosting of their confidence, subsequently leading to a higher self-esteem. According to another study, fanship can help one feel more confident (Frederika, 2015). The desire for individuals to feel competent and worthwhile drives them to engage in the behavior of comparing themselves to others. When individuals believe they have a positive image in comparison to their idols or the members of their favorite band, they are more likely to follow them in order to increase their self-esteem. When choosing their own favorite idols, people tend to have this trait. a. Fanship as A Predictor of Happiness This study aims to examine if K-pop fanship predicts happiness. The hypothesis stating that K- pop fanship predicts happiness is accepted. Results found that indeed fanship is a predictor of happiness among K-pop fans. The result of this study is in line with past studies (Reysen & Branscombe, 2010; Jang & Park, 2021). The findings of this study may be explained by the fact that K-pop provides its worldwide fans with a pleasant and motivating hobby to be passionate about. Individuals gather and discuss how much they love Korean pop and media instead of engaging in negative behaviors. Being a fan of something enables individuals to meet and connect with others who share their passions in addition to focusing on what they love. Being a member of a social group, anticipating updates each day, and taking part in enjoyable activities can all contribute to an individual's happiness over time. Psychology has long recognized the significance of social relationships and affiliations for wellbeing. Being a fan or being a part of a fandom can provide some people with a safe place to feel, cry, laugh, or display indications of affection in a society where people frequently feel they must suppress their inner emotions. Some people who are fans of anything also get to experience the sense of achievement, even though it was done by someone or some group they admire rather than themselves. Implication The yielded results highlight that fanship predicts psychosocial outcomes, depending on the domains. K-pop has been constantly tied up to its negative effects on individuals and the cons of it. Some people are worried about the enormous growth of K-pop and how it plays a vital role in everyone’s life. While it might be true that excessive obsession towards K-pop leads to negative effects of psychosocial outcomes, it is not the sole reason for it. This study would increase the awareness among the Malaysian community on addressing K-pop as the rising phenomenon and how it influences psychosocial outcomes. Adding into it, this study would provide a vision to parents too. Parents can learn how K-pop has been influencing their children’s psychosocial outcomes on a daily basis. Particularly if the children are still students or in the age range of 18 to 22 years old, parents can take action to 950 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 support their interests so that they achieve the most positive outcomes, and they can also assist them in overcoming any unwanted influences. support their interests so that they achieve the most positive outcomes, and they can also assist them in overcoming any unwanted influences. Moreover, this study will provide information for the future researchers that intend to study about this topic or any relatable topics that involves fanship and psychosocial outcomes among fans of anything. This information will encourage them to further study the variables and their relationships. It will inspire them to study other factors that may be related to the variables in this study. Not only that, but the findings would be useful to the advancement of Psychology in Malaysia, especially in the field of Social Psychology, as this will contribute to the limited local research on K-pop fanship and psychosocial outcomes. This study also would be helpful to develop the relationship between the Malaysian and South Korean Government. The findings on positive psychosocial outcomes among fans in Malaysia would encourage Malaysia to host more K-pop based events and this will eventually benefit the economy of both governments which can lead to gain trust between each other. This study has added more evidence into existing literature related to K-pop. Implication If promoting the hobby of watching K-pop is considered as one of the solutions to good psychosocial outcomes, then this aspect must be looked into deeply to acknowledge the extent of its effects. Interventions should be specifically designed for each individual to cater to their needs and circumstances. Limitation Limitation in studies will certainly be present no matter how perfectly a research is designed, same goes for this study too. The first limitation of this study is that the samples are only taken from Malaysia. Hence, the results of this study cannot be generalized to other fans that reside in any other part of the world. Secondly, the main focus of this study is on K-pop fans that are in the age category of 18 to 35 years old. Hence, results obtained are not suitable to be generalized to populations that belong to different age groups. Adding on, there are no moderating or mediating variables involved in this study. Not only that, demographic variables such as academic intelligence, socioeconomic status, sexual orientation and physical or mental health problems are also not inquired in this study. Thus, future research can focus on these variables too. Another limitation of this study is that the data was collected by using a self-report questionnaire on an online basis. The downside of this method includes participant bias in which participants might not remember certain events, exaggerate their feelings or just do not want to reveal the truthful information as they are worried that they would be judged by others. Finally, the desired sample size was 384 in total, but only 203 responses were collected. Thus, future study can attempt to elicit more responses in order to produce broader and different outcomes. Conclusion In this study, the major aim was to assess if there is any sort of association in fanship with psychosocial outcomes. This research utilized three instruments namely Fanship Scale, Subjective Happiness Scale (SHS), and Rosenberg Self-Esteem Scale intending to measure the independent variable and dependent variable accordingly. The findings of this current study proved that there are associations between the variables. INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 an in-depth understanding of the relationship. Adding on, this research only has a total sample number of 203. Future research should recruit a much bigger number of participants to generalize the results to a larger population. an in-depth understanding of the relationship. Adding on, this research only has a total sample number of 203. Future research should recruit a much bigger number of participants to generalize the results to a larger population. Moreover, the context of study relating to fanship may delve into other aspects such as problematic fanaticism which is shown through acts of spending an unusual amount of money on merchandise or engaging in an invasion of privacy of idols. Moderating and mediating variables ought to be assessed as well to learn more about their connection between the variables. Recommendation This issue about the association of fanship with psychosocial outcomes is an intriguing one which should be explored in further research. First and foremost, future studies must consider using more diverse samples that represent a wider population. In this current study, the focus is only on K-pop fans as a sample, but it does not reflect other age groups or socioeconomic backgrounds. Secondly, the setting of the research can be wider rather than restricting it to Malaysia to find out how one’s lifestyle affects these variables. This is due to the fact that individuals living in different countries will have different patterns of fanship, and chances of them influencing psychosocial outcomes. Further research on this can help researchers gain 951 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 INTERNATIONAL JOURNAL OF ACADEMIC RESEARCH IN PROGRESSIVE EDUCATION AND DEVELOPMENT Vol. 13, No. 1, 2024, E-ISSN: 2226-6348 © 2024 Residents at a Higher Learning Institution. Jurnal Psikologi Malaysia, 25. Retrieved from https://spaj.ukm.my/ppppm/jpm/article/viewFile/8/7 Residents at a Higher Learning Institution. Jurnal Psikologi Malaysia, 25. Retrieved from https://spaj.ukm.my/ppppm/jpm/article/viewFile/8/7 Kim, M., & Kim, M. (2017). 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https://openalex.org/W2768124859
https://discovery.ucl.ac.uk/id/eprint/10039629/1/1-s2.0-S0016236117313492-main.pdf
English
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Pressure effects on flame structures and chemical pathways for lean premixed turbulent H2/air flames: Three-dimensional DNS studies
Fuel
2,018
cc-by
8,589
A R T I C L E I N F O This paper presents three-dimensional direct numerical simulations of lean premixed turbulent H2/air flames over a range of pressures using a detailed chemical mechanism. Effects of pressure on flame front structures and heat release from pressure-dependent pathways are analysed. Under the same initial turbulence at different pressures, the Kolmogorov length scale and local flame thickness decrease with increasing pressure. Thinner and sharper structures are found on the flame front at elevated pressures. As the pressure is increased from 1 atm to 5 atm, heat release is greatly enhanced at convex regions but weakened at concave regions of the flame fronts, which indicates that the effect of Darrieus-Landau instability is becoming stronger. The correlation of heat release and fuel consumption is also strengthened as pressure is elevated. A main pressure-dependent heat release reaction, H + O2(+M) = HO2(+M), is found to contribute less to the total heat release with increasing pressure for turbulent flames, which is contrary to the trend in laminar flames. In the low temperature zones, this is due to the decreased H radical pool at elevated pressure. In the high temperature regions, the reaction is less competitive compared with H + OH+M = H2O+M, thereby reducing its contribution to the heat release. Keywords: Lean premixed turbulent flame Elevated pressure Direct numerical simulation (DNS) Flame structure Pressure-dependent reaction propagation in premixed flames are widely investigated. Im and Chen [13] studied the effects of strain rate on local displacement speed of H2/ air triple flames with detailed chemistries. Cecere et al. [14] simulated a turbulent slot flame and discussed the effects of H2 enrichment on flame structures. The flames at different combustion regimes are also explored extensively. Aspden et al. [15] conducted a series of DNSs to study lean premixed H2/air combustion in a range of turbulence in- tensities and explored distributed burning. Carlsson et al. [16] in- vestigated flame structures of lean premixed flames at high Karlovitz numbers and compared the influence of small-scale turbulence on H2/ air and CH4/air flames. In addition, turbulence-chemistry interactions of lean premixed combustion have been addressed by some researchers. Aspden et al. [17] presented the decorrelation of fuel consumption and heat release at high turbulence intensity and Dasgupta et al. [18] dis- cussed the modification of chemical pathways based on his results. Xujiang Wanga, Tai Jina,b, Yongliang Xiec, Kai Hong Luoa,⁎ Xujiang Wanga, Tai Jina,b, Yongliang Xiec, Kai Hong Luoa,⁎ a Department of Mechanical Engineering, University College London, Torrington Place, London WC1E 7JE, UK b State Key Laboratory of Clean Energy Utilization, Zhejiang University, Hangzhou 310027, China c School of Mechanical Engineering, Southwest Jiaotong University, Chengdu 610031, China Fuel 215 (2018) 320–329 Fuel 215 (2018) 320–329 Contents lists available at ScienceDirect https://doi.org/10.1016/j.fuel.2017.10.094 Received 24 August 2017; Received in revised form 18 October 2017; Accepted 24 October 2017 0016-2361/ © 2017 Published by Elsevier Ltd. ⁎ Corresponding author. E-mail address: k.luo@ucl.ac.uk (K.H. Luo). ⁎ Corresponding author. 1. Introduction Flame/turbulence interactions are unsolved fundamental problems that have significant impact on the efficiency, emissions and overall performance of a wide range of combustion engines. The interactions are typically two-way: On the one hand, chemical reactions affect the flow properties and turbulence structures through gas expansion due to heat release [1,2]; on the other hand, turbulence affects chemical re- actions both directly and indirectly [3]. The majority of studies on flame/turbulence interactions are conducted at atmospheric pressure. Nevertheless, the operation of combustion engines such as gas turbines and internal combustion engines is typically at elevated pressures. There has been a growing body of experimental work on combustion at elevated pressures [4–12]. However, it is still difficult and expensive to conduct experiments under high pressures. With the availability of increasingly powerful supercomputers, di- rect numerical simulation (DNS) of turbulent reacting flow has become feasible and affordable. DNS with detailed chemical mechanisms pro- vides a powerful tool for studying the fundamentals of turbulent com- bustion, and aids the development and validation of turbulent com- bustion models for engineering applications. Flame structures and Most of the above work has been dedicated to flame structures and turbulence/chemistry interactions at the atmospheric pressure. An in- creasing effort of DNS studies of turbulent premixed flames under ele- vated pressures has been reported in the literature. Zhang et al. [19,20] conducted several two-dimensional (2D) DNSs to elucidate the com- plicated chemical and physical processes in a constant volume X. Wang et al. Fuel 215 (2018) 320–329 ⎜ ⎟ = − ⎛ ⎝ ∇ + − ∇⎞ ⎠ V D X X X Y p p ( ) 1 k k k k k k (2) enclosure relevant to homogeneous charge compression ignition en- gines. Yu et al. [21] compared 2D and three-dimensional (3D) DNS results of the auto-ignition process of a lean H2/air mixture with tem- perature stratification in a constant volume enclosure. A delayed but more rapid ignition of the mixture has been found in the three-di- mensional case than two-dimensional simulation. Most of them have employed periodic boundary conditions and are limited to 2D cases, due to the practical difficulties of performing DNS at elevated pressures and the excessive demand for computational resources as a result of very thin reaction layers that require extraordinarily fine spatial and temporal resolution. Dinesh et al. 1. Introduction [22–24] extended 3D DNS to lean premixed expanding spherical flames at elevated pressures to address the effects of preferential diffusion, turbulence intensity and equiva- lence ratio on flame structure, propagation and instability. Although these studies provide much valuable information, DNS studies of tran- sient flame characteristics with detailed chemical mechanisms at ele- vated pressures are far and few between. In particular, pressure-de- pendent reactions play an important role in heat release which is addressed for flames at atmospheric pressure [17,18,25], but the effects of pressure on theses reactions are not presented. It is thus critical to investigate the structures and chemical pathways of turbulent premixed flames at elevated pressures. (2) where Dk and Xk are, respectively, the diffusion coefficient and mole fraction for species k. The species reaction rate is given by [31]: ∑ ∏ ∏ = ⎛ ⎝ ⎜ − ⎞ ⎠ ⎟ = = ′ = ″ ω K X K X ̇ [ ] [ ] j M fj k N k v rj k N k v 1 1 1 kj kj (3) (3) where Kfj and Krj are forward and reverse reaction rates for reaction j. ′ vkj and ″ vkj are forward and reverse stoichiometric coefficients for specie k in reaction j. The rate constant for pressure-dependent reactions (unimolecular/recombination fall-offreactions) is expressed as [32]: ⎜ ⎟ = ⎛ ⎝ + ⎞ ⎠ ∞ K K P P F 1 r r (4) (4) where K∞is the limit of high-pressure rate constant and Pr is the pressure scale. F is determined with the TROE approach which is more sophisticated than the Lindemann form (where F is unity). The TROE approach is computationally more expensive but more realistic and compact enough in analysing large reaction systems [33,34]. In the present DNS of turbulent H2/air flames, three-dimensional, time-dependent, compressible Navier-Stokes equations and species transport equations are solved with a six-order explicit finite difference scheme. For time advancement, the low-storage third-order Runge- Kutta (RK3-2N) scheme is used for transport equations and third-order Livermore Solver for Ordinary Differential Equations (LSODE) is used for chemistry calculation. The time step in the simulations is controlled under 5 × 10−9 s. This code was extensively used in astrophysics and magneto-hydrodynamics. Its application in premixed turbulent com- bustion could be found in [26,35]. 3. Simulation cases = − = − − + = − − + = − − + = ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ + ∂ ∂ ∂ ∂ ∂ ω p ̇ ρ t ρu x ρu t ρu u x p x τ x ρY t ρu Y x ρV Y x E t E p u x q x u τ x ρRT MW [( ) ] ( ) i i i j i j i ij j k j k j k k j T T j j j j j ij i (1) Four 3D DNS cases were set up to simulate lean premixed H2/air flames under three different pressures in an inflow-outflow configura- tion with an aspect ratio 2:1:1. These cases have the same equivalence ratio = φ 0.6 and unburned gas temperature Tu = 298 K. Table 1 lists the detailed parameters of the studied cases. Firstly, the domain was initialized by superimposing a one-dimensional laminar flame on a pre- generated turbulence. The flames were therefore developing in turbu- lence. The initial turbulence was isotropic and homogeneous which was generated with helical forcing functions [37]. Periodic boundary con- ditions were imposed in the span-wise and lateral directions. The (1) where ρ is the mixture density, ui velocity component in xi direction, p pressure and τij stress tensor. Vk, Yk and ω̇ are respectively diffusion velocity, mass fraction and reaction rate for specie k. ET is the total energy, qj jth-component of heat flux, R universal gas constant, T temperature and MW mixture molecular weight. Mixture averaged approximation is adopted for species diffusion, where Soret and buoy- ancy effects are neglected. Buoyancy has many effects on the propa- gation and instability of premixed and non-premixed flames [27,28]. However, thermo-diffusive and Darrieus-Landau instabilities play the most important roles in lean premixed combustion [23]. It has been demonstrated that buoyancy has a significant influence on laminar flames, but the effects on turbulent flames diminish with increased turbulence intensity [29] and the influence is limited for flames in small-scales [30]. Moreover, the characteristic time of flame propaga- tion is much smaller than that of buoyant convection. As the considered cases are in small scales and under high turbulence intensities, the buoyancy effects should be small for the lean premixed H2/air flames in this study. 2. Direct numerical simulation The DNS is performed using the time-dependent compressible Navier-Stokes equations coupled with detailed transport properties and chemical kinetics [26]. The governing equations are: 1. Introduction The aim of the present work is to enhance our fundamental un- derstanding of the effects of pressure on turbulent flame structures and pressure-dependent reactions based on DNS of turbulent lean premixed H2/air flames. As following, the numerical method is described in section 2 and the computational cases at different pressures are given in section 3. DNS results are analysed in detail to elucidate the effects of pressure on turbulent flame structures and statistical properties in section 4, followed by conclusions in section 5. The chemical mechanism for hydrogen oxidation adopted was de- veloped by Li. et al. [36]. This mechanism contains 21 reversible re- actions and 9 species (H2, O2, H2O, H, O, OH, HO2, H2O2, N2). The chemistry implementation was validated quantitatively with Chemkin. ** Reynolds number, = ′ Re u l v / t . * Laminar flame thickness, = − ∂ ∂ δ T T T x ( )/| / | L b u max. *** Karlovitz number, ′ = Ka u S δ l ( / )( / ) L L t 2 3 3 . 3. Simulation cases Species diffusion velocity is expressed as [26]: where ρ is the mixture density, ui velocity component in xi direction, p pressure and τij stress tensor. Vk, Yk and ω̇ are respectively diffusion velocity, mass fraction and reaction rate for specie k. ET is the total energy, qj jth-component of heat flux, R universal gas constant, T temperature and MW mixture molecular weight. Mixture averaged approximation is adopted for species diffusion, where Soret and buoy- ancy effects are neglected. Buoyancy has many effects on the propa- gation and instability of premixed and non-premixed flames [27,28]. However, thermo-diffusive and Darrieus-Landau instabilities play the most important roles in lean premixed combustion [23]. It has been demonstrated that buoyancy has a significant influence on laminar flames, but the effects on turbulent flames diminish with increased turbulence intensity [29] and the influence is limited for flames in small-scales [30]. Moreover, the characteristic time of flame propaga- tion is much smaller than that of buoyant convection. As the considered cases are in small scales and under high turbulence intensities, the buoyancy effects should be small for the lean premixed H2/air flames in this study. Species diffusion velocity is expressed as [26]: Table 1 Parameters of simulation cases. For all the cases, the equivalence ratio is 0.6 and the temperature of the unburned gas is 298 K. 4.1. Flame structures and local properties Flame front is identified with the iso-surface which corresponds to the peak heat release rate in the turbulent flames. Fig. 3 shows in- stantaneous snapshots of flame fronts coloured by heat release rate (HRR) for the four cases. The convex structures (protruding to un- burned mixture) are taken to be positive curvature and the concave structures (protruding to products) are taken to be negative one. When the flames are under the identical Reynolds number but different pressures (Case A and D), the flame structure shows significant differ- ences. At p = 5 atm (Case D), the flame surface is slightly wrinkled, which corresponds well with Fig. 1 where Case D locates near the corrugated flamelets region. However, Case A shows a flame surface with considerable wrinkling. In this study, we will focus on the cases with the same initial velocity fluctuation but different pressures, which is representative of practical combustion [38,39]. Due to the hydro- dynamic instability, the flames all display large scale wrinkling struc- tures which are similar to those reported in [23]. It is noted that there are more small and sharp structures when pressure increases from 1 atm to 2 atm to 5 atm. This is due to the increased ′ u S / L ratio and decreased Kolmogorov length scale [39]. An interesting observation is that in high positive curvature regions the heat release rate is much lower than that in the surroundings at p = 1 atm, but the phenomenon is not obvious at p = 2 atm and 5 atm. In addition, high negative curvature areas show ultra-low heat release rate at elevated pressures. Fig. 1. Regime diagram for turbulent premixed combustion. The four cases are denoted with *(Case A), +(Case B), °(Case C) and ■(Case D). Navier-Stokes Characteristics Boundary Conditions (NSCBC) were ap- plied at the inlet/outlet to maintain a constant pressure. The domain length is 10 mm and width is 5 mm corresponding to 11 times the in- tegral length scale lt in the crossflow directions. As shown in Fig. 1, four cases are within the thin reaction zone in the premixed combustion regime diagram. From Cases A to C, the pressure increases from 1 atm to 5 atm, while the root-mean-square turbulent fluctuation velocity ′ u is maintained the same. 4.1. Flame structures and local properties It is found from Table 1, when pressure is elevated, the laminar flame speed SL, thick- ness δL and kinematic viscosity of mixture v decrease significantly. As a result, the initial Reynolds numbers increase from 153 to 763. How- ever, the Karlovitz numbers are at the same level. Case D is set up with the same Reynolds number as Case A, while the corresponding Karlo- vitz number is much lower and the case is located close to the corru- gated flamelets region. Comparing concave and convex structures shown in Fig. 3, it is found that they have different characteristics. As the pressure increases, the concaves are becoming much thinner and sharper. To further in- vestigate this phenomenon, probability density functions of mean flame surface curvature are plotted in Fig. 4. It is clear that there are more positive curvature areas and surface curvature is increasing when pressure is increased. For example, at p = 1 atm, the ratio of cumulative distribution functions (CDFs) for positive and negative curvature is 1.15:1, which is much smaller than 1.74:1 at p = 5 atm. This suggests more intermediates protrude into the unburned mixture at elevated pressures. The grids for each case are 1024 × 512 × 512 with uniform mesh resolution = x μ Δ 9. 77 m. Correspondingly, the number of grids across the flame thickness decreases from 38 to 8. In order to check that the cases are sufficiently resolved, a grid-independent-test case is per- formed. Case C with the thinnest flame is chosen as the baseline case. If the grid resolution is sufficient for Case C, then the resolution is good enough for the other three cases. All properties for the grid-in- dependent-test case are the same as the baseline case except for re- solution which decreases from 9.77 μm to 4.89 μm. Fig. 2 shows the comparison of the average heat release rate in the computational do- main for the two cases. The two simulations agree with each other very well, which indicates that the mesh used here can produce grid- To further analyse the flame structures, two-dimensional slices of normalized heat release rate (NHRR, normalized by the peak heat re- lease rate of corresponding 1D flame) taken from three-dimensional simulations for Case A, B and C are plotted in Fig. 5. Table 1 Table 1 Parameters of simulation cases. For all the cases, the equivalence ratio is 0.6 and the temperature of the unburned gas is 298 K. Case A B C D Pressure (atm) 1 2 5 5 SL (cm/s) 88.5 69.9 47.9 47.9 δL (mm)* 0.375 0.17 0.0758 0.0758 ′ u (cm/s) 656.8 656.8 656.8 131.4 v (cm2/s) 0.1923 0.0962 0.0385 0.0385 lt (mm) 0.447 0.447 0.447 0.447 x Δ (μm) 9.77 9.77 9.77 9.77 Re** 153 305 763 153 Ka*** 18.5 17.8 20.9 1.87 * Laminar flame thickness, = − ∂ ∂ δ T T T x ( )/| / | L b u max. ** Reynolds number, = ′ Re u l v / t . *** Karlovitz number, ′ = Ka u S δ l ( / )( / ) L L t 2 3 3 . 321 Fuel 215 (2018) 320–329 X. Wang et al. X. Wang et al. Fig. 1. Regime diagram for turbulent premixed combustion. The four cases are denoted with *(Case A), +(Case B), °(Case C) and ■(Case D). independent results. 4.1. Flame structures and local properties Note only the regions that cover the flame fronts are plotted and local structures at p = 5 atm are also given. Fig. 5 also shows iso-lines of progress variable CT = 0.1–0.9 and NHRR = 35%NHRRpeak which separately define the reaction zone and inner reaction zone [40]. The non-dimensional pro- gress variable CT is given by: Fig. 2. Temporal evolution of average heat release rate in the domain for Case C (red line) and grid-independent-test Case (black line). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) = − − C T T T T T u p u (5) = − − C T T T T T u p u (5) where T, Tu and Tp are the local, unburned and peak temperatures, re- spectively. It can be seen in Fig. 5 that the vorticity structures for the three cases are similar because these simulations are initialized with the same velocity field. However, the vorticity gradient is increasing with pres- sure due to the decreased kinematic viscosity, which tends to distort the flame front seriously. Even if the laminar flame thickness is decreased significantly with increased pressure, the flame front position variance is not obvious. It is also noted that, locally, the thinner reaction zone at elevated pressures makes it harder for small turbulence structures to penetrate into it. This observation confirms that the three cases are in the thin reaction zone as shown in Fig. 1. Fig. 2. Temporal evolution of average heat release rate in the domain for Case C (red line) and grid-independent-test Case (black line). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 2. Temporal evolution of average heat release rate in the domain for Case C (red line) and grid-independent-test Case (black line). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) A striking observation from Fig. 5 is that the flame front shows more finger structures with intense heat release rate when pressure is 322 elevated. In the low-pressure case, a cellular burning pattern is shown, with moderate heat release in regions of both positive and negative curvature. 4.1. Flame structures and local properties When pressure is increased to 5 atm, the cellular structures appear thinner and sharper; heat release in the convex regions is be- coming quite different from that in the concave regions. It is clear from the zoomed-in views for p = 5 atm, heat release rate in regions of po- sitive curvature is intense, but local extinction happens in regions of negative curvature. This is mainl instability. In turbulent flames, disturbance for the onset of hyd the wrinkling flame structure, tog flame reaction zone at elevated Darrieus-Landau instability on l pological complexity of the loca Fi (T Ca A1 tiv pr gi X. Wang et al. X. Wang et al. Fuel 215 (2018) 320–329 Fig. 3. Instantaneous snapshots of flame front (T = 1000 K) coloured by heat release rate for Case A-D at three pressure values. The view for A1-D1 is protruding to unburned mixture (posi- tive curvature regions) and the view for A2-D2 is protruding to products (negative curvature re- gions). Fig. 3. Instantaneous snapshots of flame front (T = 1000 K) coloured by heat release rate for Case A-D at three pressure values. The view for A1-D1 is protruding to unburned mixture (posi- tive curvature regions) and the view for A2-D2 is protruding to products (negative curvature re- gions). elevated. In the low-pressure case, a cellular burning pattern is shown, with moderate heat release in regions of both positive and negative curvature. When pressure is increased to 5 atm, the cellular structures appear thinner and sharper; heat release in the convex regions is be- coming quite different from that in the concave regions. It is clear from the zoomed-in views for p = 5 atm, heat release rate in regions of po- sitive curvature is intense, but local extinction happens in regions of negative curvature. This is mainly due to the effect of Darrieus-Landau instability. In turbulent flames, any initial vortex serves as an initial disturbance for the onset of hydrodynamic instability, which leads to the wrinkling flame structure, together with turbulent eddies [41]. Thin flame reaction zone at elevated pressure will enhance the effect of Darrieus-Landau instability on local flame structure and increase to- pological complexity of the local flame. In convex regions, thermal 323 X. Wang et al. Fuel 215 (2018) 320–329 Fig. 4. Probability density function (a) and cumulative distribution function (b) of mean curvature of flame front. 4.1. Flame structures and local properties 7 shows the scatter plots and joint probability density function of temperature and local equivalence ratio for Cases A and C. The local equivalence ratio φ is given by: diffusion to the external streamlines is weaker than the chemical energy due to the deficient reactant transportation [42,43]. Local flame in- tensity will be enhanced. However, it is interesting to note that the peak heat release rate does not happen in regions of highest positive curva- ture, but in flank regions. This explains why the regions with highest positive curvature in Fig. 3 are related to lower heat release rate values. It can be seen from the iso-lines that when pressure is increased, the correlation between heat release rate and temperature is enhancing in regions with peak positive curvature; regions with peak temperature higher than burnt gases show in the centre of finger structures. These observations show the effects of pressure on flame structures and local heat release. 4.1. Flame structures and local properties The insets in (a) are PDFs with large positive and negative curvature. Fig. 4. Probability density function (a) and cumulative distribution function (b) of mean curvature of flame front. The insets in (a) are P d cumulative distribution function (b) of mean curvature of flame front. The insets in (a) are PDFs with large positive and negative curvature Fig. 4. Probability density function (a) and cumulative distribution function (b) of mean curvature of flame front. The insets in (a) are PDFs with large positive and negative curvature. diffusion to the external streamlines is weaker than the chemical energy due to the deficient reactant transportation [42,43]. Local flame in- tensity will be enhanced. However, it is interesting to note that the peak heat release rate does not happen in regions of highest positive curva- ture, but in flank regions. This explains why the regions with highest positive curvature in Fig. 3 are related to lower heat release rate values. adiabatic temperatures are close to 1850 K, however the corresponding temperature of peak heat release region is increasing with pressure. For example, the peak heat release happens at a position near T = 1000 K at p = 1 atm. When pressure is elevated to 5 atm, the corresponding temperature region is at T = 1300 K. This observation is much more obvious for the 3D simulations in turbulent fields. In the two cases, the regions with T < 800 K are referred to as the low temperature zone of the reaction. There is noticeable heat release in the low temperature zone for the flame at p = 1 atm where the NHRR exceeds unity and even reaches a peak value as high as 2 in the domain. The intensified heat release in the low temperature zone is also observed in high Ka combustion [16] and this study suggests that the phenomenon is not unique for high Ka flames. It is interesting to note that when pressure is elevated, the heat release at low temperatures is decreased and the scatter distribution is enhanced towards the direction of deviating from 1D reference flames. It is reported in [25] that differential diffusion can enhance the heat release in the low temperature zone. This suggests that the influence of differential diffusion is obviously decreased for lean premixed flames when pressure is elevated. Fig. 4.2. Variations of heat release and species distributions The red dashed lines correspond to the distribution in 1D 3D Cases are normalized by peak values of corresponding 1D flames. where YH and YO are the molar mass of elements H and O, respectively. h h l d h d b h clearly reduced. For Case A, more H radical diffuses from reaction zone to preheat zone. The consumption of H in the low temperature zone is associated with an exothermic reaction [25]: where YH and YO are the molar mass of elements H and O, respectively. When the pressure is elevated to 5 atm, the distribution in the T −φ field becomes more scattered in the thin reaction zone due to the larger maximum curvature and stronger preferential diffusion for the tip shown in Fig. 4, but the data is more concentrated, towards the direc- tion of yielding a unity effective Lewis number. However, findings in laminar flames show the opposite phenomenon: the deviation of local equivalence ratio from unburned mixture is stronger at p = 5 atm compared with that at p = 1 atm. It indicates that the rates of turbulent transport of energy and species are getting closer at the elevated pres- sure. Furthermore, at p = 5 atm, there is a high degree of scatter in the region T > 1850 K. This corresponds well with the high temperature regions in Fig. 5. + + = + H O ( M) HO ( M) 2 2 (7) (7) + + = + H O ( M) HO ( M) 2 2 Owing to the above reaction, intensified heat release, which reaches peak values, is observed at low temperatures for Case A in Figs. 5 and 6. For Case C, the reduced decorrelation of heat release and fuel con- sumption is mainly due to the reduced mobility of atomic hydrogen, which is consistent with the analysis of Fig. 7. Comparing Case C and Case D, it is interesting to find that the correlation of heat release and fuel consumption shows a very similar trend. It implies the influence of turbulence on local flame chemistry is dramatically reduced when pressure is elevated. It becomes harder for turbulence to disrupt the flame inner structures. To examine the influence of pressure on fuel consumption and heat release, the scatter plots and joint probability density function of fuel consumption rate and heat release rate are shown in Fig. 8. 4.2. Variations of heat release and species distributions The two variables are normalized by peak values of 1D flames, respectively. Owing to the decreased flame thickness, the distribution of scattered data changes from a triangle to an acute angle shape when pressure increases from 1 atm to 5 atm. Aspden et al. [17,44] observed a dec- orrelation of fuel consumption and heat release at high positive cur- vature regions and attributed it to the high mobility of atomic hydrogen at high turbulence intensity. In this study, when pressure is elevated, there are more regions of high curvature, but the decorrelation is 4.2. Variations of heat release and species distributions It is known that flame wrinkling is affected by pressure and re- lationships among flame properties are changed by pressure. To quantitatively investigate the phenomenon, we look at the scatter plot of heat release rate and fuel consumption rate. The two quantities are normalized by the peak values of the corresponding 1D flames at dif- ferent pressures. Fig. 6 shows the joint probability density function of temperature and normalized heat release rate at various pressures. The dashed lines provide references of 1D flames at the corresponding pressures. The distributions of 1D flames are close to normal distribution and the = φ Y W Y W local /2 / H H O O (6) d lines) for Cases A-C and local structures for Case C. The reaction zone is bounded by at release equals to 35% the local maximum value. = φ Y W Y W local /2 / H H O O (6) Fig. 5. Instantaneous snapshots of normalized heat release rate with vorticity (white solid lines) for Cases A-C and local structures for Case C. The reaction zone is bounded by CT = 0.1–0.9 with black lines. The thin reaction zone is bounded by the red lines where heat release equals to 35% the local maximum value. Fig. 5. Instantaneous snapshots of normalized heat release rate with vorticity (white solid lines) for Cases A-C and local structures for Case C. The reaction zone is bounded by CT = 0.1–0.9 with black lines. The thin reaction zone is bounded by the red lines where heat release equals to 35% the local maximum value. 324 Fig. 6. Scatter plot and joint probability density function of temperature and normalized heat release rate for Cases A and C. The red dashed lines correspond to the distribution in 1D laminar flames. The heat release rates of 3D Cases are normalized by peak values of corresponding 1D flames. X. Wang et al. Fuel 215 (2018) 320–329 X. Wang et al. Fuel 215 (2018) 320–32 Fuel 215 (2018) 320–329 Fig. 6. Scatter plot and joint probability density function of temperature and normalized heat release rate for Cases A and C. The red dashed lines correspond to the distribution in 1D laminar flames. The heat release rates of 3D Cases are normalized by peak values of corresponding 1D flames. ensity function of temperature and normalized heat release rate for Cases A and C. 4.3. The contribution of pressure-dependent reactions In laminar flames, pressure-dependent reactions make a great con- tribution to total heat release. When the pressure is elevated, these reactions play a more important role. This section focuses on heat re- lease from pressure-dependent reactions in turbulent flames. There are two pressure-dependent reactions in the Li et al. mechanism. Table 2 lists the main heat release reactions in which reaction 3 is a pressure- Fig. 7. Scatter plot and joint probability density function of temperature and local equivalence ratio for Cases A and C. The red lines correspond to the distribution in 1D laminar flames. Fig 7 Scatter plot and joint probability density function of temperature and local equivale Fig. 7. Scatter plot and joint probability density function of temperature and local equivalence ratio for Cases A and C. The red lines corre nsity function of temperature and local equivalence ratio for Cases A and C. The red lines correspond to the distribution in 1D laminar flames Fig. 7. Scatter plot and joint probability density function of temperature and local equivalence ratio for Cases A and C. The red lines correspond to the distribution in 1D laminar flames. 325 X. Wang et al. Fuel 215 (2018) 320–329 Fig. 8. Scatter plot and joint probability density function of normalized fuel consumption rate and normalized heat release rate for Cases A–D. The fuel consumption rate of 3D cases is normalized by peak values of corresponding 1D flames. X. Wang et al. Fuel 215 (2018) 320–329 Fig. 8. Scatter plot and joint probability density function of normalized fuel consumption rate and normalized heat release rate for Cases A–D. The fuel consumption rate of 3D cases is normalized by peak values of corresponding 1D flames. Fig. 9. Heat release contribution of reaction 3 to total heat release at different pressures. The blue and red lines correspond to 1D laminar flames and 3D turbulent flames, re- spectively. Table 2 Table 2 The main heat release reactions from Li et al. mechanism and the contribution of single reaction to total heat release for 1D laminar flame at 1 atm and 5 atm. The main heat release reactions from Li et al. mechanism and the contribution of single reaction to total heat release for 1D laminar flame at 1 atm and 5 atm. # Reaction Chr 1 atm Chr 5 atm 1 H2 + OH = H2O + H 0.201 0.203 2 H + OH + M = H2O + M 0.254 0.213 3 H + O2(+M) = HO2(+M) 0.286 0.309 4 HO2 + H = OH + OH 0.125 0.101 5 HO2 + OH = H2O + O2 0.096 0.157 dependent reaction. The heat release from another pressure-dependent reaction is very weak which is not discussed in this paper. The con- tribution of a given reaction to total heat release is computed as: Fig. 9. Heat release contribution of reaction 3 to total heat release at different pressures. The blue and red lines correspond to 1D laminar flames and 3D turbulent flames, re- spectively. = ∑ ∑ < < < < C q q hr T T T i T T T tot u p u p (8) (8) For a clear comparison, the heat release contribution of reaction 3 in laminar and turbulent flames is plotted in Fig. 9. A striking observation is that the contribution of reaction 3 shows different variation with pressure for laminar and turbulent flames. For the laminar flames, the contribution increases from 0.286 at 1 atm to 0.309 at 5 atm. A reverse trend is observed for the turbulent flames: the contribution drops from 0.304 to 0.282. This observation suggests a possible change in the local chemical pathways with pressure. where qi is heat release from reaction i and qtot is total heat release in the domain. where qi is heat release from reaction i and qtot is total heat release in the domain. Table 2 The ratio of cumulative heat release from reaction i to total hear release is calculated as: = ∑ ∑ < < < < C q q hr T T T i T T T tot u c u p (9) (9) To investigate the reasons why the heat release contribution trend is reversed, we firstly plot the scatter distribution of temperature and heat where < < T T T u c is the temperature range in which heat release from reaction i is cumulated. where < < T T T u c is the temperature range in which heat release from reaction i is cumulated. 326 X. Wang et al. Fuel 215 (2018) 320–329 Fig. 10. Scatter plot of temperature and heat release rate of reaction 3 for Cases A and C. Fig. 10. Scatter plot of temperature and heat release rate of reaction 3 for Cases A and C. release rate of reaction 3 at 1 atm and 5 atm in Fig. 10. It is noted that heat release in the low temperature zone (T < 800 K) at p = 1 atm is very strong, and even reaches peak values. At P = 5 atm, the scatter distribution shifts towards the high temperature zone with the peak heat release value at around 1100 K. The reaction mainly occurs in high temperature regions and the distribution is similar to the total heat release shown in Fig. 6 because reaction 3 is the main exothermal re- action in the mechanism. stays at a very low level in low temperatures. It is enhanced for tur- bulent flames, but the scatter data density is much lower than that at p = 1 atm in the low temperature zone; most of the distribution is lo- cated in high temperature regions for the turbulent flame. There is also a high OH radical pool in the high temperature zone, which means reaction 2 is more competitive than reaction 3 at the high pressure. Correspondingly, the cumulative heat release ratio of the turbulent flame at 5 atm is enhanced greatly in high temperature regions. It is clear that the increased H distribution in the low temperature zone contributes to a higher heat release from reaction 3 at the low pressure and the enhanced H distribution at high temperatures results in more heat release from reaction 2 at the elevated pressure. Fig. Table 2 11 further shows the ratio of cumulative heat release to total heat release of two main H consumption reactions for both laminar and turbulent flames. For reference, Fig. 12 presents the scatter plot of H radical mass fraction at the two pressures for laminar and turbulent flames. For reaction 3 at p = 1 atm, the major increase of cumulative heat release ratio happens in the low temperature regions where the distribution of most H radical for the turbulent flame is above the level of the 1D laminar flame. The higher fraction of H can contribute to an intense heat release from reaction 3; this is the main factor responsible for the higher heat release contribution for the turbulent flame. Simi- larly, the heat release ratio is also increased for another H consumption reaction #2 in the low temperature zone. However, it is not enhanced to the same extent because there is excess O2 in the low temperature regions and reaction 3 is more competitive. For reaction 3 at p = 5 atm, the contribution of heat release is apparently reduced in the low tem- perature zone. However, the ratio soars sharply when T > 1200 K for the laminar flame. In contrast, the ratio increases slowly for the tur- bulent flame, although a slight increase is observed in the low tem- perature regions. As can be seen in Fig. 12, the fraction of H radical 5. Conclusions A series of three-dimensional direct numerical simulations have been performed for lean premixed turbulent H2/air flames in the thin reaction zone, with pressure ranging from 1 atm to 5 atm. Four flames are considered: Cases A-C are initialized with the same turbulence at three different pressures to study the influence of pressure on flame structures and chemical pathways; Case D is presented with the same Re number with Case A but a different pressure to isolate the effect of pressure on flames under the same turbulence intensity. First, comparing Case D to Case A, an obvious decrease in flame surface areas and wrinkled structures is observed. Comparing Cases A–C where planar flames interact with the same turbulence, more sharp structures are observed at elevated pressures on the flame fronts. Heat Fig. 11. Cumulative heat release ratio of reactions 3 and 2 for Cases A (red lines) and C (blue lines). The solid lines correspond to turbulent flames and the dashed lines correspond to 1D laminar flames. Fig. 11. Cumulative heat release ratio of reactions 3 and 2 for Cases A (red lines) and C (blue lines). The solid lines correspond to turbulent flames and the dashed lines correspond to 1D laminar flames. actions 3 and 2 for Cases A (red lines) and C (blue lines). The solid lines correspond to turbulent flames and the dashed lines correspond to 1D Fig. 11. Cumulative heat release ratio of reactions 3 and 2 for Cases A (red lines) and C (blue lines). The solid lines correspond to turbulen laminar flames. 327 X. Wang et al. Fuel 215 (2018) 320–329 Fig. 12. Scatter plot of temperature and H mass fraction. The red lines correspond to the distribution in 1D laminar flames. Fig. 12. Scatter plot of temperature and H mass fraction. The red lines correspond to the distribution in 1D laminar flames. [3] Hamlington PE, Poludnenko AY, Oran ES. Interactions between turbulence and flames in premixed reacting flows. Phys Fluids 2011;23(12):125111. [3] Hamlington PE, Poludnenko AY, Oran ES. Interactions between turbulence and flames in premixed reacting flows. Phys Fluids 2011;23(12):125111. release is enhanced in concave regions, while reduced in convex re- gions, which can be attributed to the Darrieus-Landau instability. With increased pressure, the flame front area with positive curvature is in- creased. 5. Conclusions It is quite interesting to observe thin finger structures pro- truding into the unburned mixture under elevated pressures due to increased flame instability. It is also found that the peak heat release does not occur in fingertips but in flank regions. [3] Hamlington PE, Poludnenko AY, Oran ES. Interactions between turbulence and flames in premixed reacting flows. Phys Fluids 2011;23(12):125111. [4] Hu E, Li X, Meng X, Chen Y, Cheng Y, Xie Y, et al. Laminar flame speeds and ignition delay times of methane–air mixtures at elevated temperatures and pressures. Fuel 2015;158:1–10. [4] Hu E, Li X, Meng X, Chen Y, Cheng Y, Xie Y, et al. Laminar flame speeds and ignition delay times of methane–air mixtures at elevated temperatures and pressures. Fuel 2015;158:1–10. [5] Wang J, Zhang M, Huang Z, Kudo T, Kobayashi H. Measurement of the in- stantaneous flame front structure of syngas turbulent premixed flames at high pressure. Combust Flame 2013;160(11):2434–41. [6] Wang J, Yu S, Zhang M, Jin W, Huang Z, Chen S, et al. Burning velocity and sta- tistical flame front structure of turbulent premixed flames at high pressure up to 1.0MPa. Exp Therm Fluid Sci 2015;68:196–204. Second, the local heat release rate of turbulent flames is examined. It is observed that the peak value is over twice of that in the corre- sponding laminar flame. With increasing pressure, peaks are moving from low temperature regions to high temperature regions, which is more obvious for turbulent flames. Differential diffusion for turbulent flames is reduced with increased pressure, which results in an effective unity Lewis number. However, laminar flames experience an opposite trend. When pressure is elevated from 1 atm to 5 atm, the transport of H radical from high temperatures to low temperatures is reduced. The consequence is the enhanced correlation of heat release rate and fuel consumption rate. [7] Li H, Li G, Sun Z, Yu Y, Zhai Y, Zhou Z. Experimental investigation on laminar burning velocities and flame intrinsic instabilities of lean and stoichiometric H2/ CO/air mixtures at reduced, normal and elevated pressures. Fuel 2014;135:279–91. [8] Hayakawa A, Goto T, Mimoto R, Arakawa Y, Kudo T, Kobayashi H. Laminar burning velocity and Markstein length of ammonia/air premixed flames at various pressures. Fuel 2015;159:98–106. [9] Bagdanavicius A, Bowen PJ, Bradley D, Lawes M, Mansour MS. Stretch rate effects and flame surface densities in premixed turbulent combustion up to 1.25 MPa. Combust Flame 2015;162(11):4158–66. 5. Conclusions [10] Bradley D, Lawes M, Liu K, Mansour MS. Measurements and correlations of tur- bulent burning velocities over wide ranges of fuels and elevated pressures. Proc Combust Inst 2013;34(1):1519–26. [11] Kumar K, Mittal G, Sung C-J, Law CK. An experimental investigation of ethylene/ O2/diluent mixtures: laminar flame speeds with preheat and ignition delays at high pressures. Combust Flame 2008;153(3):343–54. Finally, contribution to heat release from the pressure-dependent reaction H + O2(+M) = HO2(+M) is analysed, considering it con- tributes around 30% of the total heat release in laminar flames at the atmosphere pressure. A striking observation is: the heat release con- tribution of this reaction increases with pressure for laminar flames, but decreases for turbulent flames. It is attributed to the decreased H radical pool in low temperature regions for turbulent flames at elevated pres- sures. In high temperature regions, the reaction is also less competitive compared with H + OH+M = H2O + M, thereby reducing its con- tribution to the total heat release. In summary, chemical pathways and heat release patterns are altered by pressure in turbulent flames com- pared with laminar flames. [12] Deng S, Han D, Law CK. Ignition and extinction of strained nonpremixed cool flames at elevated pressures. Combust Flame 2017;176:143–50. [13] Im HG, Chen JH. Effects of flow strain on triple flame propagation. Combust Flame 2001;126(1–2):1384–92. [14] Cecere D, Giacomazzi E, Arcidiacono NM, Picchia FR. Direct numerical simulation of a turbulent lean premixed CH4/H2–Air slot flame. Combust Flame 2016;165:384–401. [15] Aspden AJ, Day MS, Bell JB. Turbulence–flame interactions in lean premixed hy- drogen: transition to the distributed burning regime. J Fluid Mech 2011;680:287–320. [16] Carlsson H, Yu R, Bai X-S. Flame structure analysis for categorization of lean pre- mixed CH4/air and H2/air flames at high Karlovitz numbers: direct numerical si- mulation studies. Proc Combust Inst 2015;35(2):1425–32. [17] Aspden AJ, Day MS, Bell JB. Turbulence-chemistry interaction in lean premixed hydrogen combustion. Proc Combust Inst 2015;35(2):1321–9. hydrogen combustion. Proc Combust Inst 2015;35(2):1321–9 [2] Luo KH. Combustion effects on turbulence in a partially premixed supersonic dif- fusion flame. Combust Flame 1999;119(4):417–35. Acknowledgements [18] Dasgupta D, Sun W, Day M, Lieuwen T. Effect of turbulence–chemistry interactions on chemical pathways for turbulent hydrogen–air premixed flames. Combust Flame 2017;176:191–201. Funding from the UK Engineering and Physical Sciences Research Council under the projects “UK Consortium on Mesoscale Engineering Sciences (UKCOMES)” (Grant No. EP/L00030X/1) and “High Performance Computing Support for United Kingdom Consortium on Turbulent Reacting Flow (UKCTRF)” (Grant No. EP/K024876/1) and funding from the National Natural Science Foundation of China (Grant No. 51576176) are gratefully acknowledged. [19] Zhang F, Liu HF, Yu R, Yao M, Bai XS. Direct numerical simulation of H2/air combustion with composition stratification in a constant volume enclosure relevant to HCCI engines. Int J Hydrogen Energy 2016;41(31):13758–70. [20] Zhang F, Yu R, Bai XS, Yao M, Peng Z. Direct numerical simulation of flame/ spontaneous ignition interaction fueled with hydrogen under SACI engine condi- tions. Int J Hydrogen Energy 2017;42(6):3842–52. [21] Yu R, Bai X-S. Direct numerical simulation of lean hydrogen/air auto-ignition in a constant volume enclosure. Combust Flame 2013;160(9):1706–16. [22] Ranga Dinesh KKJ, Shalaby H, Luo KH, van Oijen JA, Thévenin D. Effects of pressure on cellular flame structure of high hydrogen content lean premixed syngas spherical flames: A DNS study. Int J Hydrogen Energy 2016;41(46):21516–31. [1] Luo KH, Bray K. Combustion-induced pressure effects in supersonic diffusion flames. Symp (Int) Combust 1998;27(2):2165–71. [1] Luo KH, Bray K. Combustion-induced pressure effects in supersonic diffusion flames. Symp (Int) Combust 1998;27(2):2165–71. [2] Luo KH. Combustion effects on turbulence in a partially premixed supersonic dif- fusion flame. Combust Flame 1999;119(4):417–35. References [23] Ranga Dinesh KKJ, Shalaby H, Luo KH, van Oijen JA, Thévenin D. High hydrogen content syngas fuel burning in lean premixed spherical flames at elevated pressures: effects of preferential diffusion. Int J Hydrogen Energy 2016;41(40):18231–49. [24] Ranga Dinesh KKJ, Shalaby H, Luo KH, van Oijen JA, Thévenin D. 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The inverse cascade and nonlinear alpha-effect in simulations of isotropic helical hydromagnetic turbulence. Astrophys J 2001;550(2):824–40. [38] C hé C H lt F Ch C Gök l I Güld ÖL F t l h t i ti f hi h [27] Bédat B, Cheng RK. Effects of buoyancy on premixed flame stabilization. Combust Flame 1996;107:13–26. [38] Cohé C, Halter F, Chauveau C, Gökalp I, Gülder ÖL. Fractal characterisation of high- pressure and hydrogen-enriched CH4–air turbulent premixed flames. Proc Combust Inst 2007;31(1):1345–52. [28] Jiang X, Luo KH. Combustion-induced buoyancy effects of an axisymmetric reactive plume. Proc Combust Inst 2000;28:1989–95. [39] Lachaux T, Halter F, Chauveau C, Gökalp I, Shepherd IG. Flame front analysis of high-pressure turbulent lean premixed methane–air flames. Proc Combust Inst 2005;30(1):819–26. [29] Cheng RK, Bédat B, Kostiuk LW. Effects of buoyancy on lean premixed V-flames Part I: laminar and turbulent flame structures. Combust Flame 1999;116:360–75. [40] Wang H, Hawkes ER, Chen JH, Zhou B, Li Z, Aldén M. Direct numerical simulations of a high Karlovitz number laboratory premixed jet flame–an analysis of flame stretch and flame thickening. J Fluid Mech 2017;815:511–36. [30] Libby PA. Theoretical analysis of the effect of gravity on premixed turbulent flames. Combust Sci Technol 1989;68:15–33. [31] Poinsot T, Veynante D. Theoretical and numerical combustion. Bordeaux: RT Edwards; 2005. References [41] Pan K, Qian J, Law C, Shyy W. The role of hydrodynamic instability in flame-vortex interaction. Proc Combust Inst 2002;29(2):1695–704. [32] Kee RJ, Rupley FM, Meeks E, Miller JA. CHEMKIN-III: A FORTRAN chemical ki- netics package for the analysis of gas-phase chemical and plasma kinetics. Sandia National Laboratories Report SAND96-8216; 1996. [42] Law CK. Combustion physics. Cambridge: Cambridge University Press; 2010. [43] Shi X Chen J Y Numerical analysis and model development for laminar flame [42] Law CK. Combustion physics. 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Influence of socio-demographic and environmental factors on childhood diarrhea in Cambodia
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F1000Research 2020, 9:303 Last updated: 02 MAY 2023 Vong Pisey 1,2, Pannee Banchonhattakit 3 The probability of diarrhea occurring is shown to be increased by maternal unemployment, the sex of the child being male, Open Peer Review Approval Status 1 2 version 5 (revision) 23 Sep 2021 view view version 4 (revision) 04 Aug 2021 view view version 3 (revision) 12 Apr 2021 version 2 (revision) 03 Jul 2020 version 1 28 Apr 2020 view Siyan Yi , National University of Singapore, Singapore, Singapore KHANA Center for Population Health Research, Phnom Penh, Cambodia Touro University California, Vallejo, USA 1. Okechukwu S. Chukwudeh, Touro 2. First published: 28 Apr 2020, 9:303 https://doi.org/10.12688/f1000research.23246.1 Second version: 03 Jul 2020, 9:303 https://doi.org/10.12688/f1000research.23246.2 Third version: 12 Apr 2021, 9:303 https://doi.org/10.12688/f1000research.23246.3 Fourth version: 04 Aug 2021, 9:303 https://doi.org/10.12688/f1000research.23246.4 Latest published: 23 Sep 2021, 9:303 https://doi.org/10.12688/f1000research.23246.5 v1 Ope App ver (rev 23 Se First published: 28 Apr 2020, 9:303 https://doi.org/10.12688/f1000research.23246.1 Second version: 03 Jul 2020, 9:303 https://doi.org/10.12688/f1000research.23246.2 Third version: 12 Apr 2021, 9:303 https://doi.org/10.12688/f1000research.23246.3 Fourth version: 04 Aug 2021, 9:303 https://doi.org/10.12688/f1000research.23246.4 Latest published: 23 Sep 2021, 9:303 https://doi.org/10.12688/f1000research.23246.5 v1 Open Peer Review Approval Status 1 2 version 5 (revision) 23 Sep 2021 view view Open Peer Review Approval Status 1 2 version 5 (revision) 23 Sep 2021 view view version 4 (revision) 04 Aug 2021 view view version 3 (revision) 12 Apr 2021 version 2 (revision) 03 Jul 2020 version 1 28 Apr 2020 view Siyan Yi National University of 1 Vong Pisey 1,2, Pannee Banchonhattakit 3 1Doctor of Public Health Program, Faculty of Public Health, Khon Kaen University, Khon Kaen, 40002, Thailand 2Office of Rural Health Care, Pursat Provincial Department of Rural Development, Ministry of Rural Development, Cambodia 3Faculty of Public Health, Khon Kaen University, Khon Kaen, 40002, Thailand Open Peer Review Approval Status 1 2 version 5 (revision) 23 Sep 2021 view view version 4 (revision) 04 Aug 2021 view view version 3 (revision) 12 Apr 2021 version 2 (revision) 03 Jul 2020 version 1 28 Apr 2020 view Siyan Yi , National University of Singapore, Singapore, Singapore KHANA Center for Population Health Research, Phnom Penh, Cambodia Touro University California, Vallejo, USA 1. Okechukwu S. Chukwudeh, Touro 2. First published: 28 Apr 2020, 9:303 https://doi.org/10.12688/f1000research.23246.1 Second version: 03 Jul 2020, 9:303 https://doi.org/10.12688/f1000research.23246.2 Third version: 12 Apr 2021, 9:303 https://doi.org/10.12688/f1000research.23246.3 Fourth version: 04 Aug 2021, 9:303 https://doi.org/10.12688/f1000research.23246.4 Latest published: 23 Sep 2021, 9:303 https://doi.org/10.12688/f1000research.23246.5 v1 Abstract Background: Diarrhea is still the leading cause of childhood death worldwide, as well as a major cause for concern in developing countries. This study was conducted to investigate the factors related to childhood diarrhea in Cambodia. Methods: A cross-sectional study was conducted using the combination of two datasets from the Cambodia Demographic and Health Survey 2014. A generalized linear mixed model was used to analyze the determinant factors of childhood diarrhea. Results: The surveys included 2,828 children, aged 12 to 35 months. The prevalence of diarrhea was 16.44% (95% CI: 14.72%-18.31%). Factors with statistically significant associations with childhood diarrhea in Cambodia were: maternal  unemployment, compared with being in employment (AOR = 1.43; 95% CI: 1.14-1.78); the child being male (AOR = 1.25; 95%CI: 1.02-1.53); the presence of unimproved toilet facilities (AOR = 1.17; 95%CI: 1.05-1.31) compared with improved toilet facilities; and unhygienic disposal of children’s stools (AOR = 1.32; 95%CI: 1.06-1.64) compared with hygienic disposal of children’s stools when controlling for other covariates. Both maternal age (one year older; AOR = 0.85; 95%CI: 0.78– 0.93) and child age (one month older; AOR = 0.86; 95%CI: 0.78-0.94) had significant negative associations with the occurrence of childhood diarrhea. Conclusion: Childhood diarrhea remains a public health concern in Cambodia. Abstract University California, Vallejo, USA Any reports and responses or comments on the article can be found at the end of the article. lack of provision of improved toilet facilities, and the unhygienic disposal of children’s stools; whereas increasing maternal age and child’s age were associated with a reduced chance of diarrhea occurring. On the basis of these results, we recommend provision of programs focusing on reducing diarrhea through the construction of improved toilet facilities and the promotion of behavior to improve hygiene, specifically targeting younger mothers. Keywords Keywords Socio-demographic, environmental, childhood diarrhea, generalized linear mixed model, Cambodia This article is included in the Global Public Health gateway. This article is included in the Global Public Health gateway. Corresponding author: Pannee Banchonhattakit (panban@kku.ac.th) Author roles: Pisey V: Conceptualization, Data Curation, Investigation, Writing – Original Draft Preparation, Writing – Review & Editing; Banchonhattakit P: Conceptualization, Methodology, Supervision, Validation Competing interests: No competing interests were disclosed. Grant information: The author(s) declared that no grants were involved in supporting this work. Copyright: © 2020 Pisey V and Banchonhattakit P. 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 work is properly cited. How to cite this article: Pisey V and Banchonhattakit P. Influence of socio-demographic and environmental factors on childhood diarrhea in Cambodia [version 1; peer review: 1 approved with reservations] F1000Research 2020, 9:303 https://doi.org/10.12688/f1000research.23246.1 First published: 28 Apr 2020, 9:303 https://doi.org/10.12688/f1000research.23246.1 Abstract Background: Diarrhea is still the leading cause of childhood death worldwide, as well as a major cause for concern in developing countries. This study was conducted to investigate the factors related to childhood diarrhea in Cambodia. Background: Diarrhea is still the leading cause of childhood death worldwide, as well as a major cause for concern in developing countries. This study was conducted to investigate the factors related to childhood diarrhea in Cambodia. Methods: A cross-sectional study was conducted using the combination of two datasets from the Cambodia Demographic and Health Survey 2014. A generalized linear mixed model was used to analyze the determinant factors of childhood diarrhea. Results: The surveys included 2,828 children, aged 12 to 35 months. The prevalence of diarrhea was 16.44% (95% CI: 14.72%-18.31%). Factors with statistically significant associations with childhood diarrhea in Cambodia were: maternal  unemployment, compared with being in employment (AOR = 1.43; 95% CI: 1.14-1.78); the child being male (AOR = 1.25; 95%CI: 1.02-1.53); the presence of unimproved toilet facilities (AOR = 1.17; 95%CI: 1.05-1.31) compared with improved toilet facilities; and unhygienic disposal of children’s stools (AOR = 1.32; 95%CI: 1.06-1.64) compared with hygienic disposal of children’s stools when controlling for other covariates. Both maternal age (one year older; AOR = 0.85; 95%CI: 0.78– 0.93) and child age (one month older; AOR = 0.86; 95%CI: 0.78-0.94) had significant negative associations with the occurrence of childhood diarrhea. Methods: A cross-sectional study was conducted using the combination of two datasets from the Cambodia Demographic and Health Survey 2014. A generalized linear mixed model was used to analyze the determinant factors of childhood diarrhea. Siyan Yi , National University of Singapore, Singapore, Singapore KHANA Center for Population Health Research, Phnom Penh, Cambodia Touro University California, Vallejo, USA 1. Siyan Yi , National University of Singapore, Singapore, Singapore KHANA Center for Population Health Research, Phnom Penh, Cambodia Touro University California, Vallejo, USA 1. Conclusion: Childhood diarrhea remains a public health concern in Cambodia. The probability of diarrhea occurring is shown to be increased by maternal unemployment, the sex of the child being male, Page 1 of 16 F1000Research 2020, 9:303 Last updated: 02 MAY 2023 University California, Vallejo, USA Any reports and responses or comments on the article can be found at the end of the article. Independent variables i d hi h Socio-demographic characteristics take the form of continu- ous variables such as maternal age, child’s age, and number of household members and categorical variables such as mater- nal education (no education/primary/secondary/higher), mater- nal occupation (employed/unemployed), mother’s knowledge of oral rehydration salts (ORS) (good/poor)27, exposure to media (yes/no)28, sex of the child, breastfeeding (ever/never), deworm- ing (yes/no)27, vaccination (ever/never), residence (urban/rural) and wealth index (poorest/poorer/middle/richer/richest)27. CDHS data were organized in 19 province domains, which we regrouped into four regions: Central Plain; Tonle Sap; Coastal and Sea; and Plateau and Mountains29. Environmental characteris- tics were also treated as categorical variables, including drinking water source (improved/unimproved)30, whether or not the same source of drinking water was used during wet and dry seasons (same/different), whether or not water was treated before drink- ing (always/no), type of toilet facility (improved/unimproved)30, hygiene (adequate/inadequate)30, and disposal of children’s stools (sanitary/unsanitary)31. It is of great importance to understand the factors related to the prevalence of diarrhea among children aged 12 to 35 months. There are no existing studies on the association in this age group, and no national studies on childhood diarrhea in Cambodia have yet been published. Dependent variable The prevalence of diarrhea is the dependent variable consid- ered in this study. This is referred to the questionnaire thus: “Has (NAME) had diarrhea in the last 2 weeks?” The dichotomous variable childhood diarrhea can take values “1” representing a response of “yes” or “0” representing “no” and “don’t know” responses. In 2014, Cambodia still had one of the highest prevalence levels of diarrhea among children under the age of five amongst coun- tries in South-East Asia, at 12.8%20. By comparison, Myanmar had a prevalence of 10.4% in 2015–1621, Malaysia 4.4% in 20167, Laos 6.5% in 201722, Philippines 6.1% in 201723, and Indonesia 14.1% in 201724. According to 2016 data from UNICEF, Cambodia had 5,947 total neonatal deaths, of which 20 were due to diarrhea; 5,248 post-neonatal deaths, of which 672 were due to diarrhea (13%); and 692 deaths of children under five due to diarrhea (6%)25. High rates of diarrhea alone account for one fifth of the deaths of children under the age of five in Cambodia, and an estimated 10,000 deaths overall each year26. This demonstrates that diarrhea is the most common cause of death in Cambodian children. According to the Cambodia Demographic and Health Survey (CDHS) 2014, the prevalence of diarrhea among children aged 12 to 35 months was high, which is known to affect for child development and growth20. Data use Two raw CDHS 2014 datasets, comprising household data and children’s data were combined for use in this study. All entries and variables in these datasets were included in this study. Ethical statement Statistical analysis Statistical data analyses were performed using STATA/SE 14.032 as follows. Statistical analysis Statistical data analyses were performed using STATA/SE 14.032 as follows. This research project received approval from the Khon Kean University Ethics Committee in Human Research (HE632097). This study uses existing CDHS data and re-analysis was done under the original consent provided by the participants. Categorical data were analyzed to provide frequency and per- centage. Continuous data were treated as means, standard devia- tions, and ranges for analysis. A weighting variable was used in the form of the woman’s individual sample weighting. Cross- tabulations were run with the appropriate sample weights to pro- vide nationally representative results19. The svyset command was used to test for complex survey sampling methods used in the original surveys, in order to adjust for differences in the prob- abilities of sample selection and to avoid using over-sampled strata within the survey data27. Corresponding author: Pannee Banchonhattakit (panban@kku.ac.th) Corresponding author: Pannee Banchonhattakit (panban@kku.ac.th) Author roles: Pisey V: Conceptualization, Data Curation, Investigation, Writing – Original Draft Preparation, Writing – Review & Editing; Banchonhattakit P: Conceptualization, Methodology, Supervision, Validation How to cite this article: Pisey V and Banchonhattakit P. Influence of socio-demographic and environmental factors on childhood diarrhea in Cambodia [version 1; peer review: 1 approved with reservations] F1000Research 2020, 9:303 https://doi.org/10.12688/f1000research.23246.1 Page 2 of 16 F1000Research 2020, 9:303 Last updated: 02 MAY 2023 F1000Research 2020, 9:303 Last updated: 02 MAY 2023 Introductioni EAs were chosen using probabilities weighted proportional to the size of the EA. In the second stage, 24 and 28 households were picked up from every urban cluster and rural cluster, respectively, through a systematic sampling process with equal probability weighting. 15,825 households, 17,578 women, and 5,190 men were interviewed between the 2nd June and the 12th December 2014; further details can be found in the CDHS 2014 report20. The final sample size comprised 2,828 children aged 12 to 35 months, providing a suitable degree of power (0.9627, 0.9682). Diarrhea is defined as the passage of loose or watery stools, three or more times each day, or more frequent passage than is normal for an individual1. Diarrhea remains a leading cause of child mortality and morbidity in the world, with an estimated 1.7 billion cases of childhood diarrhea and 525,000 deaths of children under five caused by diarrhea each year. Diarrhea is the second leading cause of death in children under the age of five years1,2. 88% of diarrhea cases globally are attributable to poor water, poor sanitation or poor hygiene3. There is not just the one single factor associated with childhood diarrhea but mul- tiple factors, including unimproved drinking water sources4–7, untreated water8–10, unimproved toilet facilities6,8,9,11, unhygienic disposal of children’s stools12–14, lack of hand washing facilities15,16, type and location of residence11,16, the child’s age4,13,16, the child’s sex (male)13, maternal illiteracy12,13,17, the mother’s occupation9,12, maternal age14,18, wealth index4,19, and whether or not the child is breastfed10,15. CDHS 2014 The CDHS 2014 collected data nationally across the country, which is subdivided into 19 province domains. Its sampling frame consisted of 28,455 eligible enumeration areas (EAs), which comprised the 2008 Cambodian General Population Census (GPC). The sample considered any domain with a power alloca- tion, including both urban and rural areas. The stratified sample was selected in two stages. In the first stage, a fixed number of Page 3 of 16 Page 3 of 16 Page 3 of 16 F1000Research 2020, 9:303 Last updated: 02 MAY 2023 Discussioni This is the first study to report factors associated with diarrhea in children aged 12 to 35 months at the national level in Cambodia. Younger maternal age, maternal unemployment, younger child age, being male, lack of unimprovement to toi- let facilities, and unhygienic disposal of children’s stools were found to be associated with childhood diarrhea. Multivariate analysis of factors associated with childhood diarrhea in Cambodia Multivariate analysis of factors associated with childhood diarrhea in Cambodia The prevalence of diarrhea was estimated as a percentage. The numerator was the number of living children aged 12 to 35 months with an occurrence of diarrhea during the two weeks preceding the interview (i.e. an answer “yes” to, “Has (NAME) had diarrhea in the last 2 weeks?”) and the denominator was the number of living children aged 12 to 35 months. The multivariate analysis (Table 3) showed that as maternal age increased by a year, the odds of the child suffering from diarrhea decreased 15% (AOR = 0.85; 95%CI: 0.78– 0.93; p=0.001). The odds of suffering from diarrhea was 43% higher (AOR = 1.43; 95% CI: 1.14-1.78; p=0.002) in children whose mother was unemployed compared to employed. As the child’s age increased by a month, the odds of the child suffering from diarrhea decreased 14% (AOR = 0.86; 95%CI: 0.78-0.94; p=0.001). The odds of suffering from diarrhea was 25% higher (AOR = 1.25; 95%CI: 1.02-1.53; p=0.031) in males compared to females. The odds of suffering from diarrhea was 17% higher (AOR = 1.17; 95%CI: 1.05-1.31; p=0.004) in children living in a house- hold with unimproved toilet facilities compared with those with improved toilet facilities. The odds of suffering from diarrhea was 32% higher (AOR = 1.32; 95%CI: 1.06-1.64; p=0.011) in children whose stools were disposed of unhygienically compared to children whose stools were disposed of hygienically. A bivariate analysis with simple logistic regression was per- formed using the svyset (svy command). A linearity test was con- ducted between the continuous variable and dependent variable. Any independent variables significant at p<0.25 were entered into the initial model33,34. Multicollinearity was performed with the independent variables-variance inflation factor (VIF)35. Finally, a multivariate analysis was performed using a generalized mixed linear model with four regions picked as ‘random effects’ corresponding to the various clusters in the sampling design36. The backward stepwise procedure was applied as the model fit- ting strategy. Statistical significance was considered at a thresh- old of p<0.05 and the adjusted odds ratio (AOR) with 95% confidence intervals (CI) was considered as the magnitude of the effect. Results A total of 2,828 children were included in the study. The major- ity of the children (84.12%) lived in rural areas. Nearly half (44.03%) lived in Central Plain and one third (33.32%) in Tonle Sap. The average maternal age was 28.27±5.89 years old. More than half the mothers (51.08%) attended primary school. Three quarters (75.10%) of the mothers were employed and the aver- age number of household members was five. More than half (51.18%) of the children were male and the mean age was 23.33±6.79 months. Almost all (96.17%) children had been breastfed; 59.60% had received deworming treatment; and 77.97% of them had never been vaccinated. Out of 2,828 households, more than half (54.07%) always had treated water to drink; 57.97% had an unimproved toilet facility; while 68.01% used adequate hygiene; and 70.25% used sanitary disposal of children’s stool (Table 1). Socio-demographic characteristics such as maternal age were sig- nificantly associated with reduced incidence of diarrhea, in line with studies conducted in Brazil and Tanzania14,18, and perhaps due to the mother having more experience in childcare and feed- ing. The association of maternal unemployment with the incidence of diarrhea is consistent with a study in Senegal9. The child’s age had a significant, negative association with incidence of diarrhea, in line with many studies in Ethiopia and Tanzania4,14,16, and potentially due to the development of the immune sys- tem throughout childhood. Males were more likely to suffer from diarrhea than females, which may simply reflect a natural predisposition of males to develop diarrhea more frequently than females37, but is also supported by a previous study conducted in India13. Bivariate analysis of factors associated with childhood diarrhea in Cambodiai Factors with a significant association with childhood diarrhea (p<0.05) were maternal age, maternal occupation, the child’s age, available toilet facilities, and the method of stool disposal (Table 2). Further, the factors of the child’s sex, their vaccina- tion history, the number of household members, wealth index, source of drinking water during dry season, whether or not the same source of drinking water is used during wet and dry sea- sons, and the treatment/non-treatment of drinking water did not reach significance but did meet the pre-determined thresh- old of p<0.25 for inclusion in the initial model. Finally, region (p<0.25) also met the criteria for inclusion in the initial model and was used as a random effect. As such, the multivari- ate analysis was conducted using a generalized mixed linear model with each of the four regions of Cambodia treated as random effects. Environmental characteristics such as the lack of improve- ments to toilet facilities were significantly associated with the incidence of diarrhea, consistent with many studies including a systematic review4,6,8,11. Finally, disposal of children’s stools was significantly associated with the incidence of diarrhea, con- sistent with previous studies in Ethiopia, India, and Tanzania12–14. These findings demonstrate that the quality of sanitation facili- ties strongly influences the prevalence of childhood diarrhea in Cambodia. A limitation of this research study was that it used a cross-sectional design with just one outcome measure (diarrhea prevalence) taken as a snapshot at a given point in time. Bivariate analysis of factors associated with childhood diarrhea in Cambodiai Future Page 4 of 16 Page 4 of 16 Page 4 of 16 F1000Research 2020, 9:303 Last updated: 02 MAY 2023 Variables Frequency Percentage   Never 2205 77.97 Household characteristics Residence   Urban 449 15.88   Rural 2379 84.12 Region   Coastal and Sea 169 5.98   Tonle Sap 942 33.32   Central Plain 1245 44.03   Plateau and Mountains 472 16.67 Number of household members   1–4 969 34.28   >4 1859 65.72   Mean±SD 5.73±2.31   Range 1 to 22 Wealth index   Poorest 672 23.76   Poorer 523 18.49   Middle 550 19.44   Richer 493 17.45   Richest 590 20.86 Environmental characteristics Drinking water during dry season   Improved 1745 61.71   Unimproved 1083 38.29 Drinking water during wet season   Improved 2320 82.02   Unimproved 508 17.98 Same source of drinking water during wet and dry season   Same 1955 69.11   Different 873 30.89 Treating water to drink   Yes, always 1529 54.07   No 1299 45.93 Toilet facility   Improved 1189 42.03   Unimproved 1640 57.97 Hygiene   Adequate 1923 68.01   Inadequate 905 31.99 Disposal of children’s stool   Sanitary 1987 70.25   Unsanitary 841 29.75 SD, standard deviation. Table 1. Socio-demographic and environmental characteristics of households in Cambodia, 2014 (n=2,828). Variables Frequency Percentage Maternal characteristics Age (years)   16–24 397 14.04   25–34 1591 56.26   35–49 840 29.70   Mean±SD 28.27±5.89   Range 16 to 49 Education   No education 366 12.96   Primary 1445 51.08   Secondary 921 32.58   Higher 96 3.38 Occupation   Employed 2124 75.10   Unemployed 704 24.90 Knowledge of oral rehydration salts   Good 2717 96.05   Poor 111 3.95 Exposure to media   Yes 1808 63.92   No 1020 36.08 Children’s characteristics Age (months)   12–23 1460 51.64   24–35 1368 48.36   Mean±SD 23.33±6.79   Range 12 to 35 Sex   Male 1448 51.18   Female 1381 48.82 Breastfeeding status   Ever 2720 96.17   Never 108 3.83 Deworming   Yes 1686 59.60   No 1142 40.40 Vaccination   Ever 623 22.03 Page 5 of 16 F1000Research 2020, 9:303 Last updated: 02 MAY 2023 Table 2. Bivariate analysis of factors associated with childhood diarrhea in Cambodia, 2014 (n=2,828). Table 2. Bivariate analysis of factors associated with childhood diarrhea in Cambodia, 2014 (n=2,828). Bivariate analysis of factors associated with childhood diarrhea in Cambodiai Variables Number Diarrhea % COR 95% CI p-value Drinking water during dry season 0.065   Improved 1745 15.12 1   Unimproved 1083 18.56 1.28 0.98-1.66 Drinking water during wet season 0.676   Improved 2320 16.27 1   Unimproved 508 17.22 1.07 0.78-1.48 Same source of drinking water during wet and dry season 0.161   Same 1955 15.56 1   Different 873 18.40 1.22 0.92-1.62 Treating water to drink 0.139   Yes, always 1529 15.28 1   No 1299 17.81 1.20 0.94-1.53 Toilet facility 0.013   Improved 1189 13.61 1   Unimproved 1640 18.49 1.20 1.04-1.39 Hygiene 0.995   Adequate 1923 16.44 1   Inadequate 905 16.43 0.99 0.74-1.34 Disposal of children’s stool 0.020   Sanitary 1987 14.99 1   Unsanitary 841 19.85 1.40 1.05-1.87 COR, crude odds ratio; CI, confidence interval. Variables Number Diarrhea % COR 95% CI p-value Drinking water during dry season 0.065   Improved 1745 15.12 1   Unimproved 1083 18.56 1.28 0.98-1.66 Drinking water during wet season 0.676   Improved 2320 16.27 1   Unimproved 508 17.22 1.07 0.78-1.48 Same source of drinking water during wet and dry season 0.161   Same 1955 15.56 1   Different 873 18.40 1.22 0.92-1.62 Treating water to drink 0.139   Yes, always 1529 15.28 1   No 1299 17.81 1.20 0.94-1.53 Toilet facility 0.013   Improved 1189 13.61 1   Unimproved 1640 18.49 1.20 1.04-1.39 Hygiene 0.995   Adequate 1923 16.44 1   Inadequate 905 16.43 0.99 0.74-1.34 Disposal of children’s stool 0.020   Sanitary 1987 14.99 1   Unsanitary 841 19.85 1.40 1.05-1.87 COR, crude odds ratio; CI, confidence interval. Table 3. Multivariate analysis of factors associated with childhood diarrhea in Cambodia, 2014 using generalized mixed linear model (n=2,828). Table 3. Multivariate analysis of factors associated with childhood diarrhea in Cambodia, 2014 using generalized mixed linear model (n=2,828). Table 3. Multivariate analysis of factors associated with childhood diarrhea in Cambodia, 2014 using generalized mixed linear model (n=2,828). Table 3. Multivariate analysis of factors associated with childhood diarrhea in Cambodia, 2014 using generalized mixed linear model (n=2,828). Variables Number Diarrhea % AOR 95% CI p-value Maternal age (years) 2828 N/A 0.85 0.78-0.93 0.001 Maternal occupation 0.002    Employed 2124 15.00 1    Unemployed 704 20.78 1.43 1.14-1.78 Child’s age (months) 2828 N/A 0.86 0.78-0.94 0.001 Child’s sex 0.031    Female 1381 14.86 1    Male 1448 17.94 1.25 1.02-1.53 Toilet facility 0.004    Improved 1189 13.61 1    Unimproved 1640 18.49 1.17 1.05-1.31 Disposal of children’s stool 0.011    Sanitary 1987 14.99 1    Unsanitary 841 19.85 1.32 1.06-1.64 AOR, adjusted odds ratio; CI, confidence interval. AOR, adjusted odds ratio; CI, confidence interval. Bivariate analysis of factors associated with childhood diarrhea in Cambodiai Variables Number Diarrhea % COR 95% CI p-value Overall 2828 16.44 14.72- 18.31 Maternal age (years) 2828 N/A 0.82 0.73-0.92 <0.001 Maternal education 0.681   Literate 2462 16.29 1   Illiterate 366 17.46 1.09 0.73-1.62 Maternal occupation 0.007   Employed 2124 15.00 1   Unemployed 704 20.78 1.49 1.11-1.98 Mother’s knowledge of oral rehydration salts 0.481   Good 2717 16.61 1   Poor 111 12.21 0.69 0.25-1.90 Mother’s exposure to media 0.502   Yes 1808 15.99 1   No 1020 17.23 1.09 0.84-1.42 Child’s age (months) 2828 N/A 0.83 0.75-0.92 <0.001 Child’s sex 0.075   Female 1381 14.86 1   Male 1448 17.94 1.25 0.97-1.61 Breastfeeding status 0.268   Ever 2720 16.64 1   Never 108 11.42 0.64 0.29-1.40 Deworming 0.504   Yes 1686 16.91 1   No 1142 15.75 0.91 0.71-1.17 Vaccination 0.232   Ever 623 14.44 1   Never 2205 17.00 1.21 0.88-1.66 Residence 0.561   Urban 449 15.39 1   Rural 2379 16.64 1.10 0.80-1.50 Region 0.203   Coastal and Sea 169 12.36 1   Tonle Sap 942 15.55 1.31 0.82-2.07   Central Plain 1245 16.92 1.44 0.92-2.25   Plateau and Mountains 472 18.40 1.60 1.02-2.51 Number of household members 0.095   >4 1859 15.38 1   1–4 969 18.47 1.25 0.96- 1.62 Wealth index 0.128   Richest 590 14.44 1   Richer 493 17.40 1.25 0.82-1.90   Middle 550 14.65 1.02 0.67-1.55   Poorer 523 14.50 1.00 0.67-1.50   Poorest 672 20.46 1.52 1.03-2.26 Page 6 of 16 F1000Research 2020, 9:303 Last updated: 02 MAY 2023 Table 3. Multivariate analysis of factors associated with childhood diarrhea in Cambodia, 2014 using generalized mixed linear model (n=2,828). Variables Number Diarrhea % AOR 95% CI p-value Maternal age (years) 2828 N/A 0.85 0.78-0.93 0.001 Maternal occupation 0.002    Employed 2124 15.00 1    Unemployed 704 20.78 1.43 1.14-1.78 Child’s age (months) 2828 N/A 0.86 0.78-0.94 0.001 Child’s sex 0.031    Female 1381 14.86 1    Male 1448 17.94 1.25 1.02-1.53 Toilet facility 0.004    Improved 1189 13.61 1    Unimproved 1640 18.49 1.17 1.05-1.31 Disposal of children’s stool 0.011    Sanitary 1987 14.99 1    Unsanitary 841 19.85 1.32 1.06-1.64 AOR, adjusted odds ratio; CI, confidence interval. Acknowledgements Th h ld Based on this finding, the authors provide the following recommendations. g The authors would like to express sincere thanks and appreciation to: National level: The WASH program managed by the Minis- try of Rural Development in Cambodia should focus on deliv- ering guidance to younger mothers with younger children and also to unemployed mothers. Guidance should include sani- tary disposal of children’s stools in addition to water treatment, sanitation and health. Health policy decision makers should design appropriate intervention programs focusing on reducing Dr. Kavin Thinkhamrop, Health and Epidemiology Geoinformat- ics Research (HEGER), Faculty of Public Health, Khon Kaen University; Dr. Wilaiphorn Thinkhamrop, Data Management and Statistical Analysis Center (DAMASAC), Faculty of Pub- lic Health, Khon Kaen University for their statistical support; and Rebecca S Dewey, University of Nottingham for language editing. Data availability Data availability Our study used raw children’s and household data from DHS, Cambodia 2014. Data are free to access for research purposes and can be obtained through the DHS Program after register- ing and obtaining an approval letter from the Inner City Fund (ICF) (https://dhsprogram.com/data/Access-Instructions.cfm). Bivariate analysis of factors associated with childhood diarrhea in Cambodiai Page 7 of 16 F1000Research 2020, 9:303 Last updated: 02 MAY 2023 F1000Research 2020, 9:303 Last updated: 02 MAY 2023 diarrhea through the construction of improved toilet facilities in addition to promoting hygienic behaviors. diarrhea through the construction of improved toilet facilities in addition to promoting hygienic behaviors. longitudinal studies may improve on this. The CDHS 2014 was not fully comprehensive in that it did not cover the WASH fac- tors of hand washing before preparing meals and after defecat- ing. The inclusion of these questions in the survey would give a more comprehensive analysis of hygiene practices in the population. Further, the CDHS 2014 captured data by house- hold, rather than by individual person, which may introduce a confound in that it has a tendency to under-estimate the quality of both drinking water source and sanitation facility available. Community level: Younger mothers should be encouraged to enroll in health education for the prevention of childhood diarrhea. Improvements should be made to the general commu- nity sanitation facilities as well as the construction of additional toilet facilities. Future study: Longitudinal studies on childhood diarrhea need to be conducted in order to measure the impact of interventions. References 1. World Health Organization: Diarrhoeal disease. 2017. Reference Source 2. 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Acharya D, Singh JK, Adhikari M, et al.: Association of water handling and child feeding practice with childhood diarrhoea in rural community of Southern Nepal. J Infect Public Health. 2018; 11(1): 69–74. PubMed Abstract | Publisher Full Text 11. Alebel A, Tesema C, Temesgen B, et al.: Prevalence and determinants of diarrhea among under-five children in Ethiopia: A systematic review and meta- analysis. PLoS One. 2018; 13(6): e0199684. References PubMed Abstract | Publisher Full Text | Free Full Text 12. Sinmegn Mihrete T, Asres Alemie G, Shimeka Teferra A: Determinants of childhood diarrhea among underfive children in Benishangul Gumuz Regional State, North West Ethiopia. BMC Pediatr. 2014; 14(1): 102. PubMed Abstract | Publisher Full Text | Free Full Text 13. Bawankule R, Singh A, Kumar K, et al.: Disposal of children’s stools and its association with childhood diarrhea in India. BMC Public Health. 2017; 17(1): 12. PubMed Abstract | Publisher Full Text | Free Full Text 14. Edwin P, Azage M: Geographical Variations and Factors Associated with Childhood Diarrhea in Tanzania: A National Population Based Survey 2015-16. Ethiop J Health Sci. 2019; 29(4): 513–24. PubMed Abstract | Publisher Full Text | Free Full Text 15. Dagnew AB, Tewabe T, Miskir Y, et al.: Prevalence of diarrhea and associated f d fi hild i B hi D i N h E hi i 2016 2. Liu L, Johnson HL, Cousens S, et al.: Global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since 2000. Lancet. 2012; 379(9832): 2151–61. PubMed Abstract | Publisher Full Text 2. Liu L, Johnson HL, Cousens S, et al.: Global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since 2000. Lancet. 2012; 379(9832): 2151–61. PubMed Abstract | Publisher Full Text 3. Prüss-Üstün A, Bos R, Gore F, et al.: Safer water, better health: costs, benefits and sustainability of interventions to protect and promote health. World Health Organization, Geneva, 2008. 2008. Reference Source 5. Kapwata T, Mathee A, Le Roux WJ, et al.: Diarrhoeal Disease in Relation to Possible Household Risk Factors in South African Villages. Int J Environ Res Public Health. 2018; 15(8): E1665. PubMed Abstract | Publisher Full Text | Free Full Text 13. Bawankule R, Singh A, Kumar K, et al.: Disposal of children’s stools and its association with childhood diarrhea in India. BMC Public Health. 2017; 17(1): 12. PubMed Abstract | Publisher Full Text | Free Full Text 6. Yaya S, Hudani A, Udenigwe O, et al.: Improving Water, Sanitation and Hygiene Practices, and Housing Quality to Prevent Diarrhea among Under-Five Children in Nigeria. Trop Med Infect Dis. 2018; 3(2): E41. PubMed Abstract | Publisher Full Text | Free Full Text 7. References Aziz FAA, Ahmad NA, Razak MAA, et al.: Prevalence of and factors associated with diarrhoeal diseases among children under five in Malaysia: a cross- sectional study 2016. BMC Public Health. 2018; 18(1): 1–8. PubMed Abstract | Publisher Full Text | Free Full Text 14. Edwin P, Azage M: Geographical Variations and Factors Associated with Childhood Diarrhea in Tanzania: A National Population Based Survey 2015-16. Ethiop J Health Sci. 2019; 29(4): 513–24. PubMed Abstract | Publisher Full Text | Free Full Text Conclusion Diarrhea still remains a public health concern among children in Cambodia. The probability of developing diarrhea is strongly associated with maternal unemployment, being male, not hav- ing access to improved toilet facilities, or practicing hygienic disposal of children’s stools. Conversely, increasing maternal and child age is associated with a reduction in the probability of developing diarrhea. Recommendationsi Acknowledgements The authors would like to express sincere thanks and appreciation to: y ; ( ) PubMed Abstract | Publisher Full Text | Free Full Text 18. 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This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1 Saw Swee Hock School of Public Health, National University of Singapore, Singapore, Singapore 2 KHANA Center for Population Health Research, Phnom Penh, Cambodia 3 Center for Global Health Research, Touro University California, Vallejo, CA, USA 1 Saw Swee Hock School of Public Health, National University of Singapore, Singapor 1 Saw Swee Hock School of Public Health, National University of Singapore, Singapore, Singapore 2 KHANA Center for Population Health Research, Phnom Penh, Cambodia 3 C t f Gl b l H lth R h T U i it C lif i V ll j CA USA 2 KHANA Center for Population Health Research, Phnom Penh, Cambodia 2 KHANA Center for Population Health Research, Phnom Penh, Cambodia 3 Center for Global Health Research, Touro University California, Vallejo, CA, USA 3 Center for Global Health Research, Touro University California, Vallejo, CA, US General comments: This study used data from Cambodia Demographic and Health Surveys to identify risk factors associated with diarrhea in children aged 12 to 35 months. Overall, the study findings are interesting and may contribute to the literature in this area given the scarcity of data in low- and middle-income countries. The analyses appeared appropriate. The quality of the writing is acceptable, although more careful proofreading is required before the paper can be published. Here are my specific comments: Open Peer Review Current Peer Review Status: Abstract: Methods: The first sentence "A cross-sectional study was conducted using the combination of two datasets from the Cambodia Demographic and Health Survey 2014" is hard to understand. The authors may want to make it clear that this study used secondary data from CDHS. What are the two data sets? 1. What was the time frame for the prevalence of diarrhea - past month or lifetime? 2. F1000Research 2020, 9:303 Last updated: 02 MAY 2023 Methods: What does this mean: '...,which comprised the 2008 Cambodian General Population Census (GPC)?' 2. What does this mean: '...,which comprised the 2008 Cambodian General Population Census (GPC)?' 2. 'The sample considered any domain...' is not understandable. 3. Although the CDHS 2014 was referred to, some variables require a clear definition; e.g., improved/unimproved water sources, toilet facilities, adequate/inadequate hygiene, sanitary/unsanitary disposal of children's stools, etc. 4. Data analyses: - It is not accurate to state this "Continuous data were treated as means, standard deviations, and ranges for analysis." Perhaps something like 'For continuous variables, mean and standard deviations were calculated..." - I am not sure what authors wanted to tell by this "A weighting variable was used in the form of the woman’s individual sample weighting." - Any independent variables significant at p<0.25 in bivariate analyses were entered into the initial model. -Multicolinearity assessment was performed... 5. -Multicolinearity assessment was performed... F1000Research 2020, 9:303 Last updated: 02 MAY 2023 It would be helpful for readers if authors could define some terminology; e.g., neonatal deaths, post-neonatal deaths, under-five deaths, etc. 3. It would be helpful for readers if authors could define some terminology; e.g., neonatal deaths, post-neonatal deaths, under-five deaths, etc. 3. Paragraph 2: - "...and 692 deaths of children under five due to diarrhea (6%)." What is the denominator of the 6%? In the following sentence, the authors stated that 'diarrhea alone account for one fifth of the deaths of children under the age of five in Cambodia." Please clarify these. 4. Paragraph 2: - "...and 692 deaths of children under five due to diarrhea (6%)." What is the denominator of the 6%? In the following sentence, the authors stated that 'diarrhea alone account for one fifth of the deaths of children under the age of five in Cambodia." Please clarify these. - "High rates of diarrhea alone account for...' Diarrhea alone? - It is confusing that this study used data from CDHS 2014, but also cited the prevalence of diarrhea in the same population and from the same data, while claiming that no national studies on childhood diarrhea in Cambodia have yet been published. - The rationale of the study needs improvement. 4. High rates of diarrhea alone account for... Diarrhea alone? - It is confusing that this study used data from CDHS 2014, but also cited the prevalence of diarrhea in the same population and from the same data, while claiming that no national studies on childhood diarrhea in Cambodia have yet been published. - The rationale of the study needs improvement. Introduction: Introduction: Please provide a reference for the statement in the second sentence, paragraph 1. 1. Avoid starting sentences with numbers. 2. Avoid starting sentences with numbers. 2. Page 10 of 16 Results: ...and one third (33.32%) in Tonle Sap region? 1. It should be mean (SD xx). 2. It should be mean (SD xx). 2. "More than half the mothers (51.08%) attended primary school." Did this include mothers who had no education? 3. Any details to define the breastfeeding - duration, exclusivity...? 4. Please check this data: "...and 77.97% of them had never been vaccinated." This could be very wrong as the immunization coverage in Cambodia has been globally recognized as very high. 5. Page 11 of 16 F1000Research 2020, 9:303 Last updated: 02 MAY 2023 Discussion: Discussion: This section can be improved by extending more in-depth literature in this area and link to the policy implication of the findings. 1. Further limitations of the study should also be included (e.g., self-reporting measures, recall bias...). 2. Conclusions and recommendations can be combined. 3. Conclusions and recommendations can be combined. 3. Recommendations can be summarized. 4. Recommendations can be summarized. 4. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Epidemiology, community-based intervention and evaluation, infectious diseases I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Author Response 29 Jun 2020 PISEY VONG Review1   Title: The term 'influence' may not be appropriate for this study as it can only tell the associations   Page 12 of 16 Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Epidemiology, community-based intervention and evaluation, infectious diseases I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Author Response 29 Jun 2020 PISEY VONG Review1   Title: The term 'influence' may not be appropriate for this study as it can only tell the associations   Page 12 of 16 Is the work clearly and accurately presented and does it cite the current literature? Partly Are sufficient details of methods and analysis provided to allow replication by Partly If applicable, is the statistical analysis and its interpretation appropriate? Yes Abstract: 1. Methods: The first sentence "A cross-sectional study was conducted using the combination of two datasets from the Cambodia Demographic and Health Survey 2014" is hard to understand. The authors may want to make it clear that this study used secondary data from CDHS. What are the two data sets? A: Taken care. "A cross-sectional study of the secondary data from the Cambodia Demographic and Health Survey (CDHS) 2014 was conducted using the combination of household data and children’s data. See Abstract, 1st sentence, para 2 on page 1 2. What was the time frame for the prevalence of diarrhea - past month or lifetime? A: Taken care. The authors added the text “in the last 2 weeks”. See Abstract, 2nd sentence, para 3 on page 1 1. Methods: The first sentence "A cross-sectional study was conducted using the combination of two datasets from the Cambodia Demographic and Health Survey 2014" is hard to understand. The authors may want to make it clear that this study used secondary data from CDHS. What are the two data sets? data from CDHS. What are the two data sets? A: Taken care. "A cross-sectional study of the secondary data from the Cambodia Demographic and Health Survey (CDHS) 2014 was conducted using the combination of household data and children’s data. See Abstract, 1st sentence, para 2 on page 1 2. What was the time frame for the prevalence of diarrhea - past month or lifetime? A: Taken care. The authors added the text “in the last 2 weeks”. See Abstract, 2nd sentence, para 3 on page 1 Introduction: 1. Please provide a reference for the statement in the second sentence, paragraph A: Taken care. “1,2”. See 2nd sentence, para 1 on page 3. 1. Please provide a reference for the statement in the second sentence, paragraph p , p g p A: Taken care. “1,2”. See 2nd sentence, para 1 on page 3. 2. Avoid starting sentences with numbers. 2. Avoid starting sentences with numbers. A: Taken care. Globally, 88% of diarrhea cases are attributable…… See 4th sentence, para 1 on page 3. 3. It would be helpful for readers if authors could define some terminology; e.g., neonatal deaths, post-neonatal deaths, under-five deaths, etc. A: We are sorry, we do not agree with the peer reviewer on this. Authors have retained our original because we think it is not much helpful to add terminology of neonatal deaths, post-neonatal deaths, under-five deaths. A: We are sorry, we do not agree with the peer reviewer on this. Authors have retained our original because we think it is not much helpful to add terminology of neonatal deaths, post-neonatal deaths, under-five deaths. 4. Paragraph 2: Are the conclusions drawn adequately supported by the results? Yes Author Response 29 Jun 2020 Page 12 of 16 F1000Research 2020, 9:303 Last updated: 02 MAY 2023 bles, not causal relationships. A: Agree with the reviewer. As suggested, we have revised the term to “The association”. See title, para 1 on page 1 Methods: 1.What 'province domains' mean? 1.What 'province domains' mean? A. Province domains means the 19 sampling domains of provinces. There are 24 provinces in Cambodia in CDHS 2014, of which fourteen individual provinces (Banteay Meanchey, Kampong Cham, Kampong Chhnang, Kampong Speu, Kampong Thom, Kandal, Kratie, Phnom Penh, Prey Veng, Pursat, Siem Reap, Svay Rieng, Takeo, and Otdar Meanchey); and five groups of provinces (Battambang and Pailin, Kampot and Kep, Preah Sihanouk and Koh Kong, Preah Vihear and Stung Treng, and Mondul Kiri and Ratanak Kiri) 2. What does this mean: '..., which comprised the 2008 Cambodian General Population Census (GPC)?' A. It means “which used the 2008 Cambodian General Population Census”  That has been mentioned in CDHS 2014 as “The sampling frame used for the 2014 CDHS was derived from the list of all enumeration areas (EAs) created for the 2008 Cambodia General Population Census (GPC), provided by NIS 3. The sample considered any domain...' is not understandable. 3. The sample considered any domain...' is not understandable. A: Taken care. We have revised the text to “The sample was allocated into urban and rural in each domain with a power allocation preventing oversample urban, and can represent Cambodia is mainly rural. See 3rd sentence, para 3 on page 3. p p g p Cambodia is mainly rural. See 3rd sentence, para 3 on page 3. Cambodia is mainly rural. See 3rd sentence, para 3 on page 3. y , p p g 4. Although the CDHS 2014 was referred to, some variables require a clear definition; e.g., improved/unimproved water sources, toilet facilities, adequate/inadequate hygiene, sanitary/unsanitary disposal of children's stools, etc. 4. Although the CDHS 2014 was referred to, some variables require a clear definition; e.g., improved/unimproved water sources, toilet facilities, adequate/inadequate hygiene, sanitary/unsanitary disposal of children's stools, etc. A: Agree with the reviewer. As suggested, we have added the text “World Health Organization (WHO) guidelines on water, sanitation and hygiene (WASH) were used to classify WASH as either improved or unimproved according to the WHO/UNICEF Joint Monitoring Programme (Table1 and Table 2). See 3rd sentence, para 2 on page 4; and we also added the Table 1 and Table 2. See on page 11 and 12. 5. 4. Paragraph 2: - "...and 692 deaths of children under five due to diarrhea (6%)." What is the denominator of the 6%? In the following sentence, the authors stated that 'diarrhea alone account for one fifth of the deaths of children under the age of five in Cambodia." Please clarify these. - "...and 692 deaths of children under five due to diarrhea (6%)." What is the denominator of the 6%? In the following sentence, the authors stated that 'diarrhea alone account for one fifth of the deaths of children under the age of five in Cambodia." Please clarify these. - "High rates of diarrhea alone account for...’Diarrhea alone? - "High rates of diarrhea alone account for...’Diarrhea alone? - It is confusing that this study used data from CDHS 2014, but also cited the prevalence of diarrhea in the same population and from the same data, while claiming that no national studies on childhood diarrhea in Cambodia have yet been published. - The rationale of the study needs improvement. A: The denominator of the 6% is the “number of under five children”, however in the data from UNICEF, they do not put it. They just put only “under five deaths due to diarrhea: 692”; and “% underfive deaths due to diarrhoea: (6%)”. According to calculation by the authors, denominator of (6%) is about 11,533. A: Please, see the reference on number 26 which mentioned as “High incidences of diarrhoeal diseases alone account for one fifth of the deaths of children age five and under in Cambodia, and an estimated 10,000 overall deaths annually, largely owing to lack of sanitation and poor hygiene practices”. A: Agree with the reviewer. As suggested, we have revised the text to “There are no existing studies on the association in this age group, and no national studies on the associated factors with childhood diarrhea in Cambodia have yet been published”. See 2nd sentence, Page 13 of 16 F1000Research 2020, 9:303 Last updated: 02 MAY 2023 Methods: Data analyses: h d d d d - It is not accurate to state this "Continuous data were treated as means, standard deviations, and ranges for analysis." Perhaps something like 'For continuous variabl - It is not accurate to state this "Continuous data were treated as means, standard deviations, and ranges for analysis." Perhaps something like 'For continuous variables, mean and standard deviations were calculated..." mean and standard deviations were calculated..." - I am not sure what authors wanted to tell by this "A weighting variable was used in the form of the woman’s individual sample weighting." - Any independent variables significant at p<0.25 in bivariate analyses were entered into the initial model. - Any independent variables significant at p<0.25 in bivariate analyses were entered into the initial model. -Multicolinearity assessment was performed... -Multicolinearity assessment was performed... A: Agree with the reviewer. As suggested, we have revised the text “Categorical variable were analyzed to provide frequency and percentage. Continuous variable were calculated as means, standard deviations, and ranges”. See 1st sentence, para 3 on page 4. A: Because it is survey data, by providing national representative, we used Woman’s individual weighting because child data was accessed by asking for their mother. A: Yes, please see in the “Result” on 2nd sentence, para 2 on page 6. A: Taken care “Multicolinearity assessment was performed...” Results: Page 14 of 16 F1000Research 2020, 9:303 Last updated: 02 MAY 2023 ...and one third (33.32%) in Tonle Sap region? A. Taken care. We have revised by adding “lived”. See on 2nd sentence, para 1 page 5 2. It should be mean (SD xx). A. Taken care. 3."More than half the mothers (51.08%) attended primary school." Did this include mothers who had no education? A: No, It did not include mothers who had no education 4. Any details to define the breastfeeding - duration, exclusivity...? A: No, in the data showing only “ever breastfed (not currently breastfeed, never breastfed, still breastfeeding”; so we group as “ever and never”. 5. Please check this data: "...and 77.97% of them had never been vaccinated." This could be very wrong as the immunization coverage in Cambodia has been globally recognized as very high. A: Agree with the reviewer. As in the data of 2828 showed that 2.15% was no, 22.03% was yes, 0.27% was don’t know, and 75.55 was missing. Methods: The authors request to delete this variable since it is too much missing, moreover according to literature review this variable is not related with childhood diarrhea, only rotavirus vaccination is associated. Discussion: 1. This section can be improved by extending more in-depth literature in this area and link to the policy implication of the findings. A: Agree with the reviewer. However, we do not extending more in-depth literature. We have retained our original. 2. Further limitations of the study should also be included (e.g., self-reporting measures, recall bias...). A. Taken care. See 4th sentence, para 3 on page 7. 3. Conclusions and recommendations can be combined. A: Authors have retained our original because it will be a good idea to separate the limitation, recommendations and conclusion. Moreover, conclusion is an important part of the paper. 4. Recommendations can be summarized. A: Taken care. As suggested, the authors have summarized the text in Recommendations part. See page 8. Competing Interests: No competing interests ...and one third (33.32%) in Tonle Sap region? A. Taken care. A. Taken care. 3."More than half the mothers (51.08%) attended primary school." Did this include mothers who had no education? A: No, It did not include mothers who had no education A: No, It did not include mothers who had no education 4. Any details to define the breastfeeding - duration, exclusivity...? A: Agree with the reviewer. As in the data of 2828 showed that 2.15% was no, 22.03% was yes, 0.27% was don’t know, and 75.55 was missing. The authors request to delete this variable since it is too much missing, moreover according to literature review this variable is not related with childhood diarrhea, only rotavirus vaccination is associated. Discussion: 2. Further limitations of the study should also be included (e.g., self-reporting measures, recall bias...). A. Taken care. See 4th sentence, para 3 on page 7. 3. Conclusions and recommendations can be combined. 3. Conclusions and recommendations can be combined. A: Authors have retained our original because it will be a good idea to separate the limitation, recommendations and conclusion. Moreover, conclusion is an important part of the paper. 4. Recommendations can be summarized. A: Taken care. As suggested, the authors have summarized the text in Recommendations part. See page 8. Methods: Competing Interests: No competing interests Page 15 of 16 F1000Research 2020, 9:303 Last updated: 02 MAY 2023 The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com The benefits of publishing with F1000Research: The benefits of publishing with F1000Research: Page 16 of 16
https://openalex.org/W4361883104
https://figshare.com/articles/journal_contribution/Supplementary_Figure_6_from_Dual_CDK4_CDK6_Inhibition_Induces_Cell-Cycle_Arrest_and_Senescence_in_Neuroblastoma/22451160/1/files/39902244.pdf
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Supplementary Figure 2 from Dual CDK4/CDK6 Inhibition Induces Cell-Cycle Arrest and Senescence in Neuroblastoma
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A B Cell Line Slope p-value BE2C Vehicle 0.3352 <0.0001 LEE011 0.0736 B A B Cell Line Slope p-value BE2C Vehicle 0.3352 <0.0001 LEE011 0.0736 B A A A A B Cell Line Slope p-value BE2C Vehicle 0.3352 <0.0001 LEE011 0.0736 1643 Vehicle 0.1159 <0.0001 LEE011 0 0540 B LEE011 0.0540 EBC1 Vehicle 0.2921 0.51 LEE011 0.2495 Figure S6 Statistical analysis of in vivo growth suppression (A) Growth rates of BE2C Figure S6. Statistical analysis of in vivo growth suppression. (A) Growth rates of BE2C, 1643, and EBC1 xenografts treated with 200 mg/kg LEE011 or vehicle, as determined by linear mixed effects analysis. (B) Summary table of statistics. B Cell Line Slope p-value BE2C Vehicle 0.3352 <0.0001 LEE011 0.0736 1643 Vehicle 0.1159 <0.0001 LEE011 0 0540 B LEE011 0.0540 EBC1 Vehicle 0.2921 0.51 LEE011 0.2495 B Figure S6 Statistical analysis of in vivo growth suppression (A) Growth rates of BE2C Figure S6. Statistical analysis of in vivo growth suppression. (A) Growth rates of BE2C, 1643, and EBC1 xenografts treated with 200 mg/kg LEE011 or vehicle, as determined by linear mixed effects analysis. (B) Summary table of statistics. Figure S6 Statistical analysis of in vivo growth suppression (A) Growth rates of BE2C Figure S6. Statistical analysis of in vivo growth suppression. (A) Growth rates of BE2C, 1643, and EBC1 xenografts treated with 200 mg/kg LEE011 or vehicle, as determined by linear mixed effects analysis. (B) Summary table of statistics.
https://openalex.org/W4381110685
https://journal.unnes.ac.id/sju/index.php/jils/article/download/61019/24435
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Registration of Copyright as Guarantee of Batik Motif Legal Protection (Comparation Study of Indonesia, Malaysia and Thailand)
JILS (Journal of Indonesian Legal Studies)
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Copyright © 2023 by Author(s). This work is licensed under a Creative Commons Attribution- ShareAlike 4.0 International License. All writings published in this journal are personal views of the authors and do not represent the views of this journal and the author's affiliated institutions. Journal of Indonesian Legal Studies Volume 8 Issue 1 (2023), pp. 1-44 DOI: https://doi.org/10.15294/jils.v8i1.61019 Available online since May 31, 2023 Submitted: 06/10/2022 Revised: 18/04/2023; 28/05/2023 Accepted: 30/05/203 Type: Research Article Registration of Copyright as Guarantee of Batik Motif Legal Protection (Comparation Study of Indonesia, Malaysia and Thailand) Agustina Balik1 , Yosia Hetharie2 , Novyta Uktolseja3 , Putri Anggia4 , Revivo Tulaseket5 1,2,3 Faculty of Law, Universitas Pattimura, Ambon, Indonesia 4 Faculty of Law, Universitat Autonoma de Barcelona, Barcelona, Spain 5 Faculty of Law, University of New South Wales, Sydney, Australia josephushetharie@gmail.com Agustina Balik1 , Yosia Hetharie2 , Novyta Uktolseja3 , Putri Anggia4 , Revivo Tulaseket5 josephushetharie@gmail.com josephushetharie@gmail.com 1 Wulandari, Ari. Batik Nusantara: Makna filosofis, Cara Pembuatan, dan Industri Batik. (Yogyakarta: Penerbit Andi, 2022). See also Trixie, Alicia Amaris. "Filosofi Motif Batik Sebagai Identitas Bangsa Indonesia." Folio 1, No. 1 (2020): 1-9; Syed Shaharuddin, Sharifah Imihezri, et al. "A review on the Malaysian and Indonesian batik production, challenges, and innovations in the 21st century." SAGE Open 11, No. 3 (2021): 21582440211040128. It is also further explained that Batik art is indeed a traditional Indonesian art form that involves drawing on cloth for clothing and other decorative purposes. It has a rich history and cultural significance in Indonesia. The word "batik" is derived from the Javanese word "amba," meaning "to write," and the Indonesian word "titik," meaning "dot." The origins of batik in Indonesia can be traced back thousands of years. It is believed to have been practiced since the 6th century in Java, and it later JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 2 copyright is very important. The Government of Indonesia, should also play an important role in terms of contributing through mentoring and empowering businesses of this kind which of course do not only provide economic and moral benefits to creators but also to the region and the people of Kei. When compared to neighboring countries such as Malaysia and Thailand which also have works of art in the form of batik, Indonesia has a variety of batik motifs with different styles between regions. Therefore, good legal protection is needed so that it is not claimed by other parties or even other countries. Keywords: Legal Protection, Copyright, Batik Motif Keywords: Legal Protection, Copyright, Batik Motif ABSTRACT This study aims to identify and analyze the legal protection of Kei batik motifs through copyright registration and the role of local governments in providing legal protection for these Kei batik motifs. This research is a sociolegal research, which is a combination research method between doctrinal legal research methods and empirical legal research methods. Based on the results of the research, Camelia Batik Kei's business raised batik motifs based on the philosophical customs and culture of the people of the Kei Islands. In the aspect of intellectual property rights, Batik Kei has not been touched at all either through the awareness of the business actors themselves or the local government. Even though the Kei Batik motif with the traditional and cultural characteristics of the Kei people is very promising from the aspect of business development, that's why legal protection through registration of intellectual property rights in the field of INTRODUCTION Batik art is the art of drawing on cloth for clothing which became one of the family cultures of the ancient Indonesian kings. Over time, this batik art was imitated by the people closest to him and subsequently expanded into the work of women in their households to fill their spare time. The white cloth used at that time was self-woven. While the coloring materials used consist of native Indonesian plants which are made by themselves, including: noni tree, tall, soga, indigo, and the soda material is made from soda ash, and the salt is made from mud.1 spread to other parts of the Indonesian archipelago. Batik was initially reserved for royalty and the nobility, and it became one of the family cultures of the ancient Indonesian kings. Batik designs often incorporate motifs inspired by nature, mythology, and cultural symbols. Different regions in Indonesia have their own distinct styles and patterns, reflecting the local traditions and influences. Some well-known styles include Javanese batik, Balinese batik, and Sumatran batik, each with its own unique characteristics. See also Mamp, Michael. "Ethel Wallace: A Forgotten History of Batik and Fashion." The Journal of Modern Craft 14, No. 3 (2021): 253-273; Selamet, Juhri. "Indonesian batik translation: A case study." The International Journal of Visual Design 12, No. 3 (2018): 11-17. JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 3 3 3 The tradition of batik was originally a hereditary tradition, so sometimes a motif can be recognized as coming from a certain family batik. Some batik motifs can show a person's status and have a symbolic meaning and full of spiritual value. Batik from the cultural aspect is a high-level art. Batik is not just a cloth written using wax (liquid wax). The patterns (motifs) in batik have a philosophy that is very close to the culture of every society. Batik is the pride of the Indonesian people, an identity that has been inherited for hundreds of years. The art of batik can be done by several people who join an association but can also be done by individuals. This individual batik business emerged because of the birth of various Micro, Small and Medium Enterprises (MSMEs) which are growing at this time. One area where there are MSMEs actors who produce batik that has regional philosophical values is in Tual City, Maluku Province. The business that was founded under the name Camellia Batik Kei since 2017 has around 10-15 motifs. These motifs have philosophical values that are regionally meaningful, especially the values of life that exist in the Maluku people, especially the Kei people. These motifs include the embal motif, the lela motif, the complete kei motif, the gong motif, the small lela motif, the lat motif, the combination embal lat motif, the embal leaf motif, the beach motif and the Tanimbar Kei carving motif. spread to other parts of the Indonesian archipelago. Batik was initially reserved for royalty and the nobility, and it became one of the family cultures of the ancient Indonesian kings. Batik designs often incorporate motifs inspired by nature, mythology, and cultural symbols. Different regions in Indonesia have their own distinct styles and patterns, reflecting the local traditions and influences. Some well-known styles include Javanese batik, Balinese batik, and Sumatran batik, each with its own unique characteristics. See also Mamp, Michael. "Ethel Wallace: A Forgotten History of Batik and Fashion." The Journal of Modern Craft 14, No. 3 (2021): 253-273; Selamet, Juhri. "Indonesian batik translation: A case study." The International Journal of Visual Design 12, No. 3 (2018): 11-17. spread to other parts of the Indonesian archipelago. Batik was initially reserved for royalty and the nobility, and it became one of the family cultures of the ancient Indonesian kings. Batik designs often incorporate motifs inspired by nature, mythology, and cultural symbols. Different regions in Indonesia have their own distinct styles and patterns, reflecting the local traditions and influences. Some well-known styles include Javanese batik, Balinese batik, and Sumatran batik, each with its own unique characteristics. See also Mamp, Michael. "Ethel Wallace: A Forgotten History of Batik and Fashion." The Journal of Modern Craft 14, No. 3 (2021): 253-273; Selamet, Juhri. "Indonesian batik translation: A case study." The International Journal of Visual Design 12, No. 3 (2018): 11-17. 4 Cornish, William Rodolph. Intellectual Property: Omnipresent, Distracting, Irrelevant?. (Oxford: Clarendon Law Lectures, 2004); See also Cornish, William Rodolph, David Vaver, and Lionel Bently, eds. Intellectual Property in the New Millennium: Essays in Honour of William R. Cornish. (Cambridge: Cambridge University Press, 2004); Khoirul Hidayah, Hukum HKI (Hak Kekayaan Intelektual) di Indonesia: Kajian Undang-Undang dan Integrasi Islam (Malang: Setara Press, 2013); Nissa, Khoirun. "Protection of Industrial Design Law in the Enhancement of Economic Development in Indonesia." Journal of Private and Commercial Law 3, No. 2 (2019): 76-81. https://doi.org/10.15294/jpcl.v3i2.19774; Raharjo, Riko, M. Khoidin, & Ermanto Fahamsyah. “Perlindungan Hukum terhadap Pencipta atas Pencatatan Suatu Ciptaan yang Sama." Lentera Hukum 5, No. 3 (2018): 437-456. 2 Etty Susilowati, Kontrak Alih Teknologi Pada Industri Manufaktur (Yogyakarta: Genta Press, 2007). ) 3 Ismail Saleh, Hukum dan Ekonomi (Jakarta: Gramedia Pustaka Utama). 4 JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 4 The business of Camelia Batik Kei is still in a small scale so that production is carried out at other batik companies outside Tual City, namely in Java. For the market itself, most of it is still in Tual City. However, there are some customers outside Tual City who also buy the batik. In addition, through collaboration with the local government of Tual City and Southeast Maluku, this batik is also marketed at bazaar stands outside Tual City as one of the products originating from Kei. The batik motif Camellia Batik Kei is interesting for research from the aspect of legal protection of Intellectual Property Rights (IPR). Because from the aspect of intellectual property rights, the Camellia Batik Kei batik motif has not received IPR legal protection at all through Copyright. According to W.R. Cornish, IPR protects the use of ideas and information that have commercial or economic value.2 Furthermore, Ismail Saleh emphasized that Intellectual Property Rights can be translated as intellectual property rights, concerning copyrights and industrial property rights.3 Intellectual Property Rights (IPR) are economic rights granted by law to a creator or inventor for a work of human intellectual ability.4 The Camelia Batik Kei motif is designed with the aim of embracing business opportunities while infusing philosophical Available online at http://journal.unnes.ac.id/sju/index.php/jils 5 Sulistyowati Irianto, “Praktik Penelitian Hukum Perspektif Sosiolegal” In Sulistyowati Irianto and Shidarta, Metode Penelitian Hukum: Konstelasi dan Refleksi. (Jakarta: Yayasan Obor Indonesia, 2009). 6 Sociolegal research refers to the interdisciplinary study of the interaction between society and law. It involves examining the social, political, economic, and cultural factors that influence legal systems and the impact of laws on society. Sociolegal researchers explore how laws are created, implemented, and enforced, and how they shape social norms, behaviors, and power dynamics. Sociolegal research draws on theories and methodologies from sociology, law, political science, anthropology, and other social sciences. It typically involves qualitative research methods such as interviews, ethnographic observations, and document analysis, as well as quantitative methods like JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 5 5 values, thus creating batik motifs that possess both commercial and economic value. In the context of the IPR, the batik motif Camelia Batik Kei can be categorized as a copyright that can get legal protection under Law Number 28 of 2014 concerning Copyright (hereinafter as Indonesian Copyrights Law). Indonesian Copyright Law grants exclusive rights to creators, which are automatically established based on declarative principles once a work is expressed in a tangible form, subject to certain limitations as prescribed by legislation. Copyright is a crucial component of intellectual property that encompasses a wide range of protected works, spanning science, art, literature, and even computer programs. Under Law No. 28 of 2014, copyright protection is granted automatically upon the realization of a tangible expression, without the need for registration (declarative). It is important to note that registering works in the General Register of Works does not imply an endorsement of the content, meaning, intent, or form of the registered works. In the further context, legal protection for the copyright of traditional batik motifs owned by individuals also needs to get legal protection so that in the future, the batik motifs owned are not taken or "stolen" by irresponsible parties. In this study, the type of research used is sociolegal research,5 which combine between doctrinal methods and empirical methods6 to identify copyright registration as JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 6 a guarantee of legal protection for Tual batik motifs. The types of data in this research are primary data and secondary data. The data collection techniques used were literature study and interview techniques which were studied and analyzed in depth in order to obtain a complete and in-depth conclusion in an analytical descriptive manner. surveys and statistical analysis. See Harrington, Christine B., and Barbara Yngvesson. "Interpretive sociolegal research." Law & Social Inquiry 15, No. 1 (1990): 135-148; Banakar, Reza, and Max Travers, eds. Theory and Method in Socio-legal Research. (London: Bloomsbury Publishing, 2005); Graham, Nicole, Margaret Davies, and Lee Godden. "Broadening law’s context: materiality in socio-legal research." Griffith Law Review 26, No. 4 (2017): 480-510.. IMPLEMENTATION OF THE AGREEMENT BETWEEN THE OWNER OF THE CAMELIA BATIK KEI MOTIF & PARTNERS Business Profile of Camelia Batik Kei Business Profile of Camelia Batik Kei Batik is one of the typical crafts from Indonesia which is now worldwide. Therefore, many craftsmen are looking to develop a batik business, one of which is Camelia Batik Kei in Tual City, Maluku Province. Camelia Batik Kei's business has been running since 2017. The name of Camelia's business comes from the name of the owner of the business. The initial idea of opening a Batik Kei development business started with the owner who has a hobby and interest in the world of drawing since he was in high school. Then after graduating from high school, he continued his undergraduate education to master's level in Available online at http://journal.unnes.ac.id/sju/index.php/jils JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 7 7 the city of Yogyakarta which was so thick with batik. During this study in Yogyakarta, the owner's interest in batik cloth increased because most of the activities used batik cloth both as clothing and as other supporting ornaments. Because of this interest in batik that the owner of Camelia Batik Kei has a business idea to create batik cloth products. This batik cloth, which was first made by the owner of Camelia Batik Kei, characterizes the typical culture of the Kei people. The owner of Camelia Batik Kei raised Kei culture with the aim that through this Kei Batik cloth people can get to know the Kei culture and area. At the beginning of realizing the idea of producing Batik Kei, the owner of Camelia Batik Kei looked for samples or samples by looking at and studying batik products from other regions in Indonesia that already produce batik, for example Betawi batik which is one example. In addition to Betawi batik, we also saw and studied examples of batik produced in Yogyakarta and surrounding areas which are well known both in Indonesia and abroad. Based on the results of the interview with the owner of Camelia Batik Kei, to be able to make batik that does point to the characteristics of a particular area such as those made by characterizing the distinctive culture of the Kei people, there must be a clear sign for the area through batik motifs, for example such as Betawi batik which has a distinctive culture, namely ondel-ondel, so that in the design of the Betawi batik motif there is a picture of ondel-ondel. From this thought, the owner of the Camelia Batik Kei business began to collect all the ideas that would be used as Kei batik motifs related to the culture of the Kei people themselves. For now, Camelia Batik Kei's business is still being produced and marketed in the area around Tual City and Southeast Maluku Regency. Those who are interested are employees such as Civil Servants and private employees in Tual City and its surroundings Available online at http://journal.unnes.ac.id/sju/index.php/jils JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 8 who really like this Camelia Batik Kei motif. There are several collaborations with office agencies to produce Camelia Batik Kei on a limited basis as official clothing worn on certain days. In addition to Tual City, there are several people in the Netherlands who are descendants of Kei who also order this Kei Batik as a souvenir when visiting Tual City. Produced Products At the beginning of the Camelia Batik Kei production process, what was made was a product in the form of a complete Batik Fabric with Kei cultural motifs such as embal, embal leaves, lolat, lela, kei gold, gongs, arrows, beach motifs, a combination of several motifs into one and complete motif. In addition to products in the form of cloth, there are also other products made from cloth products such as scarves, wallets, men's and women's clothing and other souvenirs. 1. Batik Fabric 8 Ayu, Bumi. "Perlindungan Hukum Hak Milik Atas Tanah “Hawear Balwirin” Terhadap Tanah Adat Larvul Ngabal Masyarakat Adat Kei di Ohoi (Desa) Wain Kecamatan Kei Kecil Timur Kabupaten Maluku Tenggara." Gloria Yuris 5, No. 2 (2017). https://jurnal.untan.ac.id/index.php/jmfh/article/view/18262 Source: Facebook Camelia Batik The Southeast Maluku region does have a culture of regulating brotherhood, with the shape of a Lolat leaf.7 Leaf Lolat plays a role in regulating community social relations, namely between two or more villages. The Southeast Maluku region does have a culture of regulating brotherhood, with the shape of a Lolat leaf.7 Leaf Lolat plays a role in regulating community social relations, namely between two or more villages. otif FIGURE 2. Lela Motif Source: Authors JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 9 9 a. Lolat Motif Lolat Motif FIGURE 1. Lolat Motif FIGURE 1. Lolat Motif Available online at http://journal.unnes.ac.id/sju/index.php/jils 7 Anton Ohoira, KEI: Alam, Manusia, Budaya, dan Beberapa Perubahan (Yogyakarta: Sibuku Media, 2016) c. Kei Gold Motif FIGURE 3. Kei Gold Motif FIGURE 3. Kei Gold Motif 9 Mansyur, Syahruddin. "Permukiman Tradisional Masyarakat Tanimbar Kei." Kapata Arkeologi (2005): 70-87. b. Lela Motif FIGURE 2. Lela Motif Source: Authors Lela is the traditional cannon of the Kei community. Lela is a traditional object that is very thick with the procession of resolving traditional violations including violating the tradition and culture of Hawear.8 These objects cannot be traded because they are an integral part of a traditional procession and are believed to have a very “expensive” Available online at http://journal.unnes.ac.id/sju/index.php/jils 9 Mansyur, Syahruddin. "Permukiman Tradisional Masyarakat Tanimbar Kei." Kapata Arkeologi (2005): 70-87. JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 10 cultural power and are upheld. Lela is a cultural object made of copper and shaped like a small cannon. It is almost certain that this object is a relic of the ancestors since time immemorial to be used only in every process of settling a case so that it can be ascertained that this is joint ownership and not the property of individuals. From the existing oral tradition, the value of this object is so high that it is not easy to display or display (read, as a means of payment) in a customary negotiation or paid for as a consequence of a violation. With this view, the indigenous people in Kei, Lela also have certain sacred values that must be fully obeyed by everyone involved in an issue or case. This means that if the results of negotiations between the parties in a litigation must be resolved using Lela, then at that time the existing problems can be considered as over and the parties can accept each other again.9 Syahruddin Mansyur, Permukiman Tradisional Masyarakat Tanimbar Kei, Kapata Arkeol. 70 (2005). 10 Sabara, Sabara. "Perempuan Dalam Kearifan Lokal Suku Kei." MIMIKRI 8.1 (2022): 88- 111. https://blamakassar.e-journal.id/mimikri/article/view/637 11 Moniharapon, Greliensia, Andrian Dektisa Hagijanto, and Bernadette Dian Arini. "Perancangan Fashion Kain Tenun Ikat Kepulauan Tanimbar dan Media Pendukungnya." Jurnal DKV Adiwarna 1, No. 12 (2018): 1-11. https://publication.petra.ac.id/index.php/dkv/article/view/7158/6493 JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 11 11 Source: Authors Kei Gold / Kei Brass is still used as a traditional means and as a form of marriage dowry when proposing to a Kei woman. Not only that, because the meaning of this "Golden Kei", is even used as a medium of apology. Kei gold is made with functional forms / decorations as jewelry, of course it will have a high economic value. This golden color is what makes the people of the Kei Islands call it "Golden".10 d. Gong Motif d. Gong Motif d. d. Gong Motif FIGURE 4. Kei Gold Motif Source: Authors FIGURE 4. Kei Gold Motif Source: Authors Gong is one of the traditional objects other than Lela (cannon) or traditional gold whose amount is calculated as offerings in traditional events and as a symbol of peace.11 Available online at http://journal.unnes.ac.id/sju/index.php/jils 10 Sabara, Sabara. "Perempuan Dalam Kearifan Lokal Suku Kei." MIMIKRI 8.1 (2022): 88- 111. https://blamakassar.e-journal.id/mimikri/article/view/637 12 Berlianty, Teng, and Yosia Hetharie. "Urgensi Pendaftaran dan Perlindungan Hukum Terhadap Embal Sebagai Indikasi Geografis Maluku Tenggara." Jurnal IUS Kajian Hukum dan Keadilan 8, No. 2 (2020): 244-255. e. Embal Motif e. Embal Motif Embal Motif FIGURE 5. Embal Motif Source: Facebook Camelia Batik FIGURE 5. Embal Motif Source: Facebook Camelia Batik Source: Facebook Camelia Batik Embal is the name of a typical food in Southeast Maluku Regency. Made from cassava, this dish is often served when relaxing with the family.12 Embal has a bland taste, hard texture, white color, and large, but varied shapes and sizes. As an alternative to staple food, Embal is eaten by dipping it in water or gravy from the food until it is soft and then consumed with side dishes, just like eating rice. f. Embal Leaf Motif FIGURE 6. Cassava Leaf Motif (Embal) f. f. Embal Leaf Motif FIGURE 6. Cassava Leaf Motif (Embal) FIGURE 6. Cassava Leaf Motif (Embal) Available online at http://journal.unnes.ac.id/sju/index.php/jils Available online at http://journal.unnes.ac.id/sju/index.php/jils JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 12 Available online at http://journal.unnes.ac.id/sju/index.php/jils 13 Laksono, P. M. "Dimakan Sayang: Turning the Common into the Exclusive, Cassava Bread (Embal) in The Kei Islands, Southeast Maluku, Indonesia." Humaniora 33, No. 2 (2021): 103-112. ( ) 14 Ellen, Roy F., ed. Modern Crises and Traditional Strategies: Local Ecological Knowledge in Island Southeast Asia. Vol. 6. (New York: Berghahn Books, 2007). Available online at http://journal.unnes.ac.id/sju/index.php/jils 15 Bandjar, Qhalfiah Hairun, B. Irwan Wipranata, and Sylvie Wirawati. "Studi Keberhasilan Pengelolaan Objek Wisata Berbasis Community Based Tourism (CBT), Objek Studi: Pantai Ngurbloat, Kabupaten Maluku Tenggara." Jurnal Sains, Teknologi, Urban, Perancangan, Arsitektur (STUPA) 3, No. 2 (2021): 3467-3478. JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 14 combination of several motifs into one product. For example, the combination of Embal motifs and gong motifs, gong motifs and Kei gold motifs, Embal motifs and Lela motifs, and various other combinations of motifs. Most of the results of this combination of motifs are also adapted to market demand or customers who are interested in Batik Kei. h. JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 13 Source: Authors In the Kei Islands, cassava (Basic Material for Embal) is the main commodity grown at the beginning of the rainy season. This type of food is very suitable as food in the famine season because it has a long shelf life, 1-2 years, if stored in good and dry conditions. Historically, the people of Maluku initially planted sago as a staple food.13 However, the reduced supply of sago in the market in the 1970s made many farmers switch to open cassava fields from the late 1970s to the 1980s. Gradually, this plant became a favorite in the eyes of the public. According to Ellen, Embal made from cassava is a "twin" of food with sago raw material called plate sago.14 FIGURE 7. Mixed Motifs (Mixed Several Motifs) Source: Authors FIGURE 7. Mixed Motifs (Mixed Several Motifs) Source: Authors Business actors Camelia Batik Kei not only make products, both whole fabrics and apparel with one motif, but there is a Business actors Camelia Batik Kei not only make products, both whole fabrics and apparel with one motif, but there is a Available online at http://journal.unnes.ac.id/sju/index.php/jils h. Beach Motif FIGURE 8. Beach Motif Source: Facebook Camelia Batik FIGURE 8. Beach Motif Source: Facebook Camelia Batik Source: Facebook Camelia Batik It is called the beach motif because this motif is made with the feel of the beach and marine life in the Kei Islands. The Kei Islands are one of the most famous in Maluku for their beach tourism, so that craftsmen are inspired to make motifs that can introduce the characteristics and advantages of the Kei Islands.15 The marine life in this beach motif, such as bia lola, shellfish, and other marine life. gy 17 Fajriani, Erni. “Pelestarian Budaya Tari Panah Sebagai Tarian Penyambutan Tamu di Kepulauan Kei Provinsi Maluku”. Thesis (Yogyakarta: Sekolah Tinggi Pariwisata Ambarrukmo STIPRAM, 2018). 16 Harbelubun, Yosep. “Gereja dan Kepemimpinan dalam Masyarakat Kei: Suatu Dialog Transformatif antara Kepemimpinan Gereja dan Kepemimpinan dalam Pemerintahan Adat Kei”. Thesis. (Yogyakarta: Universitas Kristen Duta Wacana, 2017). Available online at http://journal.unnes.ac.id/sju/index.php/jils Available online at http://journal.unnes.ac.id/sju/index.php/jils JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 15 JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 15 15 i. Bow and Arrow Motif FIGURE 9. Bow and Arrow Motif Source: Facebook Camelia Batik i. Bow and Arrow Motif FIGURE 9. Bow and Arrow Motif Source: Facebook Camelia Batik In addition to the motifs above, there is also a bow and arrow motif. The Kei Islands have the motto "Ain ni Ain" which means We are One which is a unity that cannot be separated.16 In ancient times, when fighting, the Kei people used bows and arrows as weapons17 and in the end the Kei people realized what they were fighting for and no one won and no one lost, that's where the Kei people gathered with each other and took an oath that no one else may start a riot there and if there is he will be subject to the oath. From the things that happened above emerged the Arrow Dance which described the situation of the riots at that time where the men held bows and arrows that symbolized the man was brave and proficient in In addition to the motifs above, there is also a bow and arrow motif. The Kei Islands have the motto "Ain ni Ain" which means We are One which is a unity that cannot be separated.16 In ancient times, when fighting, the Kei people used bows and arrows as weapons17 and in the end the Kei people realized what they were fighting for and no one won and no one lost, that's where the Kei people gathered with each other and took an oath that no one else may start a riot there and if there is he will be subject to the oath. From the things that happened above emerged the Arrow Dance which described the situation of the riots at that time where the men held bows and arrows that symbolized the man was brave and proficient in Available online at http://journal.unnes.ac.id/sju/index.php/jils JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 16 using the tool of war. The arrow dance at this time is often performed during guest welcoming events, and important events on the island of Kei.18 18 Aladjai, Erni. Kei: Kutemukan Cinta di Tengah Perang. (Yogyakarta: GagasMedia, 2013). j. Complete Motif j. Complete Motif Complete Motif FIGURE 10. Complete Motif (Combination of all motifs) Source: Facebook Camelia Batik FIGURE 10. Complete Motif (Combination of all motifs) Source: Facebook Camelia Batik The last motive is the complete motif. It is called with the full motif name, because all the motifs are combined into one whole fabric product which is then made into desired products such as men's and women's clothes, scarves, and other forms according to market demand. Scarf FIGURE 11. Scarves With Embal Motifs and Complete Motifs Source: Facebook Camelia Batik FIGURE 11. Scarves With Embal Motifs and Complete Motifs Source: Facebook Camelia Batik Available online at http://journal.unnes.ac.id/sju/index.php/jils Available online at http://journal.unnes.ac.id/sju/index.php/jils JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 17 3. Wallet FIGURE 12. Embal Motif Wallet with Various Colors Source: Facebook Camelia Batik 4. Clothes FIGURE 13. School Uniforms, Men's Shirts and Semi Suits Source: Facebook Camelia Batik 5. Souvenirs FIGURE 14. Necklace with Kei Batik Fabric Basic Material Source: Authors JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 17 17 3. Wallet FIGURE 12. Embal Motif Wallet with Various Colors Source: Facebook Camelia Batik Available online at http://journal.unnes.ac.id/sju/index.php/jils 3. Wallet FIGURE 12. Embal Motif Wallet with Various Colors Source: Facebook Camelia Batik FIGURE 12. Embal Motif Wallet with Various Colors Source: Facebook Camelia Batik 4. Clothes FIGURE 13. School Uniforms, Men's Shirts and Semi Suits Source: Facebook Camelia Batik FIGURE 13. School Uniforms, Men's Shirts and Semi Suits Source: Facebook Camelia Batik 5. Souvenirs 5. Souvenirs FIGURE 14. Necklace with Kei Batik Fabric Basic Material Source: Authors . Souvenirs FIGURE 14. Necklace with Kei Batik Fabric Basic Material Source: Authors FIGURE 14. Necklace with Kei Batik Fabric Basic Material Source: Authors Available online at http://journal.unnes.ac.id/sju/index.php/jils Available online at http://journal.unnes.ac.id/sju/index.php/jils Available online at http://journal.unnes.ac.id/sju/index.php/jils JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 18 19 Azhari, Reyhan Nabillah. "Understanding the Contents of Indonesian Civil Law: A Book Review Perkembangan Hukum Perdata di Indonesia, Sudikno Mertokusumo, Genta Publishing Yogyakarta, 2019, 206 Pages, ISBN 978-602-0757-08-7." Journal of Indonesian Legal Studies 5, No. 1 (2020): 263-266. 20 Hetharie, Yosia. "Perjanjian Nominee sebagai Sarana Penguasaan Hak Milik atas Tanah oleh Warga Negara Asing (WNA) Menurut Kitab Undang-Undang Hukum Perdata." SASI 25, No. 1 (2019): 27-36. Camelia Batik Kei Business Cooperation Agreement Civil law regulates the rights and obligations of individuals or individuals to one another in social life.19 One of the regulated rights and obligations is created through an agreement. An agreement occurs through or by a statement of the will of the person or party acting, which is aimed at the emergence of legal consequences or because the acting party creates confidence in the other party that his will is aimed at the occurrence of the agreement. The statement of will of the person acting includes the offer and acceptance prior to the conclusion of an agreement.20 An important moment in the process of forming or closing the agreement is the encounter of wills, the interlocking of each statement of will and the existence of reciprocal relationships.21 Also contained in Article 1338 paragraph (3) of the Civil Code an agreement must be based on the principle of good faith. Good faith can be divided into subjective good faith and objective good faith. Good faith is subjective, namely whether the person concerned himself is aware that his actions are contrary to good faith, while objective good faith is if public opinion considers such actions to be contrary to good faith.22 ( ) 21 Tjoanda, Merry, et al. "Covid-19 sebagai Bentuk Overmacht dan Akibat Hukumnya Terhadap Pelaksanaan Perjanjian Kredit." Sasi 27, No. 1 (2021): 93-101. p j j 22 Hetharie, Yosia. "Kepemilikan Tanah Oleh Warga Negara Asing Melalui Perjanjian Pinjam Nama Sebagai Bentuk Penyelundupan Hukum dalam Hukum Perdata Internasional." Balobe Law Journal 2, No. 1 (2022): 12-20. Available online at http://journal.unnes.ac.id/sju/index.php/jils 23 Pesulima, Theresia L., and Yosia Hetharie. "Akibat Hukum Pencantuman Klausula Baku dalam Perjanjian Endorsement." Lutur Law Journal 1, No. 1 (2020): 37-41. 24 R. Subekti, Aneka Perjanjian. (Bandung: Citra Aditya Bakti, 1992). 25 Hetharie, Yosia. "Default in Sea Transportation Agreement." Law Research Review Quarterly 6, No. 2 (2020): 181-184. 26 Balik, Agustina, and Yosia Hetharie. "Aspek Keadilan Klausula Baku Dalam perjanjian Kerja Outsourcing." Jurnal Dinamika Hukum 9, No. 2 (2009): 67-75. 23 Pesulima, Theresia L., and Yosia Hetharie. "Akibat Hukum Pencantuman Klausula Baku dalam Perjanjian Endorsement." Lutur Law Journal 1, No. 1 (2020): 37-41. JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 19 An agreement is a legal act based on an agreement to cause a legal consequence.23 It also agrees with Sudikno, "agreement is a legal relationship between two or more parties based on an agreement to cause a legal consequence".24 The birth of an agreement in civil law is known as the principle of consensualism, namely that to give birth to an agreement it is enough to just agree and that the agreement has been born at the time of reaching the consensus as intended above. At that second the agreement is finished and binding, not at other seconds later or before,25 and also in the agreement the principle of freedom of contract applies, meaning that the parties are free to make any contract/agreement, both those that have been regulated and those that have not been regulated and are free to determine the contents of the contract themselves.26 This principle provides the widest possible freedom to the public to enter into agreements containing anything and to arrange their own agreements that will bind the makers with restrictions that are not contrary to law, decency, and public order. As for the cooperation agreement, it is an unnamed agreement which is regulated outside the Civil Code, but occurs in the community. The birth of a cooperation agreement in practice is based on Article 1338 of the Civil Code. Based on Article 1338 Paragraph (1) of the Civil Code, this provision reads "all agreements made legally are valid as law for those who make them". The word "all" means covering all agreements, both those whose names are known and those that are not known by law. The principle of freedom of contract is related to the agreement, namely the freedom to determine "what" and with j j J ( ) 24 R. Subekti, Aneka Perjanjian. (Bandung: Citra Aditya Bakti, 1992). 26 Balik, Agustina, and Yosia Hetharie. "Aspek Keadilan Klausula Baku Dalam perjanjian Kerja Outsourcing." Jurnal Dinamika Hukum 9, No. 2 (2009): 67-75. 25 Hetharie, Yosia. "Default in Sea Transportation Agreement." Law Research Review Quarterly 6, No. 2 (2020): 181-184. 28 Purwanto, Harry. "Keberadaan asas Pacta Sunt Servanda dalam perjanjian internasional." Mimbar Hukum 21, No. 1 (2009): 155-170. 27 Berlianty, Teng, Yosia Hetharie, and Ronald Saija. "Implementation of The Plantation Production Sharing Agreement in The Unwritten Agreement Form." Jambura Law Review 4, No. 1 (2022): 73-88. Available online at http://journal.unnes.ac.id/sju/index.php/jils Available online at http://journal.unnes.ac.id/sju/index.php/jils JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 20 "who" the agreement is made and has binding power for the parties to the agreement. Cooperation Agreement itself is the agreement of the parties to make achievements, and give rise to a contractual relationship (rights and obligations) of the parties in achieving common goals.27 In running the Camelia Batik Kei business, the owner cooperates with other parties through cooperation agreements, especially in producing the desired Batik Kei motif in fabrics with certain basic colors. In this cooperation agreement Camelia Batik Kei collaborates with other business actors in Yogyakarta who make canting (a tool to print batik motifs as desired). Based on the results of an interview with the owner of Camelia Batik Kei that the beginning of the making of this canting, the owner tried to find people in Tual City to draw but did not find a suitable person to be able to draw the desired pattern of motifs so the owner looked to Java, namely in Yogyakarta. After finding someone who can draw, then the owner of Camelia Batik Kei is looking for another party to make canting with the concept made. The form of a cooperation agreement that is carried out both with the party who draws the motif and the party who makes the canting is carried out only using an oral agreement on the basis of trust. An oral agreement is an agreement made by the parties in oral form (enough with the agreement of the parties).28 This is because according to the owner of Camelia Batik Kei, because it is still in small production, the collaboration is carried out through oral communication without having to do it in writing. So far, there has been no breach of promise (default) from the parties, meaning that the Available online at http://journal.unnes.ac.id/sju/index.php/jils 1) Principle of Freedom of Contract 1) Principle of Freedom of Contract The principle of freedom of contract is an important principle in making an agreement. This principle relates to the content of an agreement which gives freedom to choose what and with whom the agreement will be made. individual freedom gives him the free right to contract. They are free to make agreements with any party they want, because there is no element of being forced to enter into this cooperation agreement based on their own will and no demands from others. The principle of freedom of contract is not absolute freedom, there are several restrictions given by the articles in the Civil Code to this principle, making the principle of freedom of contract an unlimited principle, namely: The principle of freedom of contract is an important principle in making an agreement. This principle relates to the content of an agreement which gives freedom to choose what and with whom the agreement will be made. individual freedom gives him the free right to contract. They are free to make agreements with any party they want, because there is no element of being forced to enter into this cooperation agreement based on their own will and no demands from others. The principle of freedom of contract is not absolute freedom, there are several restrictions given by the articles in the Civil Code to this principle, making the principle of freedom of contract an unlimited principle, namely: a) Article 1320 paragraph (1), an agreement or contract is invalid if it is made without a consensus or agreement from the parties who made it. This provision implies that the freedom of a party to determine the contents of the agreement is limited by the agreement of the other party. b) Article 1320 paragraph (2), the freedom of people to make agreements is limited by their abilities, for someone who according to the provisions of the law is not capable of making agreements does not have the freedom to make agreements. c) Article 1320 paragraph (3), the object of the agreement must be determined. A certain thing is the subject of the agreement, namely the achievements that must be fulfilled in an agreement. What is agreed upon must clearly specify the type, the amount may not be stated as long as it can be calculated or determined. JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 21 21 cooperation carried out is going well and smoothly in accordance with the agreed rights and obligations. The cooperation agreement entered into by the parties is based on several principles, namely: Available online at http://journal.unnes.ac.id/sju/index.php/jils 29 Hariri, Wawan Muhwan. Hukum Perikatan. (Bandung: CV. Pustaka Setia, 2011). g 30 Hernoko, Agus Yudha. Hukum Perjanjian Asas Proporsionalitas dalam Kontrak Komersial. (Jakarta: Kencana, 2010). JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 22 d) Article 1330, minors and persons under guardianship do not have the ability to make an agreement. d) Article 1330, minors and persons under guardianship do not have the ability to make an agreement. d) Article 1330, minors and persons under guardianship do not have the ability to make an agreement. This principle is clearly related to the cooperation agreement made by the owner of Camelia Batik Kei with the party who draws and makes the canting, where they are free to determine what will be mutually agreed upon.29 2) Principle of Trust The principle of trust is that the parties to an agreement must carry out the substance of the contract or achievement based on firm trust or belief and the good will of the parties in order to achieve the objectives of the agreement. The principle of trust includes the principle of good faith which is also an important principle in an agreement.30 In conducting a transaction, most people only rely on trust or trust each other without any standard agreement or contract that has binding legal force for both parties. The owner of Camelia Batik Kei is based on the principle of trust by believing and trusting that the other party, namely the party who draws and makes the canting as the executor of the work, is a party that can be trusted to cooperate. 2) Principle of Trust The principle of trust is that the parties to an agreement must carry out the substance of the contract or achievement based on firm trust or belief and the good will of the parties in order to achieve the objectives of the agreement. The principle of trust includes the principle of good faith which is also an important principle in an agreement.30 In conducting a transaction, most people only rely on trust or trust each other without any standard agreement or contract that has binding legal force for both parties. The owner of Camelia Batik Kei is based on the principle of trust by believing and trusting that the other party, namely the party who draws and makes the canting as the executor of the work, is a party that can be trusted to cooperate. JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 3) Principles of Equality and Justice In the implementation of the agreement, this principle is important to be implemented by both parties because they have the same or equal position with each other. This principle is closely related to the principle of justice. The principle of justice is the basis for fair treatment of fellow human beings. The word fair is not one-sided or balanced, equal, and equal. Justice gives everyone a share according to their services or a distribution according to their 3) Principles of Equality and Justice In the implementation of the agreement, this principle is important to be implemented by both parties because they have the same or equal position with each other. This principle is closely related to the principle of justice. The principle of justice is the basis for fair treatment of fellow human beings. The word fair is not one-sided or balanced, equal, and equal. Justice gives everyone a share according to their services or a distribution according to their 29 Hariri, Wawan Muhwan. Hukum Perikatan. (Bandung: CV. Pustaka Setia, 2011). 31 Susanti, Dyah Ochtorina, and IGN Parikesit Widiatedja. Asas Keadilan Konsep dan Implementasinya dalam Perspektif Hukum Islam dan Hukum Barat. (Malang: Bayumedia, 2011). Available online at http://journal.unnes.ac.id/sju/index.php/jils Available online at http://journal.unnes.ac.id/sju/index.php/jils JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 23 respective rights.31 In the formation of a rule of law using the principle of justice for the sake of common interests and goals, it does not burden one party in a regulation, and of course there is a balance of human rights among the community. The agreement between the owner of Camelia Batik Kei and the service provider is based on the rules they set or have agreed upon, which of course has an element of justice so as not to harm one of the parties. The implementation of this cooperation agreement aims to open up the market because so far there has been no sales of batik cloth characterized by the Kei area. It also seeks to improve the economy and introduce the culture of the Kei people to all corners of the country and even to other countries. In carrying out an agreement, of course, there must be reciprocity between the parties who do it, where reciprocity is an achievement made by one party that creates a counter-achievement from the other party. The birth of a cooperation agreement if between the parties there has been a valid agreement. The agreement here is a statement of the will that is appropriate between one or more people with other parties who will be involved in a cooperation agreement. The parties who will enter into a cooperation agreement have rights and obligations, where the rights and obligations of each party must be equally fulfilled. Broadly speaking, the party who drew and the canting maker has an obligation to draw and make the canting according to the concept that has been created and determined by the owner of Camelia Batik Kei. The rights to be received are related to payments that are also mutually agreed upon by the parties. Meanwhile, the owner of Camelia Batik Kei is obliged on the contrary to pay JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 24 according to the agreed amount and has the right to receive the desired fabric motifs and images according to the concept that has been given. according to the agreed amount and has the right to receive the desired fabric motifs and images according to the concept that has been given. 1. Malaysia Malaysian Batik is the art of Batik textiles from Malaysia, especially in the east coast of Malaysia namely Kelantan, Terengganu and Pahang. In line with the concept of Malaysia, the Malaysian government also recognizes Malaysian batik as the national dress for every general population, by providing local designers to create new batik designs that reflect Malaysia's identity. 32 Tantyo Bangun, National Geographic: Traveler Vol1 No.6 2009 (Jakarta: KOMPAS Gramedia, 2006). Available online at http://journal.unnes.ac.id/sju/index.php/jils Available online at http://journal.unnes.ac.id/sju/index.php/jils a. History of Malaysian Batik Batik comes from the Javanese word "amba" which means to write and "nitik" which means to make a point. The origin of batik production in Malaysia is quite difficult to know. Some historical artifacts exist, but it is more about the trade relationship between the Malay Kingdom in Jambi and the coastal cities of Java that has developed since the 13th century, namely the batik production areas of the northern coast of Java (Cirebon, Lasem, Tuban, and Madura) greatly influence Jambi batik. Jambi batik and Javanese batik greatly influence the batik craft in Peninsular Malaysia.32 33 Akhir, Noor Haslina Mohamad, et al. "Traditional craftsmanship: The origin, culture, and challenges of batik industry in Malaysia." Islamic perspectives relating to business, arts, culture and communication: Proceedings of the 1st ICIBACC 2014. (Singapore: Springer 2015). JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 25 According to the Museum of Cultural History in Oslo, it is known for sure that Java influenced Malay batik making technically and also in the production of patterns. In the early stages, the Malaysian Malay community used wooden blocks to produce textiles similar to batik. In the late 1920s, Javanese batik entrepreneurs introduced the use of candles and copper blocks to the east coast of Malaysia. The production of hand- painted batik in Malaysia today is related to Javanese written batik.33 Batik is mentioned in Malay history in the 17th century. It is about Admiral Hang Nadim who was ordered by Sultan Mahmud to sail to India to get 140 pieces of batik cloth with 40 types of flowers on it. As a result of not find it as controlled by the Sultan, Hang Nadim acted by making it himself. However, on the way home, his ship had sunk and he could only bring four sheets and caused the wrath of the sultan. The use of Malaysian batik among men is usually for dinner parties. While for women, Malaysian batik is often made into formal wear by combining batik and modern fashion. In addition, to encourage the use of batik in Malaysia, from January 17, 2008, all civil servants in Malaysia are required to wear Malaysian batik clothes every Thursday. Previously, the rule was to wear batik clothes only on Saturdays. The day of wearing this batik shirt was later changed to the 1st day of the month and the 15th day of the month. However, people easily forget to cause a rollback to the old system based on the day. 34 Ramli, Harozila, et al. "Creative and innovative thinking in the holistic development of students’ potentials through “sekolah bitara” visual art program,”." Turkish Journal of Computer and Mathematics Education (TURCOMAT) 12, No. 3 (2021): 406-417. Available online at http://journal.unnes.ac.id/sju/index.php/jils Available online at http://journal.unnes.ac.id/sju/index.php/jils p 35 Chong, Jinn Winn. ""Mine, yours or ours?": the Indonesia-Malaysia disputes over shared cultural heritage." SOJOURN: Journal of Social Issues in Southeast Asia 27, No. 1 (2012): 1- 53. 34 Ramli, Harozila, et al. "Creative and innovative thinking in the holistic development of students’ potentials through “sekolah bitara” visual art program,”." Turkish Journal of Computer and Mathematics Education (TURCOMAT) 12, No. 3 (2021): 406-417. 35 Chong, Jinn Winn. ""Mine, yours or ours?": the Indonesia-Malaysia disputes over shared cultural heritage " SOJOURN: Journal of Social Issues in Southeast Asia 27 No 1 (2012): 1 JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 26 Batik refers to fabric that has a bright and attractive pattern.34 Batik comes in various forms, including written batik, screen batik, and applied batik. The batik technique has been known for over a thousand years, possibly originating from ancient Egypt or Sumer. It is found in several countries in West Africa such as Cameroon, Mali and Nigeria or in Asia such as Bangladesh, India, Indonesia, Iran, Malaysia, Sri Lanka and Thailand. Batik has become a part of world culture, especially in the Malay world and since a long time that includes Southeast Asian countries. Nowadays there are new patterns known as modern batik to meet the changing tastes of consumers.35 Batik is one of the unique and famous traditional handicrafts in Malaysia. It is produced through the process of wax application and color dyeing. The history of the development of batik in Malaysia is believed to begin in the 15th century AD. Early development shows the impact and influence of batik from Java. From the 1930s until after the Second World War, the batik industry in Malaysia has grown rapidly. Batik entrepreneurs in the states of Kelantan and Terengganu are pioneers in the development of batik in this country. Local batik products are of high quality and design. This symbolizes a creative handcraft skill among the batik makers of the country. The attractiveness of batik is not only famous among the local people but it also receives a welcome from foreign tourists who come from abroad. 38 Kaewareelap, Suvita, Yaowarat Sirisathitkul, and Chitnarong Sirisathitkul. "Modernizing Batik Clothes for Community Enterprises Using Creative Design and Colorimetry." Emerging Science Journal 5, No. 6 (2021): 906-915. y g g ( ) 39 Baeren, Erren Jossie, and Humin Jusilin. "The Uniqueness of Mangrove Forests in State of Sabah as a Source of Natural Color on Batik Fabric." 5th International Conference on Arts Language and Culture (ICALC 2020). Atlantis Press, 2021. 37 Tresnadi, Chandra, and Agus Sachari. "Identification of values of ornaments in Indonesian batik in visual content of Nitiki game." Journal of Arts and Humanities 4, No. 8 (2015): 25-39. 36 Saddhono, Kundharu, et al. "The Study of Philosophical Meaning of Batik and Kimono Motifs to Foster Collaborative Creative Industry." Asian Social Science 10, No. 9 (2014): 52- 61. b. Characteristics of Malaysian Batik b. Characteristics of Malaysian Batik Malaysian batik uses flora motifs and rarely uses motifs of living creatures such as humans and animals in line with the prohibition in Islam of using animal images as decoration.36 However, the butterfly theme is still widely used in Malaysian Batik. Malaysian batik is also famous for its geometric designs. The production method of Malaysian batik is quite different from Javanese batik in Indonesia, namely the pattern is larger and simpler.37 In addition, canting is also rarely or not used in creating complicated patterns. The colors used in Malaysian batik are also brighter than Javanese batik.38 In Malaysia, batik is produced through a number of specific techniques, processes and styles, one of which is Block Batik. On the East Coast it is better known as Batik Cap or Batik Terap.39 The initial process in the development of Batik Blok is to use wood block nest carving to stamp a type of natural black dye material. This early technique was called 'Terap Hitam'. In the 1920s, the use of wax in the application process was first introduced by batik makers on the East Coast. Correspondingly, blocks made of Juda steel sheets were also introduced. The patterns of these steel blocks are more neat and Available online at http://journal.unnes.ac.id/sju/index.php/jils Available online at http://journal.unnes.ac.id/sju/index.php/jils JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 27 Available online at http://journal.unnes.ac.id/sju/index.php/jils 41 Lanoszka, Anna. "The global politics of intellectual property rights and pharmaceutical drug policies in developing countries." International Political Science Review 24, No. 2 (2003): 181-197. 40 Kari, Rabiatuadawiyah, Mohd Azhar Samin, and Rafeah Legino. "The Flora Motif as Design Identity in Local Traditional Block Batik." Environment-Behaviour Proceedings Journal 5, No. SI3 (2020): 123-127. JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 28 refined. Special blocks are provided to apply wax on the Head of Cloth, Kapit of Cloth and Body of Cloth. Blok Batik fabric is a favorite of the locals. It is used as sarong batik fabric and ela batik. In addition to being used as clothing, ela batik fabric can be modified to produce bed sheets, curtains, mattress pads, cushion covers, pillow covers, handbags and various types of beautiful and beautiful everyday items. The motifs that are often used on block batik fabric are some patterns of plants and local flowers such as Roses, Orchids, Cloves, Chinese Flowers, creeping roots, ferns and geometric patterns, especially Pucuk motifs Bamboo shoots.40 These motifs are applied in various arrangements and layouts, such as horizontal, vertical, diagonal, patches and brick arrangement. c. Copyright Legal Protection of Batik in Malaysia The Industrial Designs Act 1996 (IDA 1996) is a legal basis that batik producers can use to protect their rights. IDA 1996 protects rights related to industrial design by granting a monopoly over the design of an item made for commercialization purposes.41 The law is intended to motivate writers to work more creatively in developing design-based industries by maintaining the aesthetic aspects of design which can result in buyers choosing to buy one product over another, especially in the case of textiles, furniture, clothing and household furnishings. 42 Pila, Justine. "Copyright and Its Categories of Original Works." Oxford Journal of Legal Studies 30, No. 2 (2010): 229-254. 43 Poon, Stephen TF. "The journey to revival: thriving revolutionary batik design and its potential in contemporary lifestyle and fashion." International Journal of History and Cultural Studies (IJHCS) 3, No. 1 (2017): 48-59. 44 Shaari, Nazlina. "Indigenous knowledge creativity in batik cultural product based on Kansei." International Conference on Social Sciences and Humanities (ICSSH'15) May 5-6, 2015 Bali. JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 29 In addition, Batik protection arrangements in Malaysia are protected through the Copyright Act 1987. Section 7(1) CA 1987 includes literary works, musical works, works of art, films, sound recordings and broadcasts as 'works' entitled to protection. Section 3 defines 'artistic works' as works of graphics, photographs, sculpture or collage, regardless of their artistic quality; an architectural work into a building or a model for a building; or art craft.42 Malaysian batik patterns qualify for protection as works of art provided under section 3 as the production of hand-painted batik patterns by an artist and the Malaysian Batik Industry.43 Protecting Local Batik Designs by Copyright and Industrial Design Act manufacture of stamped batik using blocks engraved with Pattern is an element of graphic work. Thus, there is no difference between printed batik and painting batik in terms of fulfilling the elements of graphic work. Available online at http://journal.unnes.ac.id/sju/index.php/jils Available online at http://journal.unnes.ac.id/sju/index.php/jils 2. Thailand The ancient textile craft of batik has been used by several cultures around the world. Batik has a long and famous history in Thailand and is still an important component of the nation's cultural heritage.44 Thai batik has established itself in the international art and fashion scene, with traditional patterns and modern developments. In the textile art 45 Khwansuwan, Worasuda. "Batik patterns in the South of Thailand." International Journal of Art & Design 5, No. 2 (2021): 23-31. 46 Lockard, Craig A. "" The sea common to all": maritime frontiers, port cities, and Chinese traders in the Southeast Asian Age of Commerce, ca. 1400-1750." Journal of World History 21, No. 2 (2010): 219-247. JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 30 of batik, wax is used to cover cloth, which is then dyed to create intricate patterns and designs. Batik Thailand, Batik Indonesia, Batik Cambodia, Batik Brunei, Batik Singapore, Batik Vietnam, and Batik Malaysia have all been practiced for a long time. Thai batik has a long history and is an important part of the nation's cultural heritage. Thai batik is known for its intricate patterns, vibrant colors, and distinctive themes. It cannot be denied that Indonesia and Thailand have a lot in common, in addition to being geographically neighboring countries in Southeast Asia. Thailand also has pride in its batik tradition. Thai batik that is done with the wax-resist dyeing technique is called "phanung". Not all types of batik clothing are made in Thailand.45 a. History of Thai Batik a. Thailand's history with batik began with the Ayutthaya era that lasted from 1350 to 1767.46 Batik was widely used by the royal palace during this period and was imported from China and India. Batik production did not begin in Thailand until the 19th century, but quickly became a favorite local specialty. Traditional Thai textiles, Indian batik, and Chinese embroidery are just a few examples of cultural and creative movements that influenced the development of Thai batik. The evolution of Thai batik has been influenced by a number of significant individuals and movements. King Rama VI established the Royal Academy of Drawing and Painting in the early 20th century, which helped educate a new generation of designers and artists. In the 1960s, Kamol Tassananchalee, who founded the Thai Crafts Center, was one of Thailand's most influential batik painters. His works 45 Khwansuwan, Worasuda. "Batik patterns in the South of Thailand." International Journal of Art & Design 5, No. 2 (2021): 23-31. 46 Lockard, Craig A. "" The sea common to all": maritime frontiers, port cities, and Chinese traders in the Southeast Asian Age of Commerce, ca. 1400-1750." Journal of World History 21, No. 2 (2010): 219-247. 49 Kaewareelap, Suvita, Yaowarat Sirisathitkul, and Chitnarong Sirisathitkul. "Modernizing Batik Clothes for Community Enterprises Using Creative Design and Colorimetry." Emerging Science Journal 5, No. 6 (2021): 906-915. 47 Claymone, Yoopin, and Watunyu Jaiborisudhi. "A study on one village one product project (OVOP) in Japan and Thailand as an alternative of community development in Indonesia." Thai Journal of East Asian Studies 16, No. 1 (2011): 51-60. Available online at http://journal.unnes.ac.id/sju/index.php/jils Available online at http://journal.unnes.ac.id/sju/index.php/jils 48 Gibson, Chris, and John Connell. "‘Bongo Fury’: tourism, music and cultural economy at Byron Bay, Australia." Tijdschrift voor economische en sociale geografie 94, No. 2 (2003): 164- 187. JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 31 bring Thai batik to a new level of perfection by combining traditional Thai themes with modern art.47 b. Thailand focuses more on batik work in the form of sarong cloth which is often used by the people of Koh Samui Island for leisure activities.48 Typically, Thai cotton or silk fabric is used to make Thai batik, which is then cleaned and bleached before being waxed. Beeswax is generally used to make candles, but paraffin or other materials can also be used. Areas of fabric covered with wax retain their original color when dyed sequentially. After the final dip, the detailed design is revealed when the wax is removed with hot water or another solvent. Thai batik is famous for its bright and strong colors, which are often taken from nature. Dark red, gold, green, and blue are common hues.49 Design themes are also influenced by nature; Floral and animal motifs are very popular. Eagle birds, lotus flowers, and other traditional Thai symbols are also often used. In addition, the most prominent batik motif in Thailand adopts animal and plant elements. Meanwhile, the color combinations used are very diverse with the choice of bright dominant colors. Unlike if found in the Capital in Bangkok, Thai batik is widely used as a shirt such as a men's shirt, which is applied to the famous Thai cotton and silk fabric. Thai batik motifs are often taken from fauna motifs such as butterflies and elephants as a 52 van der Borght, Kim, and Saurav Ghimire. "Seeds & Intellectual Property Rights: Bad Faith and Undue Influence Undermine Food Security and Human Rights." Law and Sustainability: Reshaping the Socio-Economic Order Through Economic and Technological Innovation. (Cham: Springer International Publishing, 2022), pp. 183-208. ( ) p g j 51 Sirisathitkul, Y., and S. Kaewareelap. "Color analysis of batik fabric by facile smartphone colorimetry." International Journal of Advanced Science, Engineering and Information Technology 11, No. 1 (2021): 84-91. 50 Utiswannakul, Patcha. "The traditional textile development in Nan region." Humanities, Arts and Social Sciences Studies (Former Name Silpakorn University Journal of Social Sciences, Humanities, and Arts) 16, No. 3 (2016): 1-18. https://doi.org/10.14456/sujsha.2016.25 JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 32 symbol of the country's beauty, nicknamed the country of the white elephant.50 Thai batik is unique in its use of colors and designs, while also having certain similarities with other batik designs.51 While Indonesian batik often uses softer colors, Thai batik is known for its bright and strong patterns. Thai batik has special design motifs that emphasize natural elements and traditional Thai symbolism. Thai batik is often used to make fashionable Thai batik dresses, sarongs, batik blouses, skirts, scarves, and other batik collections. Thai Batik fabric is popular for home design products such as tablecloths, curtains, and pillowcases in addition to being used for clothing collections. The use of Thai Batik patterns in modern art and textiles has gained popularity in the last few years, especially Bangkok textiles. To create unique and eye-catching creations, artists and designers experiment with new techniques and add contemporary motifs and colors. c. Copyright Legal Protection of Batik in Thailand As a member of the World Trade Organization (WTO) and World Intellectual Property Organization (WIPO).52 Thailand generally complies with international intellectual property standards established by the Agreement on Trade-Related c. Copyright Legal Protection of Batik in Thailand As a member of the World Trade Organization (WTO) and World Intellectual Property Organization (WIPO).52 Thailand generally complies with international intellectual property standards established by the Agreement on Trade-Related Available online at http://journal.unnes.ac.id/sju/index.php/jils Available online at http://journal.unnes.ac.id/sju/index.php/jils Available online at http://journal.unnes.ac.id/sju/index.php/jils y 54 Garcia, Gabriel, and Qinqing Xu. "China’s international cooperation: assisting developing countries to build intellectual property systems." Queen Mary Journal of Intellectual Property 13, No. 1 (2023): 52-74. 53 Tenni, Brigitte, et al. "What is the impact of intellectual property rules on access to medicines? A systematic review." Globalization and health 18, No. 1 (2022): 1-40. JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 33 Aspects of Intellectual Property Rights (TRIPS).53 Thailand is also a party to the Patent Cooperation Treaty (PCT) and the Madrid Agreement Concerning the International Registration of Marks (known as the Madrid System). Patent and trademark applicants may use these international systems for filing international patent and trademark applications for requesting protection in Thailand.54 Copyrights are protected in Thailand without any registration requirement. However, formal recordation of copyrights at The Department of Intellectual Property (DIP) Copyright Office is recommended as it would be useful as evidence of ownership in the event of a dispute. A copyright notice should also be affixed to the copyrighted work. Thailand maintains its efforts in upholding intellectual property rights (IPR), the number of counterfeit and pirated goods remains a concern, especially in the online market, including with regard to counterfeiting of traditional Thai cloth and batik motifs. In January 2021, DIP launched a Memorandum of Understanding on Protection of IPR on the Internet aimed at fighting online IPR Violations, which was signed by three Thai government agencies, major e-commerce platforms in Thailand, and IPR owners or representatives. In Thailand, the Central Intellectual Property, and International Trade Court (CIPITC) is the special court adjudicating IP disputes, including infringement and invalidity issues in Thailand. These violations were also related to counterfeiting and theft of traditional Thai batik and cloth motifs. Available online at http://journal.unnes.ac.id/sju/index.php/jils Available online at http://journal.unnes.ac.id/sju/index.php/jils JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 34 55 Saliman, Abdul R., Hukum Bisnis Untuk Perusahaan Edisi V. (Jakarta: Prenada Media, 2016). THE IMPORTANCE OF COPYRIGHT REGISTRATION FOR KEI BATIK MOTIFS Copyright is an exclusive right for the creator or copyright holder to publish or reproduce his work, which arises automatically after a work is born without reducing restrictions.55 Previously, copyright was regulated in Law Number 19 of 2002 concerning Copyright, but the law was revoked and has been replaced by Law Number 28 of 2014 concerning Copyright (here in after referred to as UUHC). According to Article 1 point 1 UUHC it is stated "Copyright is the exclusive right of the creator that arises automatically based on declarative principles after a work is manifested in a tangible form without reducing restrictions in accordance with the provisions of laws and regulations." In the UUHC applicable in Indonesia, the application for registration of works is regulated starting from Article 64 – Article 79 in Chapter X concerning the Registration of Works and Related Rights Products. If you look at the provisions in Article 64 paragraph (1), it can be seen that the recording of works and related rights products is carried out by the Minister. However, in paragraph (2) of the article it is stated "Recording of works and related rights products as referred to in paragraph (1) is not a requirement to obtain copyrights and related rights." Based on these two paragraphs in Article 64, it is clear that it is not obligatory for the author to register a creation. However, it is also important to record creations, this is to create legal certainty for the work (product). Even according to Prof. Mariam Darus, registration does not only mean to provide strong evidence, but also to create property rights. Furthermore, Article 66 paragraph (2) explains that applications for registration of creations and related JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 35 rights products shall be made electronically and/or non-electronically. However, neither Article 66 nor the explanatory part of the article provides further explanation on this matter. If we look closely, Article 64 – Article 79 of UUHC only regulates basic matters regarding the recording of works and related rights products. As stated in Article 70, namely "Further provisions regarding the procedure for recording Works and Related Rights products are regulated by Government Regulations." However, it seems that this government regulation has not yet been issued. Copyright protection lasts for the lifetime of the Author plus 70 (seventy) years after the Author dies. The economic rights of the Creators and/or Related Rights Owners, including limiting the transfer of economic rights in the form of a sale (sold flat). Craftsmen as Business Actors need to understand the importance of registering Copyright for their creations. In current legal developments, Copyrights which are classified in the form of intangible movable objects can also be used as objects of fiduciary guarantees. This privilege as an award as well as an encouragement to the public's price has an understanding of the importance of registering Copyrights and then registering Copyrights for each of their copyrighted works. Authors, Copyright Holders and Related Rights Owners become Members of the Collective Management Institute in order to collect compensation or royalties, which are made in an official relationship and used commercially. Based on all the explanations related to the registration of Copyrights mentioned above, from now on Batik craftsmen, including Camelia Batik Kei in Tual City, are expected to immediately have the awareness to register their creations in order to obtain legal protection and obtain economic benefits in the form of increased sales turnover, rising prices. sales, as well as the reputation of Kei batik Available online at http://journal.unnes.ac.id/sju/index.php/jils Available online at http://journal.unnes.ac.id/sju/index.php/jils JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 36 products sold can even earn royalties from the Collective Management Institute. CONCLUSION This research concluded and highlighted that implementation of the agreement between the owner of the Kei batik motif and the work partner in this case the canting maker through a cooperation agreement which is carried out verbally on the basis of trust between the two parties. Because it is carried out through an oral agreement, the rights and obligations of the parties are also agreed upon through oral communication between the two parties. This research also confirmed that the registration of copyrights for Kei batik motifs is very important because it is related to legal protection efforts for Kei batik itself. Legal protection for the copyright of Kei batik motifs generally exists in Law No. 28 of 2014 concerning copyright. Currently, the registration of copyright for Kei batik motifs has not been carried out even though Law Number 28 of 2014 concerning copyright also emphasizes the importance of protecting copyrights because every creator, who in this case is the creator of contemporary batik motifs, has the right to moral rights and economic rights. The government, in this case the Tual City government, has collaborated with Camelia Batik Kei several times to display Batik Kei products in official activities and in the form of exhibitions. However, in relation to the responsibilities of local governments in legal protection efforts in the field of copyright, nothing has been done at all. Available online at http://journal.unnes.ac.id/sju/index.php/jils JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 37 37 ACKNOWLEDGMENT Acknowledgments and deepest appreciation are conveyed to: Acknowledgments and deepest appreciation are conveyed to: 1. Faculty of Law, University of Pattimura Ambon, Maluku, Indonesia 1. Faculty of Law, University of Pattimura Ambon, Maluku, Indonesia 2. Lembaga Pengelola Dana Pendidikan (LPDP) Ministry of Finance of the Republic of Indonesia 3. Faculty of Law, Semarang State University, Central Java, Indonesia Available online at http://journal.unnes.ac.id/sju/index.php/jils REFERENCES Akhir, Noor Haslina Mohamad, et al. "Traditional craftsmanship: The origin, culture, and challenges of batik industry in Malaysia." Islamic perspectives relating to business, arts, culture and communication: Proceedings of the 1st ICIBACC 2014. (Singapore: Springer, 2015). Aladjai, Erni. Kei: Kutemukan Cinta di Tengah Perang. (Yogyakarta: GagasMedia, 2013). Anton Ohoira, KEI: Alam, Manusia, Budaya, dan Beberapa Perubahan (Yogyakarta: Sibuku Media, 2016) Ayu, Bumi. "Perlindungan Hukum Hak Milik Atas Tanah “Hawear Balwirin” Terhadap Tanah Adat Larvul Ngabal Masyarakat Adat Kei di Ohoi (Desa) Wain Kecamatan Kei Kecil Timur Kabupaten Maluku Tenggara." Gloria Yuris 5, No. 2 (2017). https://jurnal.untan.ac.id/index.php/jmfh/article/view/18262 Azhari, Reyhan Nabillah. "Understanding the Contents of Indonesian Civil Law: A Book Review Perkembangan Hukum Perdata di Indonesia, Sudikno Mertokusumo, Genta Publishing Yogyakarta, 2019, 206 Pages, ISBN 978-602-0757-08-7." Journal of Indonesian Legal Studies 5, No. 1 (2020): 263-266. https://doi.org/10.15294/jils.v5i1.34809 Available online at http://journal.unnes.ac.id/sju/index.php/jils Available online at http://journal.unnes.ac.id/sju/index.php/jils JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 38 Baeren, Erren Jossie, and Humin Jusilin. 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(Cham: Springer International Publishing, 2022), pp. 183-208. http://dx.doi.org/10.1007/978-3-030-92620-5_7 Available online at http://journal.unnes.ac.id/sju/index.php/jils Funding Statement This research funded by Universitas Pattimura, Ambon Indonesia and Educational Fund Management Institution (LPDP Indonesia). JILS (JOURNAL OF INDONESIAN LEGAL STUDIES) VOLUME 8(1) 2023 44 Wulandari, Ari. Batik Nusantara: Makna filosofis, Cara Pembuatan, dan Industri Batik. (Yogyakarta: Penerbit Andi, 2022). Open Data Statement All data and information in this article were available without any restriction Declaration of Conflicting Statements The author(s) stated that this work is original and has not been previously published in another journal or publication. The author(s) also declared that there is no conflict of interest in the publication of this article. Available online at http://journal.unnes.ac.id/sju/index.php/jils Available online at http://journal.unnes.ac.id/sju/index.php/jils Reproducibility Statement The author(s) stated that reproducibility of this article should be based on the Copyrights notice of this Journal under license a Creative Commons Attribution 4.0 International (CC BY-SA 4.0). Available online at http://journal.unnes.ac.id/sju/index.php/jils Available online at http://journal.unnes.ac.id/sju/index.php/jils
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https://figshare.com/articles/conference_contribution/Characterising_the_relationship_between_force_and_catheter_stability_during_manual_and_robotic_paroxysmal_AF_ablation/10162307/1/files/18314522.pdf
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29Characterising the relationship between force and catheter stability during manual and robotic paroxysmal AF ablation
Europace
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CHARACTERISING THE RELATIONSHIP BETWEEN FORCE AND CATHETER STABILITY DURING MANUAL AND ROBOTIC PAROXYSMAL AF ABLATION 0.15 0.10 0.05 0.00 Displacement /mm per 1 / 60th sec 0–10 10–20 20–30 30–40 40–50 50–60 60–70 70–80 80–90 RWACA Human Robot G.S. Chu1, T.P. Almeida2, S.H. Chin1, S.S. Varanasi1, S. Siddiqui1, S.H. Man1, P.D. Brown1, P.J. Stafford3, A.J. Sandilands3, F.S. Schlindwein2, and G.A. Ng1 1Dept of Cardiovascular Sciences, University of Leicester, Leicester; 2Dept of Engineering, University of Leicester, Leicester; and 3Dept of Cardiology, Glenfield Hospital, Leicester Introduction: Optimal catheter contact force (CF) is associated with better lesion creation and improved outcomes for the ablation of paroxysmal atrial fibrillation (pAF). However it is unknown whether this is in part attributable to improved catheter stability. Method: MAST-AF is a prospective randomized controlled trial (NCT01583855) of Manual versus robotic control with the Amigo Remote Catheter System (Catheter Robotics Inc.), using the SmartTouch catheter and Carto 3 (Biosense Webster), in first time pAF ablation. All patients had point-by-point L and R WACA (wide area circumferential ablation) around ipsilateral pairs of pul- monary veins, with the operator blinded to CF. Energy was delivered at 40W throughout. 0.15 0.10 0.05 0.00 Displacement /mm per 1 / 60th sec 0–10 10–20 20–30 30–40 40–50 50–60 60–70 70–80 Force/g LWACA The 3D location of the ablation catheter tip was recorded during RF delivery, and tip dis- placement calculated as the Euclidean distance between each consecutive sample. The corre- sponding CF at that time point was also extracted. All data was grouped according to Human / Robot and L / RWACA, and sorted by CF. Data was analysed visually by percentile, and the correlation coefficient relating force and catheter displacement was calculated on log-trans- formed data using Matlab (R2014a, Mathworks). Results: 6452 ablation points were analysed from 14 manual and 16 robotic subjects, totalling 294,099 CF/displacement data pairs. Visual evaluation (see figure 1) shows that when considering human or robot in isolation, overall variation in displacement across CF appears similar. However in both L and RWACA (essentially independent groups), a complex but consistent relationship for the displacement spread (IQR) is shown; specifically, at the extremes of force, robotic displacement variation is similar to or more than that of the human, with the reverse occurring in the central force range. The correlation coefficient, its confidence intervals, and statistical significance (r, CIs, p) were calculated for: Human LWACA (0.022, 0.014-0.029, ,0.0001); Human RWACA (0.031, 0.021-0.038, ,0.0001); Robot LWACA (0.011, 0.0040-0.018, 0.0022); and Robot RWACA (-0.045, -0.038 to -0.052, ,0.0001). Ventricular tachycardia & sudden death Conclusions: CF is inversely correlated with catheter stability during WACA for pAF, except for Amigo ablation of the RWACA, where higher CF may be achievable due to superior robotic stability. Overall, for typical target CF ranges, Amigo assistance may improve catheter stability compared to manual ablation. iii9 ABSTRACTS FOR ORAL PRESENTATION, SESSION 2, HRC 2014 TATION, SESSION 2, HRC 2014 Conclusions: CF is inversely correlated with catheter stability during WACA for pAF, except for Amigo ablation of the RWACA, where higher CF may be achievable due to superior robotic stability. Overall, for typical target CF ranges, Amigo assistance may improve catheter stability compared to manual ablation. 0.15 0.10 0.05 0.00 Displacement /mm per 1 / 60th sec 0–10 10–20 20–30 30–40 40–50 50–60 60–70 70–80 80–90 RWACA Human Robot 0.15 0.10 0.05 0.00 Displacement /mm per 1 / 60th sec 0–10 10–20 20–30 30–40 40–50 50–60 60–70 70–80 Force/g LWACA Figure 1 CF vs catheter displacement between human and robotic L and R WACA. Box and whisker plots show 10th, 25th, 50th (median), 75th and 90th percentiles. Dotted lines indicates IQRs. CF groups of ,100 measurements were excluded. CHARACTERISING THE RELATIONSHIP BETWEEN FORCE AND CATHETER STABILITY DURING MANUAL AND ROBOTIC PAROXYSMAL AF ABLATION The robot RWACA group uniquely demonstrates a signifi- cant negative correlation, suggesting that robotic RWACA tends to provide greater stability as CF rises when compared with a manual procedure, and is concordant with our previous analysis showing higher CF application patterns for this particular group. F Figure 1 CF vs catheter displacement between human and robotic L and R WACA. Box and whisker plots show 10th, 25th, 50th (median), 75th and 90th percentiles. Dotted lines indicates IQRs. CF groups of ,100 measurements were excluded. Figure 1 CF vs catheter displacement between human and robotic L and R WACA. Box and whisker plots show 10th, 25th, 50th (median), 75th and 90th percentiles. Dotted lines indicates IQRs. CF groups of ,100 measurements were excluded.
https://openalex.org/W3048247075
https://www.researchsquare.com/article/rs-16528/latest.pdf
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Spectrum of antibiotic resistant bacteria and fungi isolated from chronically infected wounds in a rural district hospital in Ghana
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Page 1/16 Spectrum of antibiotic resistant ba isolated from chronically infected district hospital in Ghana Ralf Krumkamp  Bernhard-Nocht-Institut fur Tropenmedizin Kwabena Oppong  Kumasi centre for collaborative research Benedikt Hogan  Bernhard-Nocht-Institut fur Tropenmedizin Ricardo Strauss  Bernhard-Nocht-Institut fur Tropenmedizin Hagen Frickmann  Bernhard-Nocht-Institut fur Tropenmedizin Charity Wiafe-Akenten  Kumasi centre for collaborative research Kennedy Boahen  Kumasi centre for collaborative research Volker Rickerts  Robert Koch Institut Ilka McCormick  Robert Koch Institut Uwe Groß  Institute for medical microbiology university medical centre Götting Marco Schulze  Institute for medical microbiology university medical centre götting Anna Jaeger  Bernhard-Nocht-Institut fur Tropenmedizin Ulrike Loderstädt  Bernhard-Nocht-Institut fur Tropenmedizin Nimako Sarpong  Kumasi centre for collaborative reserarch Ellis Owusu-Dabo  Kumasi centre for collaborative research Jürgen May  Bernhard-Nocht-Institut fur Tropenmedizin Spectrum of antibiotic resistant bacteria and fungi isolated from chronically infected wounds in a rural district hospital in Ghana Charity Wiafe-Akenten  Kumasi centre for collaborative research Charity Wiafe-Akenten  Kumasi centre for collaborative research Kennedy Boahen  Kumasi centre for collaborative research Ilka McCormick  Robert Koch Institut Research article Keywords: Ghana, Western Africa, epidemiology, chronic wound, infection, antibiotic resistance Posted Date: March 10th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-16528/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License DOI: https://doi.org/10.21203/rs.3.rs-16528/v1 DOI: https://doi.org/10.21203/rs.3.rs-16528/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 2/16 Abstract Background: Chronic infected wounds are generally difficult to manage and treatment can be particularly challenging in resource-limited settings where diagnostic testing is not readily available. In this study, the epidemiology of microbial pathogens in chronically infected wounds in rural Ghana was assessed to support therapeutic choices for physicians. Methods. Culture-based bacterial diagnostics including antimicrobial resistance testing were performed on samples collected from patients with chronic wounds at a hospital in Asante Akim North Municipality, Ghana. Fungal detection was performed by broad-range fungal PCR and sequencing of amplicons. Results: In total, 105 patients were enrolled in the study, from which 207 potential bacterial pathogens were isolated. Enterobacteriaceae (n=84, 41%) constituted the most frequently isolated group of pathogens. On species level, Pseudomonas aeruginosa (n=50, 24%) and Staphylococcus aureus (n=28, 14%) were predominant. High resistance rates were documented, comprising 29% methicillin resistance in Staphylococcus aureus as well as resistance to 3rd generation cephalosporins and fluoroquinolones in 33% and 58% of Enterobacteriaceae, respectively. One Pseudomonas aeruginosa strain with carbapenem resistance was identified. The most frequently detected fungi were Candida tropicalis. Conclusions: The pathogen distribution in chronic wounds in rural Ghana matched the internationally observed patterns with a predominance of Pseudomonas aeruginosa and Staphylococcus aureus. Very high resistance rates discourage antibiotic therapy but suggest an urgent need for microbiological diagnostic approaches, including antimicrobial resistance testing to guide the management of patients with chronic wounds in Ghana. Ethical Considerations The Committee on Human Research, Publications and Ethics, School of Medical Science, Kwame Nkrumah University of Science and Technology in Kumasi, Ghana, approved this study. Study participants were informed about the purpose of this study and the study procedures. Written informed consent was obtained before enrolment. Study site and study participants The study was conducted at the Outpatient Department of the APH, in the Asante Akim North Municipality of Ghana. Patients aged ≥ 15 years with an infected wound (at least one of the signs and symptoms of infection: purulent discharge from wound/pain or tenderness, localised swelling or redness/heat, loss of function (functio laesa)), which has failed to proceed through a reparative process over a period of one month, with or without antibiotic treatment, were eligible for enrolment. Excluded were patients with burn-, surgical- bite- or Buruli ulcer associated wounds in order to minimise associations of particular types of wounds with specific pathogens. Sample Collection For sample collection two sterile swabs (ESwab™, COPAN Diagnostics, Murrieta, USA), one with and one without transport medium, were used. Necrotic material was removed with a sterile cotton swab (CLASSIQSwab™, COPAN Diagnostics). For sample collection, the wound was cleaned with sterile 0.9% saline. Following this, the active part of the wound below the necrotic tissue at the edge of the wound and the wound base was swabbed. For this purpose, the swab was rolled deep into the wound. When there was more than one wound at the same location, the largest wound was sampled. Within six hours, the samples were transported in a cool box to the microbiology laboratory of the Kumasi Centre for Collaborative Research in Tropical Medicine, Ghana. Background Chronic wound infections related to injuries from daily life activities, e.g. farming are common medical problems in sub-Saharan Africa (SSA) [1]. Chronicity is often a result of an underlying medical condition such as diabetes, blood flow disorders or due to late presentation to the clinic. Long persisting infected wounds cause morbidity and suffering and are typically associated with large expenditures, e.g. on medication and affect economic productivity [2]. Besides bacteria, fungi may be part of polymicrobial communities on wounds. In addition, specific fungal pathogens may cause chronic skin infections, including mycetoma and chromoblastomycosis often manifesting as slow-healing wounds [3, 4]. In resource-limited countries with inadequate diagnostic facilities, the spectrum of microorganisms causing such infections and their antibiotic resistance patterns are only superficially understood [5–8]. Pathogen- specific treatment for severe wounds is difficult if the disease-causing agent(s) remain(s) unknown. In addition, treatment success is under serious threat due to the rise of antimicrobial resistances [9]. Poor quality medicine and (over)- use of antibiotics without being able to consider the antimicrobial sensitivity patterns of causative pathogens does not only lead to treatment failures but also foster the development of Page 3/16 Page 3/16 antibiotic resistance further. These limitations hamper specific treatment, which especially applies to chronic wounds, for which standard treatment strategies are poorly defined and ineffective. This study aims to investigate the microbial composition (both bacterial and fungal) of infected chronic wounds in adults presenting to the Agogo Presbyterian Hospital (APH) in rural Ghana. Fungal detection and identification DNA was extracted from the swab (without transport medium) within 12 hours according to manufacturer’s guidelines using the BIOstic Bacteremia DNA Isolation kit (QIAGEN, Hilden, Germany). DNA extracts were stored at -80 °C until polymerase chain reaction (PCR) amplification for clinically important fungi. Fungal DNA was amplified using a broad-range fungal PCR targeting the 28S rRNA gene using Eva Green with melt curve analysis in an ABI 7500 qPCR machine as described previously [10]. Samples were tested in duplicate. Positive fungal PCR was defined as amplification of DNA of identical melt curves in both duplicates with a threshold cycle below 45. Template controls (n = 8) were included in each run to document potential contamination with fungal DNA. Amplicons of PCR positive samples were sequenced by Sanger sequencing. Fungi were identified by BLAST search using Genbank. Identification of fungi required identity > 98% over the amplicon length. In addition, a qPCR assay targeting the human 18S rRNA was performed to document successful sampling and DNA extraction. Furthermore, potential inhibition of the PCR reaction was screened by an internal amplification control, amplifying an artificial plasmid. Bacterial detection and identification Bacterial detection and identification The swab with transport medium was streaked on Columbia agar enriched with 5% sheep blood, Columbia CNA agar with 5% sheep blood and MacConkey III (Oxoid, Hampshire, United Kingdom). Aerobic blood agar was incubated in CO2. MacConkey and CNA agar were incubated in normal atmosphere at 35–37 °C. All plates were incubated for 18–24 hours. CNA agar was incubated until positive or for a maximum period of five days. Bacterial strains were identified by colony morphology, Gram stain and standard biochemical Page 4/16 Page 4/16 methods and stored in microbanks at -80 °C until transportation on dry ice to the Bernhard Nocht Institute in Hamburg, Germany for further analysis. Environmental bacteria and bacteria belonging to the skin flora (e.g. coagulase-negative staphylococci, Micrococcus spp., coryneform bacteria and Bacillus spp. other than Bacillus anthracis) were classified as contaminants. At the Institute for Medical Microbiology in Göttingen, Germany, species identification was confirmed using the MALDI Biotyper 3.0 (Bruker Daltonics, Bremen, Germany). Antibiotic susceptibility testing Susceptibility to locally-available antibiotics was tested by the disk diffusion method and interpreted following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines v.6.0 (http://www.eucast.org). Quality control of susceptibility testing was performed according to EUCAST (QC table v.5). At the University Medical Centre, Göttingen, Germany antibiotic susceptibility testing was confirmed using the automated VITEK 2 system (bio-Mérieux, Marcy-l’Étoile, France). Quality control was performed with the following ATCC reference strains: Pseudomonas aeruginosa 27853, Staphylococcus aureus 29213 and Escherichia coli 25922. Statistical analyses Categorical variables were described using frequencies and their proportion and continuous variables using the median and the interquartile range (IQR). Prevalence ratios (PRs) along with their 95%-confidence intervals (CIs) were calculated to show associations between dichotomous variables. Missing values were excluded from the analysis; hence, in some calculations the denominator differs. All analyses were conducted using Stata Statistical Software 14 (StataCorp LLC, College Station, TX). Study group One hundred and five outpatients were enrolled between January 2016 and November 2016. Characteristics of study patients are summarised in Table 1. Study participants had a median age of 54 years (IQR: 36–69) and half of the patients (n = 49; 47%) were females. From 47 (45%) patients, underlying diseases were reported, with the most frequent being hypertension (n = 27; 26%) and diabetes mellitus (n = 22; 21%). The majority of wounds were located on the leg (n = 76; 72%) followed by the foot (n = 13; 12%). Most wounds were ≥ 7 months old (n = 48; 46%), followed by below 8 weeks (n = 33; 31%). Wounds had a median area size of 15 cm2 (IQR: 5–44). Patient’s wounds showed the following characteristics: pain (n = 97, 92%), indurated border (n = 56; 53%), presence of pus (n = 43; 41%), functio laesa (n = 34; 32%) and local swelling (n = 15; 14%). In 55 (53%) patients the grade of the wound was stage two or higher. Most common reported causes of the wound were cuts (n = 36; 34%) followed by abscesses/swollen tissues (n = 30; 29%), traumata/accidents (n = 12; 11%) and blisters (n = 11;10%). In 70 (67%) of the patients the wound was previously treated with antibiotics and in 38 (36%) with traditional medicine. Patients using herbal treatment were less likely to take antibiotics (PR = 0.7; 95%-CI: 0.5–1.0). Patients who used herbal treatment were more likely to have wounds for 9 weeks to 6 months (PR = 1.8; 95%-CI = 1.2–2.6). Table 1 Page 6/16 Table 1 Patient and wound characteristics for Gram-positive and Gram-negative bacteria. Results Page 5/16 Page 5/16 Bacterial isolates from chronic wounds In total, 207 potential bacterial pathogens were isolated from 105 wounds and polymicrobial infections were frequent. In 7 (7%) of the patients no bacterial pathogen was isolated. In 34 (32%) one, in 31 (30%) two, in 21 (20%) three and in 12 (11%) patients four, bacterial pathogens were isolated. Wounds ≥ 5 cm2 were more likely to carry multiple pathogens (PR = 1.5; 95%-CI: 1.0–2.3). The detected potential pathogens are listed in Table S1. The majority of isolates were Gram-negative (n = 143; 69%) bacteria. Most were Enterobacteriaceae (n = 84; 41%) and Pseudomonas aeruginosa (n = 50; 24%) followed by Staphylococcus aureus (n = 28; 14%). In general, the bacterial composition was comparable between mono- and polymicrobial infections (Fig. 1). The most frequently detected pathogen in mono- and polymicrobial infections was Pseudomonas aeruginosa. In patients with one or two isolates detected, Staphylococcus aureus was the second most common pathogen found. Patient and wound characteristics stratified by the presence or absence of Gram-negative bacteria are summarised in Table 1, indicating that characteristics were comparable between both groups. However, the size of wounds that contained Gram-positive bacteria were larger (median = 15 cm2; IQR: 5–44) compared to wounds from which only Gram-negative bacteria were isolated (median = 5 cm2; IQR: 5–15). Study group Characteristic All (N =  105) Gram-negative bacteria present (N = 85) Only Gram-positive bacteria (N = 20) Female sex [n (%)] 49 (47) 6 (30) 43 (51) Age (years) of patient [median (IQR)] 54 (36– 69) 49 (24–59) 56 (38–69) Previous antibiotic use [n (%)] 70 (67) 57 (67) 13 (65) Area size (cm2) of wound [median (IQR)] 15 (5– 44) 5 (5–15) 15 (5–44) Duration of wound [n (%)] < 8 weeks 9 weeks–6 months ≥ 7 months 33 (31) 24 (23) 48 (46) 28 (33) 20 (24) 37 (44) 5 (25) 4 (20) 11 (55) Location of wound [n (%)] leg/ankle foot other 76 (72) 13 (12) 16 (15) 63 (74) 10 (12) 12 (14) 13 (65) 3 (15) 4 (20) Aetiology of wound [n (%)] cut abscess/tissue swelling trauma/accidents other 36 (34) 30 (29) 12 (11) 27 (26) 29 (34) 25 (29) 10 (12) 21 (25) 7 (35) 5 (25) 2 (10) 6 (30) nt and wound characteristics for Gram-positive and Gram-negative bacteria. Page 6/16 Antibiotic susceptibility testing Antibiotic susceptibility results of the most common bacterial species using the VITEK 2 system are shown in Table 2 and Fig. 2. Among all Staphylococcus aureus, 29% (n/N = 8/28) were methicillin resistant Staphylococcus aureus (MRSA). Inducible clindamycin resistance was found in 1/28 (4%) Staphylococcus aureus isolate only. Extended-spectrum beta-lactamase (ESBL) producing Escherichia coli and Klebsiella pneumoniae were 33% (n/N = 4/12) and 24% (n/N = 5/21), respectively. Fluoroquinolone resistance was seen in 58% (n/N = 7/12) Escherichia coli, 14% (n/N = 3/21) Klebsiella pneumoniae and 9% (n/N = 4/47) Pseudomonas aeruginosa. Two of Klebsiella pneumoniae isolates and one Escherichia coli exhibited resistance to three antibiotic groups: penicillins, 3rd generation cephalosporin and fluoroquinolones. One Pseudomonas aeruginosa strain exhibited resistance to the same groups of antibiotics in addition to resistance to carbapenems. Antibiotic resistance profiles of Enterobacteriaceae and Pseudomonas aeruginosa are shown in Fig. 2. Page 7/16 Page 7/16 Table 2 Table 2 Antibiotic Resistance [n/N (%)] for selected bacterial species in chronic wound infections, Ghana, 2016 Isolate Enterococcus faecalis Escherichia coli Klebsiella pneumoniae Proteus mirabilis Staphylococcus aureus Streptococcus pyogenes GEN - 2/12 (17) 6/21 (29) 1/20 (5) 1/28 (4) - CIP - 7/12 (58) 3/21 (14) 1/20 (5) 0/28 (0) - CAZ - 3/12 (25) 5/21 (24) 0/21 (0) - - MER - 0/12 (0) 0/21 (0) 0/20 (0) - - PEN 12/12 (100) - - - 28/28 (100) 0/5 (0) ERY 12/12 (100) - - - 2/28 (7) 2/5 (40) CLI 12/12 (100) - - - 1/28 (4) 0/5 (0) SXT 12/12 (58) 11/12 (92) 10/21 (48) 9/21 (43) 11/28 (39) - TET 12/12 (100) - - 1/1 (100) 16/28 (57) - CHL 4/9 (44) 10/12 (83) 12/13 (92) 9/14 (64) 15/21 (71) 5/8 (63) AMP 0/12 (0) 12/12 (100) 21/21 (100) 19/21 (90) - 5/5 (100) SAM 0/12 (0) 12/12 (100) 21/21 (100) 19/21 (90) 8/28 (29) - CXM - 12/12 (100) 21/21 (100) 21/21 (100) 8/28 (29) - CRO - 3/11 (27) 4/21 (19) 0/19 (0) - - GEN, gentamicin; CIP, ciprofloxacin; CAZ, ceftazidime; MER, meropenem; PEN, penicillin; ERY, erythromycin; CLI, clindamycin; SXT, trimethoprim/sulfamethoxazole; TET, tetracycline; CHL, chloramphenicol; AMP, ampicillin; SAM, ampicillin/sulbactam; CXM, cefuroxime; CRO, ceftriaxone. Amplification of fungal DNA from chronic wound swabs In 23 (22%) wounds, fungal DNA was amplified by broad-range PCR. Most frequent fungi were Candida tropicalis (n = 6; 6%) and Candida spp. (n = 5; 5%). Antibiotic susceptibility testing A complete list of isolated fungi is presented in Table 3. Patients with multiple bacterial infections had a higher risk to be infected or colonized with a mycotic isolate (PR = 2.4; 95%-CI: 1.0–5.3). The broad-range PCR did not amplify DNA of typical fungal pathogens Amplification of fungal DNA from chronic wound swabs In 23 (22%) wounds, fungal DNA was amplified by broad-range PCR. Most frequent fungi were Candida tropicalis (n = 6; 6%) and Candida spp. (n = 5; 5%). A complete list of isolated fungi is presented in Table 3. Patients with multiple bacterial infections had a higher risk to be infected or colonized with a mycotic isolate (PR = 2.4; 95%-CI: 1.0–5.3). The broad-range PCR did not amplify DNA of typical fungal pathogens causing chronic skin infections including chromoblastomycosis and mycetoma. Page 8/16 Table 3 Fungal isolates from 23 (22%) patient samples tested by PCR Isolate Frequency (%) Candida tropicalis 6 (6) Candida spp. 5 (5) Candida mixed sequence 4 (4) Candida albicans 3 (3) Cladophiolophora spp. 1 (1) Fusarium solani 1 (1) Geotrichium candidum 1 (1) Purpureocillium/Acremonium 1 (1) Zygosaccaromyces spp. 1 (1) Table 3 Fungal isolates from 23 (22%) patient samples tested by PCR Table 3 Discussion In this study, we assessed the microbial composition of chronically infected wounds in rural Ghana. The bacterial spectrum seen was in line with findings from previous studies conducted in another region of Ghana, in Ethiopia and in Tanzania [1, 11–15]. Altogether, Gram-negative bacteria dominated quantitatively. Enterobacteriaceae, Pseudomonas aeruginosa and Staphylococcus aureus were the most frequently isolated organisms. Previous assessments both from America and Europe as well as from Africa confirm Staphylococcus aureus and Pseudomonas aeruginosa as the leading pathogens isolated from chronic wounds. Both species express virulence factors and surface proteins negatively affecting wound healing [1, 9, 16, 17]. Pathogenicity in chronic wounds is further increased by co-infections of Staphylococcus aureus and Pseudomonas aeruginosa [16]. Biofilms caused by Pseudomonas aeruginosa play important roles by maintaining chronic wound infections thus preventing healing. Such biofilms are also known to show high adherence to biological surfaces, a phenomenon which limits the sensitivity of swabbing-based sampling with subsequent culture- based diagnostic approaches in comparison to molecular biological approaches [18]. Consequently, it is likely that the true prevalence of Pseudomonas aeruginosa was even higher. Although Pseudomonas aeruginosa followed by Staphylococcus aureus were the most common bacteria identified on species level, the most frequent isolates were Enterobacteriaceae. The estimation of their clinical relevance is much more difficult. Although it is generally accepted that Enterobacteriaceae like Escherichia coli and others might play a role in wound infections [17, 19]. inhabitants of tropical or subtropical climate zones were shown to have high colonization rates of skin and mucous membranes with Page 9/16 Gram-negative rod-shaped bacteria [20]. High frequencies were also reported from patients, students, and health-care workers in Madagascar [21]. This makes the discrimination between wound colonization and wound infection challenging. From the less frequently isolated bacteria, beta-hemolytic streptococci including Streptococcus pyogenes are highly likely to be etiologically relevant for the assessed chronic wound infections [22], while enterococci are frequent colonizers of uncertain clinical relevance in wounds [23]. In mono- and polymicrobial infections, Pseudomonas aeruginosa was the most common bacterial specie. Others, including Enterobacteriaceae and enterococci were rarely observed in cases in which only one microorganism was isolated, stressing the likelihood that they are probably contamination flora or minor components of polymicrobial infections. The observed antimicrobial resistance patterns are of interest. The recorded 29% MRSA is in discordance with a Ghanaian study on wound infections from 2014, where no MRSA were identified from infected wounds [14]. Conclusions As in other studies elsewhere, Pseudomonas aeruginosa and Staphylococcus aureus were the most frequently detected isolates and thus the most important species to be considered by physicians for therapeutic decisions. In addition, high rates of antibiotic resistance are to be expected for isolates from chronic wounds in Ghana. This stresses the need for routine bacterial diagnostics including antimicrobial resistance testing prior to targeted antimicrobial therapy if antibiotic treatment is required and locally disinfecting or surgical procedures are considered insufficient. If routine bacteriology is impossible for logistical or infrastructural reasons, the observed lack of carbapenem-resistance suggests that antibiotic substances, which are available for intravenous application only, may still have good chances of showing clinical effect. However, decision-makers should be aware that empiric treatment further increases antibiotic resistance and is thus a less sustainable approach. In addition, high rates of antibiotic resistance are to be expected for isolates from chronic wounds in Ghana. This stresses the need for routine bacterial diagnostics including antimicrobial resistance testing prior to targeted antimicrobial therapy if antibiotic treatment is required and locally disinfecting or surgical procedures are considered insufficient. If routine bacteriology is impossible for logistical or infrastructural reasons, the observed lack of carbapenem-resistance suggests that antibiotic substances, which are available for intravenous application only, may still have good chances of showing clinical effect. However, decision-makers should be aware that empiric treatment further increases antibiotic resistance and is thus a less sustainable approach. Consent for publication Not applicable Competing interests The authors declare that they have no competing interests. The publication is based on research supported by institutional funds of the BNITM Discussion This suggests a considerable increase of MRSA within less than a decade; although it has to be considered that selection processes in chronic wounds after repeated antimicrobial therapy are likely. High frequencies of 3rd generation cephalosporin and fluoroquinolone resistance but absence of carbapenem resistance in Enterobacteriaceae isolated from wounds in Ghana has, in contrast, been described previously [14]. The increase of fluoroquinolone resistance in Ghana has recently been described [24]. significantly reducing the value of this group of antibiotics for treatment of severe infections. In addition, we found one carbapenem-resistant Pseudomonas aeruginosa. Carbapenem-resistance is still comparatively rare in Ghana: 2.9% in Gram-negative rod-shaped bacteria, predominantly in Pseudomonas aeruginosa and Acinetobacter baumannii [25]. In summary, the observed resistance patterns make any rational antimicrobial therapy challenging, indicating a need for routine diagnostics including antibiotic susceptibility testing. However, antibiotic resistance seen in bacterial isolates from chronic wounds is likely to overestimate the true resistance rate in clinical isolates, because selection processes due to repeated attempts of antibiotic treatment have to be considered as likely. In regards to fungi isolated from chronic wounds, information on the immune status of the patients was not assessed. Hence potential etiological relevance remains speculative. In general, fungal infections have been seen to cause skin infection including mycetoma that may manifest as chronic wounds after trauma [26]. Detection of causative fungal pathogens requires prolonged culture on fungus-specific media or demonstration of fungal elements in tissues. The performance of broad-range fungal PCR to amplify fungal DNA for the diagnosis of these infections has not been evaluated [27]. Our results suggest that the applied approach may be more likely to amplify DNA of yeasts that may be normal skin flora or microbial communities of chronic wounds. Specific PCR assays may be more sensitive for the amplification of specific fungal pathogens such as Madurella mycetomatis [26]. Limitations of this study include geographic restrictions of sampling, reducing the representativeness of the study for the whole of Ghana. In addition, swabbing of neighboring sites of intact skin to better differentiate infection from colonization [28] was not performed. Also, more sensitive molecular diagnostic methods were not applied, making it likely that several components of mixed infections will have been missed [18]. Also, despite their potential involvement in chronic wound infections, no anaerobic bacterial culture was performed. Page 10/16 Availability of data and materials The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate All participants were informed about the study’s purpose and procedures. Prior to sample collection, written informed consent was obtained. Ethical approval for the study was attained from the Committee on Human Research, Publications and Ethics, School of Medical Science, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana Acknowledgements We are grateful to all patients wo participated in this study and to the personnel at the Agogo Presbyterian Hospital. Without their efforts, this research study would not have been possible. Abbreviations APH: Agogo Presbyterian Hospital BNITM: Bernhard Nocht Institute for Tropical Medicine CIs: Confidence Intervals DNA: Deoxyribonucleic Acid DZIF: German Centre for Infection Research EUCAST: European Committee on Antimicrobial Susceptibility Testing IQR: Interquartile Range KCCR: Kumasi Centre for Collaborative Research in Tropical Medicine MRSA: Methicillin Resistant Staphylococcus aureus qPCR: Quantitative Polymerase Chain Reaction PRs: Prevalence Ratios PSM: Phenol-Soluble Modulins PVL: Panton Valentine Leukocidin RKI: Robert Koch Institute SSA: sub-Saharan Africa References 1. Kumburu HH, Sonda T, Mmbaga BT, Alifrangis M, Lund O, Kibiki G, A infections, aetiological agents and antimicrobial resistance at a terti Authors’ contributions DD, RK, JM and BH designed and managed this study. HF, UL, RS supported writing and revising this manuscript. KYB, CWA; UG, MS were involved in bacterial diagnostics. IMS and VR were carrying out fungal Page 11/16 diagnostics. AJ designed the database for this study. NS, EOD and KO supported planning and managing the study in Ghana. All authors critically reviewed and approved the final manuscript. diagnostics. AJ designed the database for this study. NS, EOD and KO supported planning and managing the study in Ghana. All authors critically reviewed and approved the final manuscript. diagnostics. AJ designed the database for this study. NS, EOD and KO supported planning and managing the study in Ghana. All authors critically reviewed and approved the final manuscript. Abbreviations APH: Agogo Presbyterian Hospital SSA: sub-Saharan Africa References 1. Kumburu HH, Sonda T, Mmbaga BT, Alifrangis M, Lund O, Kibiki G, Aarestrup FM. Patterns of infections, aetiological agents and antimicrobial resistance at a tertiary care hospital in northern Tanzania. Trop Med Int Health 2017; 22: 454-64. 1. Kumburu HH, Sonda T, Mmbaga BT, Alifrangis M, Lund O, Kibiki G, Aarestrup FM. 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Micheel V, Hogan B, Rakotoarivelo RA, Rakotozandrindrainy R, Razafimanatsoa F, Razafindrabe T, et al. Identification of nasal colonization with β-lactamase-producing Enterobacteriaceae in patients, health care workers and students in Madagascar. Eur J Microbiol Immunol (Bp) 2015; 5: 116-25. 22. Rantala S. Streptococcus dysgalactiae subsp. equisimilis bacteremia: an emerging infection. Eur J Clin Microbiol Infect Dis 2014; 33: 1303-10. 23. Frickmann H, Köller K, Veil I, Weise M, Ludyga A, Schwarz NG, Warnke P, Podbielski A. On the Role of Enterococci in the Bloodstream: Results of a Single-Center, Retrospective, Observational Study at a German University Hospital. Eur J Microbiol Immunol (Bp) 2017; 7: 284-95. 24. Aldrich C, Hartman H, Feasey N, Chattaway MA, Dekker D, Al-Emran HM, Larkin L, McCormick J, Sarpong N, Le Hello S, Adu-Sarkodie Y, Panzner U, Park SE, Im J, Marks F, May J, Dallman TJ, Eibach D. Emergence of phylogenetically diverse and fluoroquinolone resistant Salmonella Enteritidis as a cause of invasive nontyphoidal Salmonella disease in Ghana. PLoS Negl Trop Dis 2019; 13: e0007485. 25. Codjoe FS, Donkor ES, Smith TJ, Miller K. Phenotypic and Genotypic Characterization of Carbapenem- Resistant Gram-Negative Bacilli Pathogens from Hospitals in Ghana. Microb Drug Resist 2019; 25: 1449-1457. 26. Zijlstra EE, van de Sande WWJ, Welsh O, Mahgoub ES, Goodfellow M, Fahal AH. Mycetoma: a unique neglected tropical disease. Lancet Infect Dis 2016; 16: 100-112. 26. Zijlstra EE, van de Sande WWJ, Welsh O, Mahgoub ES, Goodfellow M, Fahal AH. Mycetoma: a unique neglected tropical disease. Lancet Infect Dis 2016; 16: 100-112. 27. Frickmann H, Loderstaedt U, Racz P, Tenner-Racz K, Eggert P, Haeupler A, Bialek R, Hagen RM. Detection of tropical fungi in formalin-fixed, paraffin-embedded tissue: still an indication for microscopy in times of sequence-based diagnosis? Biomed Res Int 2015a; 2015: 938721. 27. Frickmann H, Loderstaedt U, Racz P, Tenner-Racz K, Eggert P, Haeupler A, Bialek R, Hagen RM. Detection of tropical fungi in formalin-fixed, paraffin-embedded tissue: still an indication for microscopy in times of sequence-based diagnosis? Biomed Res Int 2015a; 2015: 938721. 28. Page 12/16 Yun HC, Murray CK, Roop SA, Hospenthal DR, Gourdine E, Dooley DP. Bacteria recovered from patients admitted to a deployed U.S. military hospital in Baghdad, Iraq. Mil Med 2006; 171: 821-5. 28. Yun HC, Murray CK, Roop SA, Hospenthal DR, Gourdine E, Dooley DP. Bacteria recovered from patients admitted to a deployed U.S. military hospital in Baghdad, Iraq. Mil Med 2006; 171: 821-5. Figures Page 14/16 Figure 1 Proportion of Enterobacteriaceae and Pseudomonas aeruginosa resistant to locally available antibiotics. Figure 1 Proportion of Enterobacteriaceae and Pseudomonas aeruginosa resistant to locally available a Figure 2 Pathogen distribution in mono- and polymicrobial bacterial wound infection Pathogen distribution in mono- and polymicrobial bacterial wound infection Figure 1 Proportion of Enterobacteriaceae and Pseudomonas aeruginosa resistant to locally available antibiotics. Proportion of Enterobacteriaceae and Pseudomonas aeruginosa resistant to locally available antibiotics. Proportion of Enterobacteriaceae and Pseudomonas aeruginosa resistant to locally available antibiotics. Page 15/16 Page 15/16 Figure 2 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. This is a list of supplementary files associated with this preprint. Click to download. coinf.pdf rresistance.pdf Page 16/16 Page 16/16
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SNP Linkage Analysis and Whole Exome Sequencing Identify a Novel POU4F3 Mutation in Autosomal Dominant Late-Onset Nonsyndromic Hearing Loss (DFNA15)
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Abstract Competing Interests: The authors have declared that no competing interests exist. * E-mail: kimjw@skku.edu Competing Interests: The authors have declared that no competing interests exist. * E-mail: kimjw@skku.edu . These authors contributed equally to this work. . These authors contributed equally to this work. Hee-Jin Kim1., Hong-Hee Won2., Kyoung-Jin Park2, Su Hanka Venselaar4, Gert Vriend4, Jong-Won Kim1* 1 Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea, 2 Samsung Biomedical Research Institute, Samsung Medical Center, Seoul, Korea, 3 Department of Otorhinolaryngology-Head and Neck Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea, 4 Centre for Molecular and Biomolecular Informatics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands SNP Linkage Analysis and Whole Exome Sequencing Identify a Novel POU4F3 Mutation in Autosomal Dominant Late-Onset Nonsyndromic Hearing Loss (DFNA15) Hee-Jin Kim1., Hong-Hee Won2., Kyoung-Jin Park2, Sung Hwa Hong3, Chang-Seok Ki1, Sang Sun Cho2, Hanka Venselaar4, Gert Vriend4, Jong-Won Kim1* Abstract Autosomal dominant non-syndromic hearing loss (AD-NSHL) is one of the most common genetic diseases in human and is well-known for the considerable genetic heterogeneity. In this study, we utilized whole exome sequencing (WES) and linkage analysis for direct genetic diagnosis in AD-NSHL. The Korean family had typical AD-NSHL running over 6 generations. Linkage analysis was performed by using genome-wide single nucleotide polymorphism (SNP) chip and pinpointed a genomic region on 5q31 with a significant linkage signal. Sequential filtering of variants obtained from WES, application of the linkage region, bioinformatic analyses, and Sanger sequencing validation identified a novel missense mutation Arg326Lys (c.977G.A) in the POU homeodomain of the POU4F3 gene as the candidate disease-causing mutation in the family. POU4F3 is a known disease gene causing AD-HSLH (DFNA15) described in 5 unrelated families until now each with a unique mutation. Arg326Lys was the first missense mutation affecting the 3rd alpha helix of the POU homeodomain harboring a bipartite nuclear localization signal sequence. The phenotype findings in our family further supported previously noted intrafamilial and interfamilial variability of DFNA15. This study demonstrated that WES in combination with linkage analysis utilizing bi-allelic SNP markers successfully identified the disease locus and causative mutation in AD-NSHL. ation: Kim H-J, Won H-H, Park K-J, Hong SH, Ki C-S, et al. (2013) SNP Linkage Analysis and Whole Exome Sequencing Identify a Nov tosomal Dominant Late-Onset Nonsyndromic Hearing Loss (DFNA15). PLoS ONE 8(11): e79063. doi:10.1371/journal.pone.0079063 Editor: Andrew Dewan, Yale School of Public Health, United States of America Editor: Andrew Dewan, Yale School of Public Health, United States of America Received June 28, 2013; Accepted September 24, 2013; Published November 18, 2013 Copyright:  2013 Kim 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. Copyright:  2013 Kim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by a grant from the Korea Health Technology R&D Project, Ministry of Health & Welfare, the Republic of Korea (A120030). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. November 2013 | Volume 8 | Issue 11 | e79063 Proband and Pedigree The detailed family history revealed a pedigree of a large Korean family (6 generations) with NSHL indicating the pattern of autosomal dominant inheritance (Figure 1A and Table S1). The age of onset of hearing loss in affected individuals ranged from early 10 s to late 50 s. No individual with hearing loss had syndromic features. PTA was obtained from 4 affected individuals including the proband (IV-9, IV-13, IV-16 and IV-20) in the family, which demonstrated bilateral minimal to moderate sensorineural hearing loss with flat to gently downward-sloping audiograms (Figure 1B). Peripheral blood samples were collected from total 34 individuals including the proband (indicated with an asterisk on the right shoulder of the symbols in Figure 1A). SNP Genotyping and Linkage Analysis yp g g y Genome-wide SNP genotyping was performed on DNA samples from 21 family members including the proband for linkage analysis (11 were affected and 10 were unaffected). The Illumina’s Linkage IV B Genetic Map v2 RevD panel was used for genome-wide SNP scanning (Illumina Inc. San Diego, CA, USA). This panel includes 6,008 SNP markers distributed evenly across the genome. The genetic map position of each SNP in the panel was determined by linear interpolation using NCBI build 35physical map position and a high-resolution STR genetic deCODE map. The mean and median intervals between markers were 488 Kb (0.62 cM) and 315 Kb (0.38 cM), respectively. Prior to genotyping, the yields of pure double strand genomic DNA samples were determined by using the Quant-iTTM PicoGreenH dsDNA Assay Kit (Eugene, Oregon, USA). Samples were then normalized to 50 ng/ul. Genotyping reactions were performed using Illumina Bead-Station kit reagents and protocols. The normalized genomic DNA (5 ul) from each sample was used as a template for Illumina GoldengateH Genotype Assays. SNP arrays were scanned using Illumina BeadArray Reader (Illumina Inc. San Diego, CA, USA) under the Illumina BeadStation 500G system version 2.3. For linkage analysis, we first excluded SNPs with a Mendelian inheritance error by using PedCheck 1.1 [8]. The Hardy-Weinberg equilibrium (HWE) test (P-value,0.01) was performed by using PEDSTATS 0.6.3 [9]. Linkage scores were calculated by MERLIN 1.1.2 [10]. Both muti-point parametric and nonparametric analyses were performed to yield LOD sores and NPL Z scores, respectively. In the parametric analysis, the disease allele frequency was set to 0.0001% and penetrance with 0, 1 and 2 copies of the disease allele was set to 0.01%, 100% and 100%, respectively. Haplotypes of the region of interest (peak linkage score) in the family were reconstructed by using PHASE 2.1.1 [11]. Introduction polymorphisms (SNPs), typically bi-allelic markers, have advan- tages in its abundance in the human genome and ease of typing with a high throughput. Recent advances in sequencing technologies have enabled us to capture variants on a whole- exome or whole-genome scale. In particular, sequencing of the whole exome (whole exome sequencing [WES]) of selected individuals in an affected pedigree holds a promise for disease gene discovery in Mendelian disorders [6,7]. However, the abundance of variants across genome or exome makes the selection of ‘‘disease-causing’’ mutations still challenging. Thus, narrowing down the genomic area in linkage with the disease significantly increases the power of WES in disease mutation identification. Hearing loss is one of the most common hereditary diseases in human, and more than 70% of the cases are nonsyndromic (NSHL) [1,2]. Most postlingual (late-onset) NSHL have an autosomal dominant pattern of inheritance (AD-NSHL), while prelingual (early-onset) NSHL is typically autosomal recessive (AR-NSHL) [3]. AD-NSHL represents one of the examples of extreme genetic heterogeneity in human with a growing list of causative genes particularly by virtue of the recent advances in genomics technology [4,5]. A total of 27 genes from 64 loci (DFNA 1–64) and 41 genes from 95 loci and (DFNB1–95) have been identified for AD-NSHL and AR-NSHL, respectively (Hereditary Hearing Loss website; http://hereditaryhearingloss. org; last access in May 2013), which poses a big challenge to direct molecular genetic diagnosis of NSHL by conventional targeted gene analyses. In this study, we investigated on the deafness mutation in a large Korean family with AD-NSHL by a combination approach of linkage analysis using SNP chip and WES. As a result, we successfully identified a novel mutation in DFNA15 in the family. Linkage analysis is one of the powerful approaches for the identification of disease-causing genes, and single nucleotide November 2013 | Volume 8 | Issue 11 | e79063 1 PLOS ONE | www.plosone.org Linkage and Exome Analyses for Diagnosis of DFNA15 variants and insertion-deletion variants were detected by the SAMtools [13]. Linkage Analysis Total 215 SNPs were excluded from linkage analyses due to Mendelian inheritance error. No markers failed the HWE test. The NPL analysis revealed 4 genomic loci with an NPL Z score .3. On parametric linkage analysis, we first obtained the LOD score without including 4 individuals in the generation VI (VI:3, VI:4, VI:6 and VI:7) whose affected status was ambiguous due to the young age. As a result, a high LOD score (2.387) was observed in the genomic region of 5q31, while the other 3 loci with NPL Z score .3 demonstrated no significant LOD score (Table 1). For those young 4 individuals (VI:3, VI:4, VI:6 and VI:7), we performed a simulation study by labeling them as unaffected or affected. The results showed that the LOD score was further increased to 3.006 when they were assigned as ‘‘unaffected’’, while the score decreased to 2.408 when they were assigned as ‘‘unknown phenotype’’. The LOD score was ,1 (loss of linkage signal) when at least one individual among them was set as ‘‘affected’’. The haplotypes reconstructed from 26 SNPs with an NPL Z score .3 revealed a single haplotype (#2) segregating with the hearing loss phenotype in the family (Table S2), including Sanger Sequencing Validation and Bioinformatic Analyses Written informed consent was obtained from all participating individuals, and the study protocol was approved by the institutional review board at the Samsung Medical Center, Seoul, Korea. y We performed Sanger sequencing for candidate variants from WES in 4 individuals (IV:3, IV:17, V:1 and V:5) and also in the remaining family members as indicated. Functional prediction, conservation and allele frequency in the 1000 Genomes Project and the Exome Sequencing Project 6500 data of missense variants were annotated using dbNSFP [14]. Functional effects of missense variants were predicted by five different programs, SIFT, PolyPhen-2, LRT, MutationTaster and MutationAssesor. In addition, sequence conservation of candidate variants was estimated by Genomic Evolutionary Rate Profiling++ (GERP) Rejected Substitution and conservation based on 29 mammals genomes. For molecular modeling, we used the structure of POU3F1 (2xsd) as a template to build a homology model for POU4F3 because the 3D-structure of POU4F3 is not yet experimentally solved. Modeling and subsequent analysis were done using an automatic script in the YASARA & WHAT IF Twinset with standard parameters [15,16]. The effect of the mutation on the protein stability was estimated using the FoldX plugin in YASARA [16]. Control studies were performed by screening variants of interest involving DNA samples from 50 control individuals of Korean descent aged .60 years who had normal hearing by PTA. Proband and Audiometric Analyses The proband (IV-20) was a 45-year-old Korean woman with NSHL since 21 years of age. Her family history revealed NSHL with an autosomal dominant inheritance. The molecular genetic test for the GJB2 gene revealed no mutations. Audiometric analyses were performed in the proband and participating family members. In particular, pure tone audiograms (PTA) were obtained in each individual to determine hearing thresholds levels (dB) of air and bone conduction at frequencies ranging from 250 to 8,000 Hz on right and left ears independently, according to the standard protocols. Peripheral blood samples were collected from the proband and family members participating in the genetic diagnosis with given written informed consent. Whole Exome Sequencing Four individuals in the family were selected for WES (3 affected including family member and 1 unaffected; IV:3, IV:17, V:1 and V:5 in the pedigree Figure 1A). The 64 million bases of exome were targeted by NimbleGen SeqCap EZ Human Exome Library v3.0 and sequenced by Illumina HiSeq. The 101-base-pair sequence reads were mapped to UCSC hg19 reference genome by using the Burrows-Wheeler Aligner [12]. Single nucleotide November 2013 | Volume 8 | Issue 11 | e79063 PLOS ONE | www.plosone.org 2 Figure 1. A large Korean family with autosomal dominant late-onset nonsyndromic hearing loss. (A) The pedigree with the proband (IV- 20) indicated by an arrow. Individuals who participated in the molecular genetic diagnosis are marked with an asterisk on the right shoulder of symbols. Whole exome sequencing was performed in 4 individuals (IV:3, IV:17, V:1 and V:5). The genotypes at theArg326Lys mutation site of POU4F3are presented for each individual (mutant allele ‘‘A’’ in red). (B) Audiograms of 4 affected individuals in the family including the proband. doi:10.1371/journal.pone.0079063.g001 Linkage and Exome Analyses for Diagnosis of DFNA15 Linkage and Exome Analyses for Diagnosis of DFNA15 Figure 1. A large Korean family with autosomal dominant late-onset nonsyndromic hearing loss. (A) The pedigree with the proband (IV- 20) indicated by an arrow. Individuals who participated in the molecular genetic diagnosis are marked with an asterisk on the right shoulder of symbols. Whole exome sequencing was performed in 4 individuals (IV:3, IV:17, V:1 and V:5). The genotypes at theArg326Lys mutation site of POU4F3are presented for each individual (mutant allele ‘‘A’’ in red). (B) Audiograms of 4 affected individuals in the family including the proband. doi:10.1371/journal.pone.0079063.g001 Figure 1. A large Korean family with autosomal dominant late-onset nonsyndromic hearing loss. (A) The pedigree with the proband (IV- 20) indicated by an arrow. Individuals who participated in the molecular genetic diagnosis are marked with an asterisk on the right shoulder of symbols. Whole exome sequencing was performed in 4 individuals (IV:3, IV:17, V:1 and V:5). The genotypes at theArg326Lys mutation site of POU4F3are presented for each individual (mutant allele ‘‘A’’ in red). (B) Audiograms of 4 affected individuals in the family including the proband. doi:10.1371/journal.pone.0079063.g001 November 2013 | Volume 8 | Issue 11 | e79063 PLOS ONE | www.plosone.org 3 Linkage and Exome Analyses for Diagnosis of DFNA15 Table 1. Chromosome regions with nonparametric linkage Z score (NPL Z).3.0. Whole Exome Sequencing and Mutation Identification q g We obtained 84.3%290.6% of the target exome with coverage of at least 10 sequence reads. A total of 153,611variants were discovered in 4 individuals (IV:3, IV:17, V:1 and V:5), and 123,135 variants thereof passed the standard variant quality control (QC). Among the passed variants, 3,395 were shown to be compatible with the phenotype filter (all 3 affected individuals were heterozygous for variants, while the other control individual was homozygous for the reference genotypes) (Table S1). Among those 3,395 variants, 21 non-synonymous SNPs were coincided with the region of the linkage peak on 5q31, and 5 of them had an allele frequency of ,1% on the variation data from the 1000 Genomes Project and Exome Sequencing Project 6500 (Table S3). Among the 5 rare variants, a missense variation Arg326Lys (c.977G.A) was occurring in the POU4F3 gene (POU domain, class 4, transcription factor 3; MIM# 602460), a previously known gene causing AD-NSHL (DFNA15). The affected Arg326 residue was shown to be evolutionarily conserved across species, and the missense change Arg326Lys was predicted to be deleterious by all 5 algorithms for functional prediction (Table 2). On the other hand, 4 missense variants in other genes were predicted to be benign. Sanger sequencing confirmed the WES findings in all 4 individuals (3 affected individuals were heterozygous for Arg326Lys [G/A] and 1 unaffected individual was homozygous Whole Exome Sequencing Chromosome region Peak SNP Position (cM)a Position (Mb) NPL Z LODb Peak region (cM) Peak region (Mb, hg18) 5p13,p14 rs2034586 48.70 30.08 3.66 28.99 16.38260.61 6.12238.07 5q31 rs1016344 136.60 134.60 3.66 3.01 135.862152.10 (135.862165.02)c 133.442148.71 (133.442160.43)c 10p15 rs713588 17.89 5.93 3.58 29.27 4.44232.09 1.96213.10 12q22,q23 rs1030048 108.27 96.16 3.73 25.98 103.492112.36 92.21299.96 aGenetic distance from the p-terminus according to the deCODE high-resolution map. bScores obtained on simulation analyses yielding the maximum value on 5q31 (see text for detail). cThe peak region of 5q31 in parenthesis means the interval corresponding to the region with LOD .3.0. doi:10.1371/journal.pone.0079063.t001 aGenetic distance from the p-terminus according to the deCODE high-resolution map. bScores obtained on simulation analyses yielding the maximum value on 5q31 (see text for detail). cThe peak region of 5q31 in parenthesis means the interval corresponding to the region with LOD .3.0. doi:10.1371/journal.pone.0079063.t001 individuals VI:3, VI:4, VI:6 and VI:7 whose haplotypes were assigned as ‘‘unaffected’’. individuals VI:3, VI:4, VI:6 and VI:7 whose haplotypes were assigned as ‘‘unaffected’’. for the reference genotype [G/G]). The sequencing analyses targeting Arg326 of POU4F3 in other family members confirmed that the mutant genotype was running together with the risk haplotype and was completely segregating with the disease phenotype (Figure 1A). The molecular modeling analyses demonstrated that the Arg326Lys mutation was predicted to destabilize the 3rd alpha-helix structure of the POU homeodomain of the protein and thereby reduce the proper protein-DNA interactions (Figures 2A and 2B). Lastly, the Arg326Lys variant was not observed in a control population aged .60 years with normal hearing (0/100 control chromosomes). Discussion The POU4F3 protein has 2 DNA binding domains, POU-specific domain and POU homeodomain. In particular, the POU homeodomain contains 2 functional nuclear localization signal sequences, monopartite (amino acids 274–278) and bipartite (amino acids 314–331 forming the 3rd alpha helix) [26]. The missense mutations Leu223Pro and Glu232Lys affected the POU- specific domain. Following the previously reported mutation number of variants obtained from WES pinpointed a missense variant Arg326Lys in POU4F3 on 5q31, a previously known NSHL gene, as a strong candidate (Table 2). The variant was running with the risk haplotype ht-2 reconstructed from SNPs with high LOD scores from linkage analysis (Figure 1). Based on the further evidence from Sanger sequencing and bioinformatic analyses, we concluded that Arg326Lys of POU4F3 on 5q31 was the candidate causative mutation. On molecular modeling analysis, the missense mutation was predicted to result in a deficiency in the proper protein-DNA interaction of the protein by destabilizing the 3rd alpha helix of the POU homeodomain. number of variants obtained from WES pinpointed a missense variant Arg326Lys in POU4F3 on 5q31, a previously known NSHL gene, as a strong candidate (Table 2). The variant was running with the risk haplotype ht-2 reconstructed from SNPs with high LOD scores from linkage analysis (Figure 1). Based on the further evidence from Sanger sequencing and bioinformatic analyses, we concluded that Arg326Lys of POU4F3 on 5q31 was the candidate causative mutation. On molecular modeling analysis, the missense mutation was predicted to result in a deficiency in the proper protein-DNA interaction of the protein by destabilizing the 3rd alpha helix of the POU homeodomain. The POU4F3 (POU domain, class 4, transcription factor 3) gene on 5q31 (DFNA15) is one of the two genes belonging to the superfamily of POU domain transcription factors that cause NSHL, along with POU3F4 on Xq21.1 (DFN3). It was first identified as a causative gene of AD-NSHL in a large Israeli Jewish family by linkage analysis [19]. POU4F3 was an excellent candidate gene because targeted deletion of both Pou5f3 alleles in mice had been reported to result in complete deafness [20,21]. Indeed, the POU4F3 protein is a member of the POU family transcription factors and is critical for the maintenance of inner ear hair cells. Following the first report, 4 additional families with Table 3. Genetics and phenotypes of DFNA15 in previously reported 5 families and in the Korean family in this report. No. Discussion The diagnosis of AD-NSHL is typically made based on the phenotype and the inheritance pattern within the family affected, and direct identification of the genetic background is not feasible due to the large number of disease genes/DNFA loci. In this study, we demonstrated that the molecular genetic diagnosis was successful in a large Korean family with DFNA by utilizing a combination approach of linkage analysis using genome-wide SNP markers and WES. The linkage analyses uncovered a 27.1 Mb region on the 5q31 band, which is one of the most gene-rich genomic segments in human, and several known disease loci (genes) have been previously identified on 5q31 in linkage with hereditary HL including DFNA1 (DIAPH1), DFNA15 (POU4F3), DFNA42, and DFNA54 [17–19]. Sequential filtering of the large Figure 2. Molecular modeling of the POU4F3 protein constructed by the 2xsd protein as the template. (A) Overview of the modeled POU4F3 domain. The protein chain is shown in grey using a ribbon representation, the position of the mutated residue is indicated in green, the DNA is shown as a ball-and-stick model and colored yellow. (B) A close-up view of the mutated residue. The side chains of the wild-type and the mutant residue are shown in green and red, respectively. The figures show that the wild-type Arginine residue interacts with the phosphate backbone of the DNA, while the mutant Lysine residue is slightly shorter than Arginine and cannot make the same interactions with DNA. doi:10.1371/journal.pone.0079063.g002 Figure 2. Molecular modeling of the POU4F3 protein constructed by the 2xsd protein as the template. (A) Overview of the modeled POU4F3 domain. The protein chain is shown in grey using a ribbon representation, the position of the mutated residue is indicated in green, the DNA is shown as a ball-and-stick model and colored yellow. (B) A close-up view of the mutated residue. The side chains of the wild-type and the mutant residue are shown in green and red, respectively. The figures show that the wild-type Arginine residue interacts with the phosphate backbone of the DNA, while the mutant Lysine residue is slightly shorter than Arginine and cannot make the same interactions with DNA. doi:10.1371/journal.pone.0079063.g002 November 2013 | Volume 8 | Issue 11 | e79063 PLOS ONE | www.plosone.org 4 Linkage and Exome Analyses for Diagnosis of DFNA15 Table 2. Five rare missense mutations in the 5q31 linkage region. Discussion Chr Position (bp, hg19) Reference allele Mutant allele Reference aa Mutant aa Gene Functional predictiona GERP+ +RSb 29way logOddsc 1000G ALL_AF 1000G ASN_AF ESP AA_AF ESP EA_AF 5 145719967 G A R K POU4F3 +++++ 4.62 16.40 0 0 0 0 5 140222014 G A A T PCDHA8 ++––– 2.66 12.17 0 0 0 0 5 139189261 A G N S PSD2 –+––– 3.99 9.55 0.0005 0.0017 0 0.0001 5 137548913 C T R Q CDC23 ––––– 4.13 9.63 0 0 0 0 5 140557626 G A S N PCDHB8 ––U –– 0.10 2.27 0.0014 0.0052 0 0 Abbreviations: Chr, chromosome; aa, amino acid; ALL, all populations; AF, allele frequency; ASN, Asian; AA, African American; EA, European American. aFunctional effects predicted by SIFT, PolyPhen-2, LRT, MutationTaster and MutationAssesor in order. Deleterious effect was denoted as+, neutral effect as –, and unpredicted effect as U. bGenomic Evolutionary Rate Profiling++ Rejected Substitution score. The larger the score, the more conserved the site. cConservation score is calculated based on 29 mammals genomes. The larger the score, the more conserved the site. doi:10.1371/journal.pone.0079063.t002 Abbreviations: Chr, chromosome; aa, amino acid; ALL, all populations; AF, allele frequency; ASN, Asian; AA, African American; EA, European American. aFunctional effects predicted by SIFT, PolyPhen-2, LRT, MutationTaster and MutationAssesor in order. Deleterious effect was denoted as+, neutral effect as –, and unpredicted effect as U. bGenomic Evolutionary Rate Profiling++ Rejected Substitution score. The larger the score, the more conserved the site. cConservation score is calculated based on 29 mammals genomes. The larger the score, the more conserved the site. doi:10.1371/journal.pone.0079063.t002 NSHL from POU4F3 mutations have been reported in the literature (Table 3) [22]. Collin et al. reported a missense mutation (c.865C.T, p.Leu289Phe) in a large Dutch family by linkage study using short tandem repeat (STR) markers and candidate gene sequencing [23]. They additionally found another family with DFNA15 from a different mutation (c.668T.C, p.Leu223Pro) by screening 30 unrelated families by targeted sequencing of POU4F3. The other 2 patients with DFNA15 were Korean families. By screening for DFNA15 in 42 unrelated Korean families with AD-NSHL by sequencing analyses of POU4F3, Lee et al. uncovered a family with a frameshift mutation by deletion of 14 nucleotides (c.662_675del14, p.Gly221Glufs*77) [24]. Another Korean family with DFNA15 from a missense mutation in POU4F3 (c.694G.A, p.Glu232Lys) was reported by targeted massive parallel sequencing of 80 genes [25]. Linkage and Exome Analyses for Diagnosis of DFNA15 Linkage and Exome Analyses for Diagnosis of DFNA15 Leu289Phe, Arg326Lys identified in our patient was the 2nd missense mutation in the POU homeodomain. Of note, Leu289Phe was located in the 1st alpha-helix, and Arg326Lys was the first missense mutation affecting the 3rd alpha-helix of the POU homeodomain, which has the bipartite nuclear localization signal. In line with our observations from molecular modeling analyses, a previous bioinformatic study on the missense mutation affecting the Arg329 residue of POU3F4 (Arg329Pro), which corresponds to the Arg325 residue of POU4F3 mutated in our family, demonstrated a detrimental functional consequence of the mutation on the 3rd alpha helix structure [27]. The precise molecular pathophysiological mechanisms would be different between POU3F4- and POU4F3- related deafness in speculation of the different modes of inheritance (X-linked recessive vs autosomal dominant, respectively) [27]. Collectively, the Arg326Lys mutation of POU4F3 in our family was predicted to exert its mutational effects by a combination of defects in the stability of its tertiary structure, DNA binding ability, transcrip- tional activity, and the nuclear localization of the protein. As for the genotype-phenotype correlations in DFNA15, the evidence collected from previously reported 5 families, albeit limited in number, have demonstrated late-onset bilateral progressive hearing loss with significant intra- and interfamilial variabilities in terms of the age of onset and severity of disease and the configuration of audiometric findings (Table 3) [19,23–25,28]. In line with this observation, the family in this report also demonstrated a wide range of onset of hearing loss (early 10 s to late 50 s). The audiograms obtained from 4 affected individuals also showed sensorineural hearing loss with an intrafamilial variability (Figure 1B). Further investigation is needed to delineate the precise molecular pathophysiological consequence of this variant in the hearing function. successful identification of novel mutations in known DFNA genes [33,34]. In particular, the study by Park et al. on DFNA20/26 (ACTG1 mutation) pinpointed the causative mutation from the list of variants obtained from WES utilizing linkage information from SNP markers as in our study [34]. Also in our family, we could decrease the number of functional candidate variants from 444 (genomewide) to 21 by applying the linkage region (5q31) (Table S3). Collectively, it is anticipated that WES in combination of linkage information can be successfully utilized in elucidating the genetic background of AD-NSHL. Acknowledgments The authors thank the proband and her family members who participated in this study. Table S2 Haplotypes in the linkage region on 5q31. (DOCX) Recently, the identification of disease-causing genes and mutations has been accelerated by the unprecedented advances and growing availability of genome research technologies includ- ing massively parallel sequencing. However, how to select and determine which variant (s) is (are) the disease-causing mutation (s) in the patient/family under investigation is a big challenge due to the large number of variants obtained from genome or exome sequencing. In order to cope with this challenge, linkage information is critically useful to narrow down the disease locus and thereby to decrease the number of candidate variants, utilizing identity-by-descent information [29]. In the field of hearing genetics, there also have been reports on novel disease genes and mutations by utilizing WES. As in other disease entities, the application of WES is more robust in AR-NSHL (DFNB) especially in consanguineous families with or without homozygos- ity mapping by SNP-array [30–32]. On the other hand, the determination of disease-causing gene/mutation is more challeng- ing in AD-NSHL, and a couple of recent studies reported on Table S3 Filtering of variants obtained from whole exome sequencing of 3 affected and 1 unaffected individuals. (DOCX) Author Contributions Conceived and designed the experiments: HJK HHW JWK. Performed the experiments: SSC. Analyzed the data: HJK HHW KJP CSK SSC HV GV JWK. Contributed reagents/materials/analysis tools: SHH. Wrote the paper: HJK HHW JWK. Linkage and Exome Analyses for Diagnosis of DFNA15 It should also be noted that the technical success and growing availability of genome tests also bring up issues of carrier detection by family study and informed genetic counseling. As in presymptomatic minors carrying the POU4F3 mutation in our family, for example, a careful approach is mandatory for pre-test and post-test counseling in late-onset diseases such as DFNA [35]. In conclusion, this study demonstrated that WES in combina- tion with linkage analysis using bi-allelic SNP markers successfully identified the candidate locus and mutation in AD-NSHL. The POU4F3 mutation identified in the Korean family was the first missense mutation affecting the 3rd alpha helix of the POU homeodomain harboring a bipartite nuclear localization signal of the protein. We believe the growing availability of molecular genomic tests opens the possibility of better understanding of molecular mechanisms and genotype-phenotype correlations in NSHL. 1. Morton NE (1991) Genetic epidemiology of hearing impairment. Ann N Y Acad Sci 630: 16–31. 11. Stephens M, Smith NJ, Donnelly P (2001) A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 68: 978–989. 12. Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows- Wheeler transform. Bioinformatics 25: 1754–1760. 10. Abecasis GR, Cherny SS, Cookson WO, Cardon LR (2002) Merlin–rapid analysis of dense genetic maps using sparse gene flow trees. Nat Genet 30: 97– 101. 13. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, et al. (2009) The Sequence Alignment/Map format and SAMtools. Bioinformatics 25: 2078–2079. 5. Steel KP, Kros CJ (2001) A genetic approach to understanding auditory function. Nat Genet 27: 143–149. 2. Toriello HV, Reardon W, Gorlin RJ (2004) Hereditary hearing loss and its syndromes. Oxford: Oxford University Press. 4. Keats BJ, Berlin CI (1999) Genomics and hearing impairment. Genome Res 9: 7–16. Discussion Ethnicity Methods Mutation Domain Phenotype Reference 1 Israeli Jewish Linkage analysis using STR markers and candidate sequencing c.884_891del8 (p.Ile295Thrfs*5) NA 18–30 y; progressive; flat to downward-sloping AC Vahara et al. 1998 (19) 2 Dutch Linkage analysis using STR markers and candidate sequencing c.865C.T (p.Leu289Phe) POU homeodomain (1st alpha-helix) 13 y–20; progressive; flat to gently downward-sloping AC; vestibular hypofunction +/2 Collin et al. 2008 (23), Pauw et al. 2008 (28) 3 Dutch? Targeted sequencing of POU4F3 in 30 URF c.668T.C (p.Leu223Pro) POU-specific domain 40 y; moderate to severe; affecting mainly high frequencies Collin et al. 2008 (23) 4 Korean Targeted sequencing of POU4F3 in 42 URF c.662_675del14 (p.Gly221Glufs*77) NA 20 y; severe; downward-sloping AC Lee et al. 2010 (13) 5 Korean Targeted massively parallel sequencing of 80 genes and Sanger validation in 8 URF c.694G.A (p.Glu232Lys) POU-specific domain 27/28 y; moderate/severe; flat/ downward-sloping AC Baek et al. 2012 (25) 6 Korean Linkage analysis using SNP markers and Sanger validation c.977G.A (p.Arg326Lys) POU homeodomain (3rd alpha-helix) Early 10 s-late 50 s; mild to moderate flat to gently downward-sloping AC Kim et al. This report NA, not applicable; y, years of age; AC, audiometric configuration; URF, unrelated families. doi:10.1371/journal.pone.0079063.t003 cs and phenotypes of DFNA15 in previously reported 5 families and in the Korean family in this report. Table 3. Genetics and phenotypes of DFNA15 in previously reported 5 families and in the Korean fam November 2013 | Volume 8 | Issue 11 | e79063 PLOS ONE | www.plosone.org 5 3. Van Camp G, Willems PJ, Smith RJ (1997) Nonsyndromic hearing impairment: unparalleled heterogeneity. Am J Hum Genet 60: 758–764. Table S1 Family members of the pedigree. (DOCX) Table S2 Haplotypes in the linkage region on 5q31. (DOCX) impairing ion transport in the distal nephron. Nat Genet 44: 456–460, S451– 453. 6. Bamshad MJ, Ng SB, Bigham AW, Tabor HK, Emond MJ, et al. (2011) Exome sequencing as a tool for Mendelian disease gene discovery. Nat Rev Genet 12: 745–755. 7. Louis-Dit-Picard H, Barc J, Trujillano D, Miserey-Lenkei S, Bouatia-Naji N, et al. (2012) KLHL3 mutations cause familial hyperkalemic hypertension by 8. O’Connell JR, Weeks DE (1998) PedCheck: a program for identification of genotype incompatibilities in linkage analysis. Am J Hum Genet 63: 259–266. 1. Morton NE (1991) Genetic epidemiology of hearing impairment. Ann N Y Acad Sci 630: 16–31. 2. Toriello HV, Reardon W, Gorlin RJ (2004) Hereditary hearing loss and its syndromes. Oxford: Oxford University Press. 3. Van Camp G, Willems PJ, Smith RJ (1997) Nonsyndromic hearing impairment: unparalleled heterogeneity. Am J Hum Genet 60: 758–764. 4. Keats BJ, Berlin CI (1999) Genomics and hearing impairment. Genome Res 9: 7–16. 5. Steel KP, Kros CJ (2001) A genetic approach to understanding auditory function. Nat Genet 27: 143–149. 6. Bamshad MJ, Ng SB, Bigham AW, Tabor HK, Emond MJ, et al. (2011) Exome sequencing as a tool for Mendelian disease gene discovery. Nat Rev Genet 12: 745–755. 7. Louis-Dit-Picard H, Barc J, Trujillano D, Miserey-Lenkei S, Bouatia-Naji N, et al. (2012) KLHL3 mutations cause familial hyperkalemic hypertension by Linkage and Exome Analyses for Diagnosis of DFNA15 Linkage and Exome Analyses for Diagnosis of DFNA15 14. Liu X, Jian X, Boerwinkle E (2011) dbNSFP: a lightweight database of human nonsynonymous SNPs and their functional predictions. Hum Mutat 32: 894– 899. 25. Baek JI, Oh SK, Kim DB, Choi SY, Kim UK, et al. (2012) Targeted massive parallel sequencing: the effective detection of novel causative mutations associated with hearing loss in small families. Orphanet J Rare Dis 7: 60. g p 26. Weiss S, Gottfried I, Mayrose I, Khare SL, Xiang M, et al. (2003) The DFNA15 deafness mutation affects POU4F3 protein stability, localization, and transcrip- tional activity. Mol Cell Biol 23: 7957–7964. 15. Vriend G (1990) WHAT IF: a molecular modeling and drug design program. J Mol Graph 8: 52–6, 29. 16. Krieger E, Koraimann G, Vriend G (2002) Increasing the precision of comparative models with YASARA NOVA–a self-parameterizing force field. Proteins 47: 393–402. 27. Lee HK, Song MH, Kang M, Lee JT, Kong KA, et al. (2009) Clinical and molecular characterizations of novel POU3F4 mutations reveal that DFN3 is due to null function of POU3F4 protein. Physiol Genomics 39: 195–201. 17. Leon PE, Raventos H, Lynch E, Morrow J, King MC (1992) The gene for an inherited form of deafness maps to chromosome 5q31. Proc Natl Acad Sci U S A 89: 5181–5184. 28. Pauw RJ, van Drunen FJ, Collin RW, Huygen PL, Kremer H, et al. (2008) Audiometric characteristics of a Dutch family linked to DFNA15 with a novel mutation (p.L289F) in POU4F3. Arch Otolaryngol Head Neck Surg 134: 294– 300. 18. Lynch ED, Lee MK, Morrow JE, Welcsh PL, Leon PE, et al. (1997) Nonsyndromic deafness DFNA1 associated with mutation of a human homolog of the Drosophila gene diaphanous. Science 278: 1315–1318. 29. Kong A, Masson G, Frigge ML, Gylfason A, Zusmanovich P, et al. (2008) Detection of sharing by descent, long-range phasing and haplotype imputation. Nat Genet 40: 1068–1075. of the Drosophila gene diaphanous. Science 278: 1315–1318. 19. Vahava O, Morell R, Lynch ED, Weiss S, Kagan ME, et al. (1998) Mutation in transcription factor POU4F3 associated with inherited progressive hearing loss in humans. Science 279: 1950–1954. 30. Walsh T, Shahin H, Elkan-Miller T, Lee MK, Thornton AM, et al. (2010) Whole exome sequencing and homozygosity mapping identify mutation in the cell polarity protein GPSM2 as the cause of nonsyndromic hearing loss DFNB82. Am J Hum Genet 87: 90–94. 20. References impairing ion transport in the distal nephron. Nat Genet 44: 456–460, S451– 453. 8. O’Connell JR, Weeks DE (1998) PedCheck: a program for identification of genotype incompatibilities in linkage analysis. Am J Hum Genet 63: 259–266. 2. Toriello HV, Reardon W, Gorlin RJ (2004) Hereditary hearing loss and its syndromes. Oxford: Oxford University Press. g yp p g y 9. Wigginton JE, Abecasis GR (2005) PEDSTATS: descriptive statistics, graphics and quality assessment for gene mapping data. Bioinformatics 21: 3445–3447. 3. Van Camp G, Willems PJ, Smith RJ (1997) Nonsyndromic hearing impairment: unparalleled heterogeneity. Am J Hum Genet 60: 758–764. 10. Abecasis GR, Cherny SS, Cookson WO, Cardon LR (2002) Merlin–rapid analysis of dense genetic maps using sparse gene flow trees. Nat Genet 30: 97– 101. 4. Keats BJ, Berlin CI (1999) Genomics and hearing impairment. Genome Res 9: 7–16. 5. Steel KP, Kros CJ (2001) A genetic approach to understanding auditory function. Nat Genet 27: 143–149. 6. Bamshad MJ, Ng SB, Bigham AW, Tabor HK, Emond MJ, et al. (2011) Exome sequencing as a tool for Mendelian disease gene discovery. Nat Rev Genet 12: 745–755. 7. Louis-Dit-Picard H, Barc J, Trujillano D, Miserey-Lenkei S, Bouatia-Naji N, et al. (2012) KLHL3 mutations cause familial hyperkalemic hypertension by PLOS ONE | www.plosone.org November 2013 | Volume 8 | Issue 11 | e79063 6 Linkage and Exome Analyses for Diagnosis of DFNA15 Erkman L, McEvilly RJ, Luo L, Ryan AK, Hooshmand F, et al. (1996) Role of transcription factors Brn-3.1 and Brn-3.2 in auditory and visual system development. Nature 381: 603–606. J 31. Yariz KO, Duman D, Seco CZ, Dallman J, Huang M, et al. (2012) Mutations in OTOGL, encoding the inner ear protein otogelin-like, cause moderate sensorineural hearing loss. Am J Hum Genet 91: 872–882. 21. Xiang M, Gan L, Li D, Chen ZY, Zhou L, et al. (1997) Essential role of POU- domain factor Brn-3c in auditory and vestibular hair cell development. Proc Natl Acad Sci U S A 94: 9445–9450. 32. Diaz-Horta O, Duman D, Foster J 2nd, Sirmaci A, Gonzalez M, et al. (2012) Whole-exome sequencing efficiently detects rare mutations in autosomal recessive nonsyndromic hearing loss. PLoS One 7: e50628. 22. BIOBASE Human Genome Mutation Database (HGMDH Professional). Available: http://www.biobase-international.com/product/hgmd Accessed 2013 Jun 28. 33. Gao J, Xue J, Chen L, Ke X, Qi Y, et al. (2013) Whole exome sequencing identifies a novel DFNA9 mutation, C162Y. Clin Genet 83: 477–481. 23. Collin RW, Chellappa R, Pauw RJ, Vriend G, Oostrik J, et al. (2008) Missense mutations in POU4F3 cause autosomal dominant hearing impairment DFNA15 and affect subcellular localization and DNA binding. Hum Mutat 29: 545–554. 34. Park G, Gim J, Kim AR, Han KH, Kim HS, et al. (2013) Multiphasic analysis of whole exome sequencing data identifies a novel mutation of ACTG1 in a nonsyndromic hearing loss family. BMC Genomics 14: 191. 24. Lee HK, Park HJ, Lee KY, Park R, Kim UK (2010) A novel frameshift mutation of POU4F3 gene associated with autosomal dominant non-syndromic hearing loss. Biochem Biophys Res Commun 396: 626–630. 35. Ross LF, Saal HM, David KL, Anderson RR (2013) Technical report: Ethical and policy issues in genetic testing and screening of children. Genet Med 15: 234–245. November 2013 | Volume 8 | Issue 11 | e79063 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 7 7
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Custom-Made Computer-Aided-Design/Computer-Aided-Manufacturing Biphasic Calcium-Phosphate Scaffold for Augmentation of an Atrophic Mandibular Anterior Ridge
Case Reports in Dentistry/Case reports in dentistry
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Hindawi Publishing Corporation Case Reports in Dentistry Volume 2015, Article ID 941265, 11 pages http://dx.doi.org/10.1155/2015/941265 Hindawi Publishing Corporation Case Reports in Dentistry Volume 2015, Article ID 941265, 11 pages http://dx.doi.org/10.1155/2015/941265 Hindawi Publishing Corporation Case Reports in Dentistry Volume 2015, Article ID 941265, 11 pages http://dx.doi.org/10.1155/2015/941265 Francesco Guido Mangano,1,2 Piero Antonio Zecca,1,2 Ric van Noort,3 Samvel Apresyan,4 Giovanna Iezzi,5 Adriano Piattelli,5 Aldo Macchi,1,2 and Carlo Mangano1,2 1Department of Surgical and Morphological Science, Dental School, University of Insubria, Via Giuseppe Piatti 10, 21100 Varese, Italy 2ITEB Research Centre, University of Insubria, Via Giuseppe Piatti 10, 21100 Varese, Italy 3Academic Unit of Restorative Dentistry, School of Clinical Dentistry, University of Sheffield, 19 Claremont Crescent, Sheffield S10 2TA, UK fi 4Academic Unit of Prosthodontics, Moscow State University of Medicine and Dentistry, 20 Delegatskaya Street, Moscow 127473, Russia fi cademic Unit of Prosthodontics, Moscow State University of Medicine and Dentistry, 20 Delegatskaya Street, Moscow 127473, Russia 5Department of Medical, Oral and Biotechnological Sciences, Dental School, G. d’Annunzio University, Via dei Vestini 31, 66100 Chieti, Italy Correspondence should be addressed to Francesco Guido Mangano; francescomangano1@mclink.net Correspondence should be addressed to Francesco Guido Mangano; francescomangano1@mclink.ne Received 6 February 2015; Accepted 21 April 2015 Received 6 February 2015; Accepted 21 April 2015 Academic Editor: Miguel Pe˜narrocha Academic Editor: Miguel Pe˜narrocha Copyright © 2015 Francesco Guido Mangano 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. This report documents the clinical, radiographic, and histologic outcome of a custom-made computer-aided-design/computer- aided-manufactured (CAD/CAM) scaffold used for the alveolar ridge augmentation of a severely atrophic anterior mandible. Computed tomographic (CT) images of an atrophic anterior mandible were acquired and modified into a 3-dimensional (3D) reconstruction model; this was transferred to a CAD program, where a custom-made scaffold was designed. CAM software generated a set of tool-paths for the manufacture of the scaffold on a computer-numerical-control milling machine into the exact shape of the 3D design. A custom-made scaffold was milled from a synthetic micromacroporous biphasic calcium phosphate (BCP) block. The scaffold closely matched the shape of the defect: this helped to reduce the time for the surgery and contributed to good healing. One year later, newly formed and well-integrated bone was clinically available, and two implants (AnyRidge, MegaGen, Gyeongbuk, South Korea) were placed. The histologic samples retrieved from the implant sites revealed compact mature bone undergoing remodelling, marrow spaces, and newly formed trabecular bone surrounded by residual BCP particles. This study demonstrates that custom-made scaffolds can be fabricated by combining CT scans and CAD/CAM techniques. Further studies on a larger sample of patients are needed to confirm these results. 2. Case Presentation An 18-year-old, nonsmoker female patient, with no history of systemic disease, was referred to the Oral Surgery Unit of the Department of Surgical and Morphological Science, Univer- sity of Varese, Italy, for a fixed implant-supported prosthetic rehabilitation of the anterior mandible. One year earlier the patient had been involved in a car accident and had fractured her anterior symphysis, losing her lower incisors; in that con- text, internal fixation of the fracture was obtained by means of two rigid plates placed along the upper and lower border of the symphysis.i t Whereas all the aforementioned surgical techniques and materials can be successful to augment bone vertically and horizontally, the number of complications and failures of these procedures is still high [4, 5, 13]. Alveolar ridge aug- mentation remains a major challenge due to anatomical limi- tations and technical problems, such as the difficulty to shape the bone graft into an appropriate three-dimensional (3D) configuration [5, 13, 16]. During the first visit, a complete clinical and radiographic examination was carried out. The patient was wearing a removable partial denture (RPD) as an interim prosthesis to replace the missing mandibular incisors and improve her aes- thetic appearance. After removal of the RPD, the first clinical assessment revealed significant contraction of the soft tissues, probably associated with a vertical and horizontal bone defect in the edentulous area, as confirmed by dental casts analysis. Clinical examination revealed unsatisfactory oral hygiene and, consequently, the patient was provided with professional oral hygiene instruction, involving reinforcement in her oral hygiene efforts, followed by a scaling and root planning of the entire dentition. Probing pocket depth (PPD) was measured using a light probing force (of approximately 25 g) with a conventional periodontal probe (PCP-UNC 15, Hu-Friedy Manufacturing, Chicago, IL, USA) at 4 sites per tooth (mesial, midbuccal, distal, and midlingual). The patient was peri- odontally healthy with PPD values ranging from 3 to 5 mm. Finally, for a better assessment of the bony anatomy, com- puted tomography (CT) datasets of the mandible were acquired in the Digital Imaging and Communication in Medicine (DICOM) format and immediately transferred to specific segmentation software (Mimics, Materialise, Leuven, Belgium). In this software, the hard tissue threshold was care- fully selected so that only bone would be reconstructed from the slices. Accordingly, it was possible to perform an accurate and complete 3D reconstruction of the mandible. 2. Case Presentation Although CT evaluation and 3D reconstruction showed healing of the fracture, they also showed severe posttraumatic atrophy of the mandibular anterior ridge. In detail, a huge vertical bone defect was present in the symphyseal area (9.3, 10.0, 8.6, and 7.8 mm of alveolar bone were lost from the right lateral incisor to the left lateral incisor area, resp.) combined with a marked reduction in the horizontal alveolar ridge width (Figures 1(a)-1(b)). i Until recently, it was common practice for surgeons to estimate the size and shape of a bone graft on plain radio- graphs, decide the final shape, and manually cut the scaffold into the desired shape during the operation [16–19]. Unfor- tunately, this approach is complex and time-consuming and the size and shape of bone graft can be highly inaccurate, as it depends heavily upon the clinicians’ ability to contour deli- cate 3D shapes manually. This may finally result in an unstable clinical outcome [19, 20].t Ideally, bone grafts should be customised to meet individ- ual patient needs, since there are individual variations among patients and differences in damaged parts. The use of grafts that are made to fit precisely according to the 3D shape of the patient’s bone defects may improve the vascularization and the biocompatibility of the scaffold following implantation [15–18]. At present, the combination of digital techniques such as model reconstruction based on medical images and com- puter-aided design/computer-aided manufacturing (CAD/ CAM) offers new solutions for planning bone reconstructive surgery in relation to the aesthetic outcomes and the final prosthetic and functional rehabilitation [19–22]. In particu- lar, owing to recent improvements in computer technology combined with advanced computer numerically controlled (CNC) milling units, it is now possible to fabricate 3D custom-made scaffolds in a biocompatible material. A block of bone substitute can be milled into the most appropriate shape that has been preoperatively calculated using 3D simu- lation [3, 19–24]. This new approach may provide a valuable alternative to conventional procedures that are based on manual intraoperatory modelling of the graft [3, 19, 23, 24]. Given this problematic anatomical situation, the place- ment of dental implants for supporting a fixed prosthetic rehabilitation was not possible without considering some form of preprosthetic bone reconstructive surgery. 1. Introduction including absorption of alveolar bone after tooth loss, peri- odontal diseases, traumatic injuries, cysts, and tumors, may result in severe alveolar bone defects, with insufficient bone volume to place the implants correctly [5]. In this situation, alveolar ridge augmentation is indicated, either before or in conjunction with implant placement, in order to attain long- term function and an aesthetic outcome [3–5].f Dental implants are a valid and predictable modality to restore function and aesthetics in completely and partially edentulous patients, with satisfactory high long-term survival rates, particularly in the mandible [1, 2].fi Sufficient alveolar bone volume is required to ensure the correct placement of implants and to achieve an aesthetically pleasing outcome [2–4]. However, a variety of processes, Different surgical techniques have been used to overcome alveolar ridge atrophy, including onlay/inlay bone grafting 2 Case Reports in Dentistry Case Reports in Dentistry Case Reports in Dentistry [6–8], guided bone regeneration (GBR) [9, 10], ridge split technique/ridge expansion [11], and distraction osteogenesis [7, 12]. The aim of the present report is therefore to document the clinical, radiographic, and histologic outcome of a custom- made, anatomically shaped CAD/CAM scaffold used for the alveolar ridge augmentation of a severely atrophic anterior mandible. Autogenous bone has always been considered the “gold standard” for alveolar ridge augmentation because of its inherent osteogenic, osteoinductive, and osteoconductive properties [5, 13]. Accordingly, bone reconstructions often involve onlay bone grafts, harvested from either intraoral or extraoral sites [5–7, 13]. However, the use of autografts as onlays has drawbacks, such as additional surgery for harvest- ing, limited availability, donor site morbidity (which includes risk of infection, bleeding, pain, swelling, and damage to nerves and blood vessels), and high resorption rate of the graft [5, 13]. To overcome these limitations, bone substitute materials such as allografts [8], xenografts [14], and synthetic bone grafts [3, 10, 15] have been introduced. 2. Case Presentation Based on the detailed clinical and radiographic examinations, a bone reconstructive procedure with a custom-made synthetic t Until now, however, only a few studies have dealt with custom-made scaffolds for alveolar ridge augmentation [3, 23, 24] and none of these has focused on bone regeneration of the anterior mandible. 3 Case Reports in Dentistry Entwicklungs GmbH, Bayreuth, Germany), was then placed in the CNC milling machine and milled into the exact shape of the 3D project. In this way, an anatomically shaped, custom-made BCP scaffold was manufactured. In addition, a scaffold replica in polytetrafluoroethylene (PTFE) was fabricated; this PTFE replica was intended as a guide for the correct positioning of the hole for the fixation screw (using the BCP scaffold as a guide for drilling the hole could have led to fracture of the fragile scaffold). It took two weeks for the design and fabrication of the BCP scaffold with its replica. The BCP scaffold and its PTFE replica were sterilized before surgery. (a) (b) Figure 1: Preoperative situation. (a) 3D reconstruction of the atrophic anterior mandible by means of specific software: a huge vertical bone defect is present in the symphyseal area, combined with a marked reduction in the horizontal alveolar ridge width. (b) Photorealistic rendering of the mandible. The severe posttraumatic atrophy of the mandibular anterior ridge is evidenced. Entwicklungs GmbH, Bayreuth, Germany), was then placed in the CNC milling machine and milled into the exact shape of the 3D project. In this way, an anatomically shaped, custom-made BCP scaffold was manufactured. In addition, a scaffold replica in polytetrafluoroethylene (PTFE) was fabricated; this PTFE replica was intended as a guide for the correct positioning of the hole for the fixation screw (using the BCP scaffold as a guide for drilling the hole could have led to fracture of the fragile scaffold). It took two weeks for the design and fabrication of the BCP scaffold with its replica. The BCP scaffold and its PTFE replica were sterilized before surgery. (a) (a) (a) (b) g y Two weeks before surgery, the patient underwent peri- odontal treatment, involving instruction and reinforcement in her oral hygiene efforts, followed by a scaling and root planing of the entire dentition. On the day of surgery the interim removable prosthesis was removed (Figure 3(a)) and local anesthesia was obtained by infiltrating articaine 4% containing 1 : 100.000 adrenaline. 2. Case Presentation Following a crestal incision with two deep lateral incisions, a mucoperiosteal flap was ele- vated with wide exposure of the mandibular symphysis. The mental neurovascular bundles were identified and protected with a retractor. The fixation plates were unscrewed and removed. Then, the PTFE replica was placed in position and used for precise positioning of the hole for the fixation screw of the scaffold (Figure 3(b)). Once the hole for the fixation screw was precisely drilled (Figure 3(c)), the PTFE replica was removed. Prior to implantation of the BCP scaffold into the alveolar bone defect area, multiple small holes were drilled through the remaining alveolar bone into the marrow cavity, with a 1 mm round bur under copious saline irrigation. This was done to enhance bleeding of the mandibular cortex (Figure 3(d)). A preparation rich in growth factor (PRGF) was prepared, in order to promote healing and tissue regen- eration. The preparation was conducted such as to obtain a platelet-rich plasma preparation, a platelet-poor plasma preparation, and a fibrin scaffold. This protocol differed from the original one described by Anitua and colleagues [25] for the lack of sodium citrate and calcium chloride used as anti- coagulant and activator, respectively. The platelet-rich plasma preparation was applied to the surgical site (Figure 3(e)). Once the site had been prepared the custom-made BCP scaf- fold was removed from its sterile packaging (Figure 3(f)) and placed in position, strictly overlapping the underlying alveo- lar crest and creating a biological rigid fixation (Figure 3(g)). Fixation of the scaffold was obtained by means of a small titanium screw (Figure 3(h)). The BCP scaffold rapidly acted as a sponge, absorbing a large amount of blood from the surgical site (Figure 3(i)). The surgical site was finally covered and protected with a fibrin membrane (Figure 3(j)). During wound closure great care was taken to obtain a tension-free suture above the scaffold, so as to avoid ischemic damage to the mucosa and suture dehiscence (Figure 3(k)). The patient was instructed to avoid hard food and received oral antibiotics, amoxicillin + clavulanic acid 2 g/d for 6 days (Augmentin, GlaxoSmithKline Beecham, Brentford, UK). Postoperative pain was controlled by administering 100 mg of nimesulide (Aulin, Roche Pharmaceutical, Basel, Switzer- land) every 12 hours for 2 days, and detailed instruction about (b) Figure 1: Preoperative situation. 2. Case Presentation (a) 3D reconstruction of the atrophic anterior mandible by means of specific software: a huge vertical bone defect is present in the symphyseal area, combined with a marked reduction in the horizontal alveolar ridge width. (b) Photorealistic rendering of the mandible. The severe posttraumatic atrophy of the mandibular anterior ridge is evidenced. scaffold, followed by delayed implant placement, was pro- posed to the patient. The patient was fully informed and received a thorough explanation about the planned treatment along with its potential risks and complications. She was also advised about the alternative treatment options of a fixed partial denture on natural teeth or a removable partial den- ture. After careful consideration, she accepted the proposed treatment and signed a written informed consent form. The study was approved by the Local Ethical Committee at the University of Varese, Italy, and was performed according to the principles outlined in the World Medical Association’s Declaration of Helsinki on experimentation involving human subjects, as revised in 2008.t j One week after the signing of the informed consent form, the process for the fabrication of the anatomically shaped, custom-made hydroxyapatite scaffold started, as previously reported [23, 24]. In brief, the 3D reconstruction of the mandible was transferred as a stereolithographic (STL) file to a 3D CAD program (Rhinoceros, Robert McNeel & Asso- ciates, Seattle, WA, USA). With this software, it was possible to reconstruct the alveolar ridge defect virtually and design an anatomically shaped, custom-made scaffold. The anatom- ically shaped, custom-made scaffold included a hole in its centre to allow the placement of a fixation screw (Figure 2). The 3D geometry of the scaffold was then imported into proprietary CAM software that is used to generate a set of tool-paths for fabrication on a CNC milling machine. A commercially available, synthetic micromacroporous bipha- sic calcium-phosphate (BCP) block, consisting of 70% beta- tricalcium phosphate and 30% hydroxyapatite (Biocer, Biocer 4 Case Reports in Dentistry Figure 2: The anatomically shaped, custom-made scaffold is designed. The scaffold includes a hole in its centre to allow the placement of a fixation screw. Figure 2: The anatomically shaped, custom-made scaffold is designed. The scaffold includes a hole in its centre to allow the placement of a fixation screw. internal connections (AnyRidge, MegaGen Implants Co., Ltd., Gyeongbuk, South Korea) were inserted under local anesthesia by the same surgeon who had performed the graft- ing procedure. 2. Case Presentation Local anaesthesia was obtained by infiltrating articaine 4% containing 1 : 100.000 adrenaline. A full-thick- ness crestal incision was made and the soft tissue overlying the reconstructed alveolar process was elevated. The patient showed significant bone augmentation, confirming the pos- sibility of placing two dental implants in the preplanned positions. Accordingly, two implants (3.75 × 11.5 mm) were placed in locations numbers 32 and 42 (Figures 6(b), 6(c), and 6(d)). The threads of the implants used in this study were designed to provide high insertion torque, by increasing their dimensions toward the coronal end of the implant. This specific macrotopographical feature may allow for axial and radial bone compression during implant insertion, and it may be particularly useful in regenerated areas, providing the increased primary stability. An insertion torque of 55 Ncm was registered. Implant stability was determined clinically as the absolute absence of axial or rotational movement by the removal of the implant driver without use of the stabilizing wrench. oral hygiene was given, along with mouth rinses with 0.12% chlorhexidine (Chlorhexidine, Oral-B, Boston, MA, USA) to be administered daily for 7 days. The patient was seen on a weekly basis during the first 4 weeks. At the first control visit, 7 days after the surgery, a clinically healthy marginal area was present, and no postoperative pain or swelling was reported. There was no bleeding or wound infection. At the second control visit, 14 days after the surgery, sutures were removed (Figure 3(l)). Monthly professional plaque control supple- mented this healing phase for 6 months. No clinical complications were observed during the 1- year healing period. In this period, the patient wore her RPD as interim prosthesis, primarily for aesthetic reasons. One year after surgery the patient underwent a postoperative CT scan. The new CT datasets were immediately transferred to the 3D reconstruction software (Mimics, Materialise, Leuven, Belgium) for the segmentation of the mandible (Figures 4(a)- 4(b)). After that, the vertical and horizontal bone gain was radiographically evaluated by comparing the 3D reconstruc- tion of the preoperative CT scan with that obtained 1 year later, using a method previously described [10, 26]. In brief, data from the preoperative and the postoperative CT scans were segmented using the aforementioned 3D reconstruction software. Based on the result of these segmentations, a surface mesh model was generated according to conventional match- ing cube algorithms, followed by automated surface mesh model generation. 2. Case Presentation The postoperative mesh model was super- imposed on the preoperative mesh model and rigidly aligned by anatomical landmarks with the help of software for the overlapping of digital images (Geomagic Studio, Geomagic, Morrisville, NC, USA). The distance between the 2 surface meshes was presented as color-coded graded figures to iden- tify zones of facial bone resorption. By overlapping digital images the hard tissue gain could be confirmed (Figure 5).fi During implant surgery, two bone core biopsies (approxi- mately 2 × 6 mm, one for each site of implant placement) were retrieved with a 2 × 10 mm trephine bur, via a transcrestal path, with the aim of performing a histologic evaluation of the augmented bone. The biopsies were immediately stored in 10% buffered formalin and were subsequently processed (Precise 1 Automated System, Assing, Rome, Italy) to obtain thin ground sections. The specimens were dehydrated in an ascending series of alcohol rinses and embedded in gly- colmethacrylate resin (Technovit 7200 VLC, Heraeus Kulzer GmbH & Co., Wehrheim, Germany). After polymerization, the specimens were sectioned lengthwise along the longer axis to about 150 𝜇m using a high-precision diamond disk saw and subsequently ground down to about 30 𝜇m. Two sections were obtained from each specimen. The sections were stained with basic fuchsin and toluidine blue and the histologic eval- uation was performed. Histological evaluation revealed com- pact mature bone undergoing remodelling, marrow spaces i Since the 3D radiographic examination showed sufficient bone increase and density for implant insertion in the treated anterior mandible, the placement of two implants was digitally planned with the aid of implant navigation software (Invivo Dental 5, Anatomage, San Jose, CA, USA) (Figure 6(a)). Two weeks later, two conical implants with Case Reports in Dentistry Case Reports in Dentistry 5 (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) (l) igure 3: Surgery on patient. 2. Case Presentation (a) Preoperative situation; (b) a PTFE replica is placed in position; (c) the PTFE replica is used for precis ositioning of the hole for the fixation screw of the scaffold; (d) multiple small holes are drilled through the remaining alveolar bone into th marrow cavity, with a 1 mm round bur, under copious saline irrigation, to enhance bleeding of the mandibular cortex; (e) a preparation rich n growth factor (PRGF) is prepared and applied to the surgical site; (f) the custom-made BCP scaffold is removed from its sterile packaging g) the scaffold is placed in position strictly overlapping the underlying alveolar crest; (h) fixation of the scaffold is obtained by means of a mall titanium screw; (i) the BCP scaffold rapidly acts as a sponge, absorbing a large amount of blood from the surgical site; (j) the surgica te is protected with a fibrin membrane; (k) care is taken to obtain a tension-free suture above the scaffold, so as to avoid ischemic damag the mucosa and suture dehiscence; (l) 2 weeks after surgery sutures are removed. (c) (b) (a) (b) (c) (a) (d) (e) (f) (d) (f) (e) (i) (g) (h) (h) (i) (g) (l) (j) (k) (k) (l) (j) Figure 3: Surgery on patient. (a) Preoperative situation; (b) a PTFE replica is placed in position; (c) the PTFE replica is used for precise positioning of the hole for the fixation screw of the scaffold; (d) multiple small holes are drilled through the remaining alveolar bone into the marrow cavity, with a 1 mm round bur, under copious saline irrigation, to enhance bleeding of the mandibular cortex; (e) a preparation rich in growth factor (PRGF) is prepared and applied to the surgical site; (f) the custom-made BCP scaffold is removed from its sterile packaging; (g) the scaffold is placed in position strictly overlapping the underlying alveolar crest; (h) fixation of the scaffold is obtained by means of a small titanium screw; (i) the BCP scaffold rapidly acts as a sponge, absorbing a large amount of blood from the surgical site; (j) the surgical site is protected with a fibrin membrane; (k) care is taken to obtain a tension-free suture above the scaffold, so as to avoid ischemic damage to the mucosa and suture dehiscence; (l) 2 weeks after surgery sutures are removed. made of residual particles of BCP surrounded by compact bone. 2. Case Presentation A poor adaptation of the graft material to the recipient site is a major problem during alveolar ridge augmentation, since the lack of mechanical stability of the scaffold may jeopardize the biological response and conse- quently the treatment outcome [4, 5, 13, 16, 17, 23, 24]. (a) (b) Nowadays, the combination of recent 3D computer simu- lation techniques, manufacturing technology, and novel bone substitutes with excellent bone tissue conductivity promises to open new interesting horizons for alveolar ridge augmenta- tion. It is now possible to produce an accurate 3D shape of the graft calculated by computer simulation and create a synthetic bone substitute cut exactly into the required shape in a 3D milling machine [23, 24]. (b) Figure 4: One year after surgery. (a) 3D reconstruction of the mandible by means of specific software: the vertical and horizontal bone gains are clearly evidenced. (b) Photorealistic rendering of the mandible. Deviazioni 3D Max ±: 5.0400/−5.0399mm Media ±: 0.8736/−0.7842mm Deviazione standard: 1.2746mm Stima RMS: 1.3353mm −5.0400 −4.2664 −3.4928 −2.7191 −1.9455 −1.1719 −0.3983 0.3983 1.1719 x 1.9455 2.7191 3.4928 4.2664 5.0400 z Figure 5: Overlapping of digital images. The DICOM (Digital Imaging and Communication in Medicine) files of the obtained CT datasets, before and 1 year after grafting, are converted into a surface mesh model with digital imaging software (Mimics, Materialise, Leuven, Belgium). The two surface mesh models are then superimposed and rigidly aligned with anatomical landmarks, with the aid of software for the overlapping of digital images (Geomagic Studio, Morrisville, NC, USA). The distance between the 2 surface meshes is presented as color-coded graded figures (blue: tissue loss; yellow/orange/red: tissue apposition; green: little or no modifications) to identify zones of apposition/resorption. Deviazioni 3D Max ±: 5.0400/−5.0399mm Media ±: 0.8736/−0.7842mm Deviazione standard: 1.2746mm Stima RMS: 1.3353mm −5.0400 −4.2664 −3.4928 −2.7191 −1.9455 −1.1719 −0.3983 0.3983 1.1719 x 1.9455 2.7191 3.4928 4.2664 5.0400 z Deviazioni 3D Max ±: 5.0400/−5.0399mm Media ±: 0.8736/−0.7842mm Deviazione standard: 1.2746mm Stima RMS: 1.3353mm −5.0400 −4.2664 −3.4928 −2.7191 −1.9455 −1.1719 −0.3983 0.3983 1.1719 x 1.9455 2.7191 3.4928 4.2664 5.0400 z In the present report, we describe an onlay technique in the anterior atrophic mandible using a synthetic calcium- phosphate bone graft, shaped with a CAD-CAM system. This approach has the benefits that it avoids the need to harvest autologous bone block and assures a perfect fit of the implant above the alveolar crest. 2. Case Presentation In some areas, traces of residual particles surrounded by mineralized bone matrix were found; multinucleated cells were in close contact with the BCP particles. In the marrow spaces, new blood vessels were evident (Figures 7(c)-7(d)). The implants were left undisturbed for a period of 3 months after which a provisional acrylic resin fixed partial denture (FPD) was provided (Figures 8(a)-8(b)) which was left in situ for a further 3 months. This was replaced with and newly formed trabecular bone surrounded by residual BCP particles. The newly formed bone appeared well orga- nized. Close to the porous BCP particles, new bone formation was observed, with newly formed osteoid matrix undergoing mineralization. In detail, the left specimen was made of compact mature bone undergoing remodelling, with a few marrow spaces; no residual biomaterial particles were found, as only traces of BCP mixed with mineralized bone matrix were evidenced (Figures 7(a)-7(b)). The right specimen was made of residual particles of BCP surrounded by compact bone. In some areas, traces of residual particles surrounded by mineralized bone matrix were found; multinucleated cells were in close contact with the BCP particles. In the marrow spaces, new blood vessels were evident (Figures 7(c)-7(d)).ht The implants were left undisturbed for a period of 3 months after which a provisional acrylic resin fixed partial denture (FPD) was provided (Figures 8(a)-8(b)) which was left in situ for a further 3 months. This was replaced with Case Reports in Dentistry 6 (a) (b) Figure 4: One year after surgery. (a) 3D reconstruction of the mandible by means of specific software: the vertical and horizontal bone gains are clearly evidenced. (b) Photorealistic rendering of the mandible. patients with extensive horizontal and vertical ridge resorp- tion [3–5]. (a) Strategies used to overcome mandibular atrophy include various techniques developed to increase bone volume, such as onlay/inlay bone grafting [6–8], GBR [9, 10], ridge expan- sion [11], and distraction osteogenesis [7, 12]. Although it has been shown that it is possible to augment bone with all these different techniques, each of these options poses a risk of complications or potential for dimensional graft loss [4, 5, 13, 16]. Moreover, all the aforementioned techniques are based on manual, intraoperative modelling of the graft. This procedure is challenging and time-consuming and may result in an unsatisfactory adaptation of the scaffold to the bony defect [4, 5, 13, 16, 17, 23, 24]. 2. Case Presentation A clinically healthy, young female patient was referred to the Oral Surgical Unit of the University of Varese for treatment with dental implants. The patient presented a severe posttraumatic atrophy of the mandibular anterior ridge, with a huge vertical bone defect in the symphy- seal area combined with a marked reduction of the horizontal alveolar ridge width. For this patient ridge augmentation was considered appropriate in order to improve soft and hard tissue volume. In particular, in this clinical situation, a strong rigid graft exceeding 3 mm in height and width was required to allow fixation to the recipient site and 3D stability to withstand muscular force. For these reasons, an onlay tech- nique was selected. Figure 5: Overlapping of digital images. The DICOM (Digital Imaging and Communication in Medicine) files of the obtained CT datasets, before and 1 year after grafting, are converted into a surface mesh model with digital imaging software (Mimics, Materialise, Leuven, Belgium). The two surface mesh models are then superimposed and rigidly aligned with anatomical landmarks, with the aid of software for the overlapping of digital images (Geomagic Studio, Morrisville, NC, USA). The distance between the 2 surface meshes is presented as color-coded graded figures (blue: tissue loss; yellow/orange/red: tissue apposition; green: little or no modifications) to identify zones of apposition/resorption. Although autologous bone, harvested from either intrao- ral or extraoral sites, is currently the most reliable material for alveolar ridge augmentation, with the highest success rate, the use of autografts as onlays has many drawbacks, such as the need for multiple interventions, limited bone availability, the risk of morbidity at the donor site, and high resorption rate of the graft [3, 5–7, 13]. Not to be underestimated, patients prefer a bone substitute block over an autograft block, harvested from an intraoral/extraoral site [3]. the definitive metal-ceramic restoration (Figures 8(c)-8(d)), which was cemented with a zinc oxide-eugenol cement (Temp-Bond, Kerr, Orange, CA, USA). Occlusion was thor- oughly checked. The implant-supported FPD showed good functional and an acceptable aesthetic result. Currently, a variety of bone substitute materials, such as allogenic [8], xenogenic [14], or synthetic materials [3, 10, 15], are available for ridge augmentation. An ideal bone substitute should be able to regenerate complex 3D anatomical defects [3, 5, 13, 16, 17, 20, 21]. It should be biocompatible, osteo- conductive, and osteoinductive, encouraging appropriate cell 3. Discussion To perform aesthetic and prosthetic rehabilitation with dental implants, alveolar ridge augmentation is often needed for 7 7 Case Reports in Dentistry (a) (b) (c) (d) Figure 6: Placement of dental implants in the regenerated area. (a) The placement of two implants is planned with the aim of implant navigation software (Invivo Dental 5, Anatomage, San Jose, CA, USA). (b, c, and d) Two AnyRidge dental implants (AnyRidge, MegaGen Implants Co., Ltd., Gyeongbuk, South Korea), 3.75 mm diameter × 11.5 mm length, are placed in locations numbers 32 and 42. (b) (a) (a) (b) (c) (d) (d) (c) Figure 6: Placement of dental implants in the regenerated area. (a) The placement of two implants is planned with the aim of implant navigation software (Invivo Dental 5, Anatomage, San Jose, CA, USA). (b, c, and d) Two AnyRidge dental implants (AnyRidge, MegaGen Implants Co., Ltd., Gyeongbuk, South Korea), 3.75 mm diameter × 11.5 mm length, are placed in locations numbers 32 and 42. (a) (b) (c) (d) Figure 7: Histological evaluation. (a, b) The left specimen is made of compact mature bone undergoing remodelling, with a few marrow spaces; no residual biomaterial particles are found, as only traces of BCP mixed with mineralized bone matrix are present. (c, d) The right specimen is made of residual particles of BCP surrounded by compact bone. In some areas, traces of residual particles surrounded by mineralized bone matrix are evidenced; multinucleated cells are in close contact with the BCP particles. In the marrow spaces, new blood vessels are evident. (b) (a) (b) (a) (c) (d) (c) (d) Figure 7: Histological evaluation. (a, b) The left specimen is made of compact mature bone undergoing remodelling, with a few marrow spaces; no residual biomaterial particles are found, as only traces of BCP mixed with mineralized bone matrix are present. (c, d) The right specimen is made of residual particles of BCP surrounded by compact bone. In some areas, traces of residual particles surrounded by mineralized bone matrix are evidenced; multinucleated cells are in close contact with the BCP particles. In the marrow spaces, new blood vessels are evident. Figure 7: Histological evaluation. (a, b) The left specimen is made of compact mature bone undergoing remodelling, with a few marrow spaces; no residual biomaterial particles are found, as only traces of BCP mixed with mineralized bone matrix are present. 3. Discussion (c, d) The right specimen is made of residual particles of BCP surrounded by compact bone. In some areas, traces of residual particles surrounded by mineralized bone matrix are evidenced; multinucleated cells are in close contact with the BCP particles. In the marrow spaces, new blood vessels are evident. 8 Case Reports in Dentistry 8 (a) (b) (c) (d) Figure 8: Prosthetic restoration. (a) The implants are left submerged for a period of 3 months, after which they are uncovered and healing abutments are placed. (b) A provisional acrylic resin fixed partial denture is provided. (c, d) Three months later, the definitive ceramometallic restoration is delivered. (b) (a) (a) (b) (d) (c) (d) (c) Figure 8: Prosthetic restoration. (a) The implants are left submerged for a period of 3 months, after which they are uncovered and healing abutments are placed. (b) A provisional acrylic resin fixed partial denture is provided. (c, d) Three months later, the definitive ceramometallic restoration is delivered. differentiation through either soluble or insoluble factor sig- nalling and allowing for delivery of pluripotent cell types [3, 10, 15, 23, 24, 27–29]. It should be structurally similar to bone, possessing mechanical properties similar to the native struc- tures and allowing for function and load bearing [3, 10, 23, 24, 27–29]. It should be synthetic and should not be derived from human cadavers or animals [30, 31]. Finally, it should be easy to shape into various forms and bioresorbable. For these purposes, 3D porous materials are currently used as bone substitutes [3, 10, 15, 23, 24, 27–29]. The 3D porous structure provides space for new bone formation, supports the pro- liferation of cells, and maintains their differential function, thus mimicking many roles of the extracellular matrix, and its architecture defines the ultimate shape of the new bone [3, 10, 15, 23, 24, 27–29]. appropriate mechanical properties; it is synthetic and cost- effective, is able to form a suitable shape easily, and ultimately replaces the bone within a short period [10, 32–35]. In the present study, a new protocol for computer-assisted surgery is introduced. 4. Conclusions Digital technology is advancing rapidly in dentistry. Comput- ers are making previously manual tasks easier, faster, cheaper, and more predictable. Personalized therapy is an emerging practice offering tailored solutions to each individual. This approach is envisioned to revolutionize healthcare, through greater cost-effectiveness, efficiency, and improved patient outcomes. In this paper, the authors have described a new dig- ital approach for alveolar ridge augmentation, represented by the use of a CAD/CAM custom-made, anatomically shaped scaffold of BCP, a biomimetic and biocompatible material with the same chemical composition as the bone mineral phase and characterised by high porosity. Despite its limita- tions, the proposed protocol for alveolar ridge augmentation using CAD/CAM to fabricate custom-made scaffolds plates may represent a viable method of reproducing the patient’s anatomical contour, giving the surgeon better procedural control and reducing theatre time. In fact, this technique allowed the successful development of a patient-specific scaf- fold from a CAD model of an alveolar bone defect obtained from CT images. The benefit is to shorten operating time, improve recovery, and achieve lower morbidity rate. Further clinical studies with longer dental implant follow-up are needed to verify these findings. In the future, the emergence of new rapid prototyping technologies for producing 3D constructs may help to modify the design of the synthetic onlays by adding geometrical features that would facilitate and enhance blood perfusion within the graft; this should improve the bone growth in these onlays in vivo. In addition, custom-made scaffolds may be preseeded with cells prior to implantation. The availability of personalized bone grafts engineered from the patient’s own stem cells would probably revolutionize the way we currently treat these defects. Nevertheless, the protocol introduced in this study has some limitations. The first limitation is dimensional and is related to the maximum size of the customized scaffold (12 mm height × 10 mm width). The development of custom- made scaffolds of large dimensions remains challenging because of the requirements for appropriate oxygen and nutrient diffusion throughout the entire construct [4, 23, 24, 36]. To obtain favorable reconstructive results, the bone grafting procedure needs suitable vascular support [36, 37]. Because the osteoblasts require high oxygen tension for bone matrix production, the higher the permeability of the graft to the vascular network, the more effective the new bone formation [36, 37]. An adequate vascular invasion of the scaffold has to be considered an important prerequisite for successful bone regeneration [37]. 3. Discussion This protocol can be divided into four phases: (1) the data acquisition phase, which includes CT scan of the patient; (2) the planning phase, which includes the importing of CT data into a software program for virtual planning and design of the anatomically shaped, custom- made scaffold; (3) the manufacture of the custom-made scaffold using CAD/CAM technology and proprietary CNC milling machine; and (4) the surgical phase, which includes utilizing CAD/CAM-derived scaffold for alveolar ridge aug- mentation of the anterior mandible. This combination of digital technologies has led to the fabrication of a customized scaffold of greater quality than what could be achieved with manual systems; the scaffold perfectly fitted the recipient site without any amendment required during surgery.hfii Among bone substitutes, synthetic calcium-phosphates materials has been suggested as being able to meet all these criteria [3, 10, 15, 23, 24]. In the present study, a syn- thetic micromacroporous biphasic calcium-phosphate (BCP) block, consisting of 70% beta-tricalcium phosphate and 30% hydroxyapatite, was selected as the scaffold material. Biphasic calcium-phosphates have been widely used for hard tissue repair and augmentation in different preclinical [32, 33] and clinical settings [10, 34, 35]. The structure and chemical composition of BCP is very similar to that of the mineral phase of bone [10, 32–35]; it is biocompatible, osteocon- ductive and possesses osteoinductive properties [32–35]; it possesses appropriate porosity for the diffusion of nutrients and the invasion of vascularity from the surrounding tissue and surface chemistry to allow cells to adhere and express the osteogenic phenotype [32–35]. It is characterised by The present protocol offers several benefits. Most signifi- cantly it simplifies the surgery, considerably as well as reduc- ing the treatment time [4, 23, 24]. The anatomically shaped, custom-made scaffolds arrive in the operating environment in sterile packaging and only need to be positioned and fixed to the recipient point in the final step of the surgery. The CNC milling process is highly precise, as it offers an extremely accurate, anatomically fitting scaffold, with the benefit of increased stability and excellent reproduction of the patient’s bony contour [4, 19, 23, 24]. Improving the precision in adapt- ing the graft is critical to its integration with the surrounding bone: a valid interface between graft and the osteogenic cell lines, and the mechanical stability of the scaffold is needed 9 Case Reports in Dentistry for new bone formation [4, 16–19, 21, 23, 24]. 4. Conclusions Without complete vascular invasion and angiogenesis new bone formation is not possible and cells, nutrients, and soluble signals (growth factors), that are mandatory for new bone formation, would be missing [4, 23, 24, 36, 37]. If the scaffold is too big, vascular invasion can be poor, and this could finally jeopardize the healing process [4, 23, 24, 36]. In our present study, PRGF was added to the surgical site. The rationale for the use of PRGF stands in the delivery of a cocktail of proteins and growth factors that may promote wound healing and tissue regeneration to the surgical site [25, 38]. As reported in previous studies, PRGF may be effective in delivering many growth factors such as platelet-derived growth factor, transforming growth factor beta, endothelial growth factor, vascular endothelial growth factor, insulin-like growth factor-1, and fibroblast growth factor. All these soluble factors are capable of promoting healing and tissue regen- eration [25, 38]. Other potential limitations of the present CAD/CAM technique include movement artifacts during CT scans and artifacts from filled teeth or metallic restorations close to the edentulous area [4, 23, 24]. In fact, if the patient moves during the radiologic exam, CT datasets can be rather inaccurate and the presence of metallic artifacts may compli- cate the CAD process and the custom-made scaffold design. Conflict of Interests The authors declare that they have no financial relationship with any commercial firm that may pose a conflict of interests regarding the publication of this study. No grants, equipment, or other sources of support were provided. 3. Discussion In the present report, we had the opportunity to use an anatomically shaped, custom-made CAD/CAM scaffold that perfectly fitted the recipient site, without any amendment required during surgery. This precision may have supported the biological integration of the scaffold, resulting in excellent clinical and histological outcomes. The treatment time is considerably reduced, with clear benefits for the patient: in fact, intraop- erative time is not consumed by repeatedly modelling the scaffold to the native bone (as in conventional procedures) [4, 23, 24]. The procedure allows a more rapid closure of the surgical wound, avoiding possible sources of contamination of the graft and reducing postoperative discomforts such as swelling and pain, which derive from long and difficult surgical procedures [4]. A consequence of this is that the entire procedure is simplified and more accessible even to less experienced surgeons [4, 23, 24]. avoid that bone remodelling processes may alter the patient’s anatomy; in fact, alteration of the residual anatomy may result in inaccuracy of the custom-made scaffold during surgery. 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Analisis Risiko Kerusakan Material Pipa Penyangga Jembatan Jalur Pengeboran Minyak dengan Metode Risk Based Maintenance
Jurnal INTECH Teknik Industri Universitas Serang Raya/Jurnal Intech Teknik Industri Universitas Serang Raya
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Available online at: http://e-jurnal.lppmunsera.org/index.php/INTECH Analisis Risiko Kerusakan Material Pipa Penyangga Jembatan Jalur Pengeboran Minyak dengan Metode Risk Based Maintenance ABSTRAK PT.PQR adalah perusahaan penyedia jasa minyak dan gas di Indonesia yang memiliki beberapa jembatan sebagai infrastruktur penunjang transportasi dalam pengeboran minyak yang tersebar di Provinsi Riau. Jembatan PCK-03- B yang dibangun pada tahun 1959 menjadi obyek penelitian dengan fokus pada pipa-pipa penyangganya yang memiliki sambungan pengelasan yang rentan mengalami korosi. Penelitian ini bertujuan untuk meminimalisasi tingkat risiko yang ditimbulkan akibat kerusakan material pada pipa penyangga jembatan sebagai bagian dari kegiatan pemeliharaan terhadap kondisi material pipa penyangga. Metode Risk-Based Maintenance (RBM) digunakan dengan menentukan Probability of Failure (PoF) dan Consequence of Failure (CoF). CoF terdiri dari Stand by Availability, Financial Model dan Location Model yang dihitung untuk mendapatkan hasil berupa Criticality Matrix. Pipa penyangga PL 2 C dijadikan acuan kondisi keseluruhan jembatan PCK-03-B karena memiliki laju korosi terkritis sebesar 0,151 mm/tahun dengan sisa umur layan jembatan paling pendek yaitu 9 tahun mendatang jika dibandingkan dengan yang lainnya. Hasil analisis menunjukkan bahwa pipa penyangga PL 2 C diperoleh nilai PoF sebesar 2 dan CoF sebesar 10 yang menunjukkan dalam kategori 2D dengan tingkat kerusakan kategori sedang. Rentang penjadwalan perbaikan jembatan PCK-03-B ditetapkan pada 8 tahun (per-2019) mendatang yaitu di tahun 2027, sebelum masa umur pakai habis, sehingga dapat menghindari risiko terjadinya kecelakaan yang tidak diharapkan. Sejarah Artikel: Artikel Masuk: 23 Januari 2023 Artikel direvisi: 02 Mei 2023 Artikel diterima: 07 Mei 2023 Artikel Masuk: 23 Januari 2023 Artikel direvisi: 02 Mei 2023 Artikel diterima: 07 Mei 2023 Pemeliharaan Pipa Penyangga Risk Based Maintenance Risk Matrix Sisa Umur Layan Analisis Risiko Kerusakan Material Pipa Penyangga Jembatan Jalur Pengeboran Minyak dengan Metode Risk Based Maintenance Fikar Ramdan, Gama Harta Nugraha Nur Rahayu* Program Studi Teknik Industri, Universitas Pancasila, Jl. Lenteng Agung Raya No.56, Daerah Khusus Ibukota Jakarta 12630, Indonesia Jurnal INTECH Teknik Industri Universitas Serang Raya Vol 9 No 1 Juni 2023, 86-94 Jurnal INTECH Teknik Industri Universitas Serang Raya Vol 9 No 1 Juni 2023, 86-94 Available online at: http://e-jurnal.lppmunsera.org/index.php/INTECH Jurnal INTECH Teknik Industri Universitas Serang Raya ISSN (Print) 2407-781X ISSN (Online) 2655-2655 * Penulis Korespondensi 1. PENDAHULUAN perawatan yang dilaksanakan disebabkan adanya indikasi terdapatnya kegiatan proses produksi secara mendadak yang mengakibatkan terhambat- nya proses produksi. Selain itu, perawatan harus dilakukan ketika terjadi kerusakan yang beralasan untuk keselamatan kerja, dapat dikatakan perawat- an tidak terencana ini dilakukan setelah mengalami kerusakan pada jembatan. PT. PQR merupakan salah satu perusahaan yang bergerak di bidang industri jasa minyak dan gas di Indonesia. Perusahaan yang berada di Provinsi Riau lebih dari 60 tahun tersebut memiliki beberapa jembatan yang tersebar di dalam provinsi, yang menghubungkan antar lokasi sumur eksplorasi (well) yang satu dengan yang lainnya. Jembatan tersebut juga dimanfaatkan sebagai infrastruktur penunjang transportasi dalam penge- boran minyak, sehingga perlu dijamin keamanan- nya ketika akan dilewati oleh kendaraan pengang- kut peralatan untuk pengeboran. Perusahaan berusaha menerapkan peningkatan efektivitas perawatan terhadap sarana jembatan (Wijaya & Sholihin, 2022). Maintenance bertujuan untuk men- jaga serta memperbaiki alat penunjang produksi hingga menjadi satu kondisi yang selayaknya (Limantoro, 2013). Gambar 1. Jembatan PCK-03-B Salah satu komponen jembatan yang perlu diperhatikan adalah material dari pipa penyangga jembatan yang memiliki sambungan pengelasan yang rentan mengalami korosi. Data hasil uji kete- balan profil pipa penyangga jembatan pada tahun 2019, didapatkan nilai laju korosi paling kritis terdapat pada pipa penyangga dengan kode PL 2 segmen C dengan nilai laju korosi sebesar 0,151 mm/tahun dan berdasarkan NACE RP-0169 dapat dikatakan laju korosi ini termasuk dalam kategori sedang. Sedangkan sisa umur layan jembatan terhitung tinggal 9 tahun lagi. Mengingat sisa umur layan jembatan tersebut adalah yang paling pendek maka pipa penyangga dengan kode PL 2 segmen C tersebut dijadikan sebagai acuan kondisi keseluruhan jembatan PCK-03-B. Gambar 1. Jembatan PCK-03-B Gambar 1 menunjukkan pipa penyangga jembatan PCK-03-B pada jalur pengeboran minyak PT. PQR. Pipa penyangga tersebut memiliki sambungan pengelasan yang rentan mengalami korosi. Korosi merupakan penghancuran paksa zat seperti logam dan bahan bangunan mineral media sekitarnya, yang umumnya berbentuk cair (agen korosif). Kehancuran tersebut dapat menyebar ke bagian dalam material. Organisme juga dapat berkontribusi pada korosi bahan bangunan (Knöfel, 1978). Korosi juga dapat didefinisikan sebagai penurunan mutu logam yang disebabkan oleh reaksi elektrokimia antara logam dengan lingkung- an sekitarnya (Afandi et al., 2015). j Penelitian ini difokuskan pada pipa penyan- gga pada jembatan PCK-03-B yang berfungsi sebagai penopang jembatan yang dilalui oleh ken- daraan alat berat serta sebagai penopang beban kendaraan truk eksplorasi pengeboran minyak dan gas. Pipa penyangga jembatan menjadi salah satu komponen penting yang diharuskan untuk dilaku- kan perawatan atau perbaikan secara berkala. ABSTRACT Keywords Maintenance Support Pipe Risk Based Maintenance Risk Matrix Remaining Life PT.PQR is an oil and gas service company in Indonesia with several bridges supporting infrastructure for transport in oil drilling spread across Riau Province. The PCK-03-B bridge, built in 1959, is the object of research focusing on supporting pipes with welding connections prone to corrosion. This research aims to minimise the risk caused by material damage to the bridge support pipes as part of maintenance activities on the condition of the support pipe material. The Risk-Based Maintenance (RBM) method is used to determine the Probability of Failure (PoF) and Consequence of Failure (CoF). CoF consists of Stand by Availability, Financial Model and Location Model, which are calculated to get the results as a Criticality Matrix. The PL 2 C support pipe is used as a reference for the overall condition of the PCK-03-B bridge because it has the most critical corrosion rate of 0.151 mm/year with the shortest remaining bridge service life of the next nine years when compared to the others. The analysis results show that the PL 2 C support pipe obtained a PoF value of 2 and CoF of 10 which indicates in the 2D category with a moderate level of damage. The repair scheduling range for the PCK-03-B bridge is set for the next eight years (per-2019), namely in 2027, before the service life expires, to avoid the risk of unexpected accidents. This is an open-access article under the CC–BY-SA license. This is an open-access article under the CC–BY-SA license. This is an open-access article under the CC–BY-SA license. © 2023. Some rights reserved 86 http://dx.doi.org/10.30656/intech.v9i1.6098 Jurnal INTECH Teknik Industri Universitas Serang Raya Vol 9 No 1 Juni 2023, 86-94 2. METODE PENELITIAN Dimana RL: Sisa Umur Layan (tahun); Corrosion Rate: Laju korosi tertinggi; tinitial(ti): Ketebalan actual awal jembatan dibangun (mm); tactual(ta) min: Ketebalan maksimum awal diukur Untuk mengatasi risiko korosi maka peng- ujian pipa penyangga jembatan diperlukan untuk mengetahui sisa umur layan (Remaining Life) dari jembatan PCK-03-B. Remaining Life adalah batasan waktu dari sebuah pipa yang diukur dari minimum ketebalannya. Bila tidak terdapat kerusakan yang signifikan, setidaknya mengeta- hui pemeliharaan yang tepat untuk jembatan tersebut. Strategi pemeliharaan yang didapat diharapkan dapat mengurangi risiko terjadinya kecelakaan kerja seperti runtuhnya jembatan sebelum masa umur pakai digunakan. Dari permasalahan tersebut, diperlukan metode Risk Based Maintenance (RBM) untuk menangani masalah korosi pada pipa penyangga. Metode RBM merupakan pendekatan terhadap penilaian risiko dalam upaya membantu proses pengambil- an keputusan tentang kebijakan perawatan kom- ponen untuk meminimalisir kegagalan alat penunjang produksi (Arunraj & Maiti, 2007; Jaderi et al., 2019). 2.2. Risk-Based Maintenance (RBM) Setiap kegiatan organisasi pasti menghadapi berbagai risiko yang mempengaruhi pencapaian target organisasi (Rahayu et al., 2018). Risiko merupakan kemungkinan terjadinya peristiwa atau situasi yang dapat menimbulkan akibat atau dampak negatif. Dengan kata lain, risiko adalah kemungkinan terjadinya sesuatu yang buruk. Jadi ada dua hal yang diukur dari sebuah risiko, yaitu kemungkinan (probability/likelihood) terjadinya peristiwa dan konsekuensi (consequence) atau dampak dari peristiwa tersebut (Rahayu, 2023). Oleh karena itu, diperlukan metode yang bersifat proaktif dalam mengidentifikasi sumber risiko/bahaya, sehingga potensi risiko/bahaya bisa dikendalikan sesuai dengan hasil penilaian dan analisis risiko (Mahardhika & Pramudyo, 2023). Terjadinya kecelakaan akibat suatu kegiat- an berpotensi memberikan dampak pada penca- paian produktivitas perusahaan. Berbagai upaya dapat dilakukan guna mengurangi dampak atau risiko tersebut. Pengurangan dampak risiko kec- elakaan kerja selayaknya memperhatikan keter- kaitan antara kriteria/faktor dalam kecelakaan kerja (Rahayu & Fitri, 2020). Kegiatan preventive maintenance juga akan mempengaruhi nilai produktivitas (Saputra & Rahayu, 2019). 1. PENDAHULUAN Proses maintenance yang kurang baik dapat menyebabkan perusahaan mengalami kerugian rusaknya material pada jembatan dan akan menurunkan jumlah produksi atau hasil produksi. Oleh karena itu diperlukan teknik perawatan, dimana terdapat dua teknik maintenance sebagai operasi produksi pada suatu perusahaan yaitu perawatan terencana dan perawatan tidak terencana (Mentari & Lie, 2017). Perawatan terencana merupakan perawatan yang dilakukan secara rutin dengan dengan terkoordinasi untuk mencegah kerusakan peralatan sebelum peralatan tersebut rusak. Perawatan ini dilaksanakan dengan mencatat sesuai dengan rencana yang telah ditetapkan. Perawatan tidak terencana merupakan y ( , ) Korosi dapat diakibatkan dari faktor geo- grafis yang berbeda dari satu tempat dengan tempat lain. Lingkungan menjadi penyebab utama yang mengakibatkan korosi (Fontana, 1986). Beberapa jenis korosi yang umum ditemukan pada suatu pipa penyangga jembatan, yaitu korosi seragam (uniform corrosion), yang terjadi pada per- mukaan logam, kelembaban (humidity) merupakan kondisi lingkungan yang mengakibatkan logam yang terpapar langsung oleh udara terjadi pengikisan pada permukaannya, korosi lubang (pitting corrosion), yang terjadi pada logam baja dan aluminium, tidak homogennya komposisi logam merupakan faktor yang mengakibatkan korosi, korosi erosi (erosion corrosion), yang terjadi pada pipa, biasanya menimbulkan bagian yang kasar hingga tajam yang terjadi karena keausan yang mengakibatkan film pelindung logam terkikis akibat pergerakan fluida yang yang cepat seperti abrasi pasir, dan korosi tegang (stress corrosion), yang terjadi pada pipa, yang diakibatkan oleh aksi http://dx.doi.org/10.30656/intech.v9i1.6098 87 Jurnal INTECH Teknik Industri Universitas Serang Raya Vol 9 No 1 Juni 2023, 86-94 lebih dari 0,381 mm/tahun. Tabel 1. Kategori Laju Korosi (National Association of Engineer, 2002) Kategori Laju Korosi (mm/tahun) Rendah < 0,127 Sedang 0,127 – 0,2032 Tinggi 0,2032 – 0,381 Parah > 0,381 lebih dari 0,381 mm/tahun. tegang (stress) akibat kondisi metalurgi yang menyebabkan kegagalan komponen (Siregar et al., 2021). Tabel 1. Kategori Laju Korosi (National Association of Engineer, 2002) Tujuan penelitian ini adalah untuk memi- nimalisasi tingkat risiko yang ditimbulkan akibat kerusakan material pada pipa penyangga jembatan sebagai bagian dari kegiatan pemeliharaan. Dengan mengetahui kondisi pipa penyangga dari jembatan PCK-03-B dan sisa umur layan (remaining life) pada jembatan PCK-03-B, maka dapat diketahui tingkat risiko pada pipa penyangga jembatan PCK-03-B yang mengalami kerusakan material akibat korosi sehingga dapat ditentukan penjadwalan perbaikan yang dibutuhkan. Perkiraan sisa umur layan konstruksi dihitung dengan menggunakan rumus: RL = tactual - trequired Corrosion Rate (2) RL = tactual - trequired Corrosion Rate (2) (2) 2.1. Sisa Umur Layan Financial Model (CoF 2) Waktu Perbaikan Kerusakan/Biaya Perbaikan (B) Skor M ≥ 7 Hari B ≥ Rp. 1M 5 5 ≤ M ≤ 7 Hari Rp.500 Juta - Rp. 1M 4 3 ≤ M ≤ 5 Hari 300 – 500 JT 3 1 ≤ M ≤ 3 Hari 100 – 300 JT 2 M ≤ 1 Hari B < 100 JT 1 Tabel 6. Location Model (CoF 3) Tabel 2. Probability of Failure Ranking Remaining of Life Probability of Failure RL ≤ 4 Tahun 5 4 < RL ≤ 6 4 6 < RL ≤ 8 3 8 < RL ≤ 10 2 RL ≥ 10 Tahun 1 Tabel 2. Probability of Failure Ranking ( ) Kelas Lokasi Skor Area Publik, Padat penduduk (lalu lintas padat) 5 Area Publik, jauh dari pemukiman (tidak terlalu padat) 4 Lokasi umum dan lalu lintas dapat dimonitor 3 Di dalam Area produksi, jauh dari lalu lintas penduduk 2 Cluster ada security (di area produksi yang termonitor) 1 Consequence of Failure adalah dampak yang terjadi akibat adanya segala kemungkinan dari kegagalan pada jembatan PCK-03-B. Penentuan nilai kemungkinan dari dampak yang terjadi akibat dari kegagalan terbagi lima kategori, dimana nilai CoF terkecil bernilai 1 (Sangat Rendah) dan nilai CoF tertinggi bernilai 5 (Tabel 3). Consequence of Failure dibagi menjadi tiga klasifikasi: Stand by Availability, Financial Model dan Location Model. Tabel 3. Nilai Parameter Consequence Tabel 3. Nilai Parameter Consequence Penentuan perhitungan matriks risiko sebagai berikut: Nilai Parameter Consequence of Failure 5 Catastrophic (Tinggi) 4 Significant (Sedang Tinggi) 3 Moderate (Sedang) 2 Minor (Rendah) 1 Insignificant (Sangat Rendah) Nilai Parameter Consequence of Failure 5 Catastrophic (Tinggi) 4 Significant (Sedang Tinggi) 3 Moderate (Sedang) 2 Minor (Rendah) 1 Insignificant (Sangat Rendah) Risk = ∑PoF x ∑CoF (3) (3) Dimana CoF: Consequences of Failure dan PoF: Probability of Failure. Dimana CoF: Consequences of Failure dan PoF: Probability of Failure. y Nilai CoF terbagi A = 0 – 3, B = 4 – 6, C = 7 – 9, D = 10 – 12 dan E = 13 – 15. Berdasarkan matriks risiko dapat diketahui tingkat risiko kegagalan pipa penyangga yang dapat ditentukan menggunakan Criticality Matrix (Tabel 7). Criticality Matrix merupakan alat umum untuk penilaian dan komunikasi kekritisan suatu material yang digunakan untuk menentukan tingkat risiko. Tabel 8 menunjukkan matriks untuk peren- Stand by availability adalah dampak yang timbul dari material yang telah ditentukan oleh kerusakan atau keparahannya (Tabel 4). http://dx.doi.org/10.30656/intech.v9i1.6098 2.1. Sisa Umur Layan Laju korosi merupakan suatu penurunan kualitas bahan terhadap waktu, peristiwa terjadi- nya korosi pada suatu material yang digunakan saat proses produksi berlangsung (Afandi et al., 2015). Perhitungan laju korosi berdasarkan standar American Petroleum Institute (API) 570 : 2003. CR= tinitial - tactual Tahun diantara umur ti dan ta (1) (1) Metode Risk-Based Maintenance (RBM) merupakan suatu metode kuantitatif yang didasar- kan dari integrasi pendekatan antara reliability dan sebuah strategi pendekatan risiko yang bertujuan untuk mengoptimumkan jadwal Main- tenance dan untuk meminimalkan risiko yang ditimbulkan akibat failure yang terjadi (Khan & Haddara, 2004). Metode RBM digunakan dengan menentukan Probability of Failure (PoF) dan Dimana CR: Laju korosi (mm/tahun); tinitial (ti): Ketebalan actual awal jembatan dibangun (mm); tactual(ta) min : Ketebalan maksimum awal diukur. Laju korosi yang telah diperhitungkan dapat didefinisikan terhadap empat kategori sebagai- mana ditunjukkan pada Tabel 1. Kategori terkecil (rendah) bernilai kurang dari 0,127 mm/tahun. Sedangkan untuk nilai tertinggi (parah) bernilai http://dx.doi.org/10.30656/intech.v9i1.6098 88 Jurnal INTECH Teknik Industri Universitas Serang Raya Vol 9 No 1 Juni 2023, 86-94 terdapat kerusakan pada jembatan PCK-03-B. Location Model adalah letak area yang berada di sekitar jembatan PCK-03-B yang dikategorikan berdasarkan kelas lokasi dari tingkat kepadatan penduduk pemukiman sekitar (Tabel 6). Consequence of Failure (CoF). CoF terdiri dari Stand by Availability, Financial Model dan Location Model yang dihitung untuk mendapatkan hasil berupa Criticality Matrix (Tulloh et al., 2019). Consequence of Failure (CoF). CoF terdiri dari Stand by Availability, Financial Model dan Location Model yang dihitung untuk mendapatkan hasil berupa Criticality Matrix (Tulloh et al., 2019). p y ( ) Probability of Failure adalah kemungkinan dari kegagalan yang berada pada bagian pipa penyangga pada jembatan PCK-03-B. Kemung- kinan ini menggunakan hasil sisa umur layan (RL/Remaining Life) sebagai penentuan kegagal- an. Nilai PoF dapat ditentukan dengan observasi, wawancara, data sekunder, dan kriteria risiko. Penentuan nilai kemungkinan dari kegagalan (PoF) berdasarkan nilai hasil sisa umur layan (Tabel 2). Terdapat lima kategori, dimana nilai PoF terkecil untuk nilai RL lebih dari sama dengan 10 tahun. Sedangkan untuk nilai PoF tertinggi untuk nilai RL kurang dari sama dengan 4 tahun. Artinya semakin besar RL, semakin kecil nilai PoF. Tabel 4. Stand By Availability (CoF 1) Tabel 4. Stand By Availability (CoF 1) Stand by Availability (s) Skor Tidak bisa dilewati sama sekali 5 Penurunan beban kendaraan s/d maksimum 20% 4 Penurunan beban kendaraan s/d maksimum 10% 3 Penurunan beban kendaraan s/d maksimum 5% 2 Tanpa Penurunan 1 Tabel 5. 2.1. Sisa Umur Layan Matrix Interval Inspection (American Petroleum Institute, 1998) Kategori Kekritisan Confidence Level (tahun) 4 3 2 1 5E High Risk 1 2 6 N/A 4E,5D 2 4 6 N/A 3E,4D,5C 3 4 6 N/A 2E,3D,4C,5B Medium Risk 4 4 8 8 1E,2D,3C,4B,5A 4 6 8 8 1D,2C,3B,4A 6 6 8 10 1C,2B,3A Low Risk 6 8 10 10 1B,2A 8 10 10 15 1A 8 10 15 15 3. HASIL DAN PEMBAHASAN Tabel 9 menampilkan data yang digunakan yaitu hasil uji ketebalan profil pipa penyangga jembatan PCK-03-B yang meliputi ketebalan maksimum dan ketebalan minimum dari elemen struktur Steel Pile dengan Tipe Profil Steel Pipe dari tiap pipa yang diperiksa pada tahun 2019. 3.1. Sisa Umur Layan Dalam perhitungan sisa umur layan dihitung terlebih dahulu laju korosi maksimal mengguna- kan hasil pengukuran ketebalan masing masing Tabel 7. Criticality Matrix (American Petroleum Institute, 2002) p g CR= 12,7-3,82 60 = 0,147 mm/tahun Institute, 2002) POF Consequence of Failure (CoF) A B C D E 5 5A 5B 5C 5D 5E 4 4A 4B 4C 4D 4E 3 3A 3B 3C 3D 3E 2 2A 2B 2C 2D 2E 1 1A 1B 1C 1D 1E 5 Catastrophic (Tinggi) 4 Significant (Sedang Tinggi) 3 Moderate (Sedang) 2 Minor (Rendah) 1 Insignificant (Sangat Rendah) Tabel 8. Matrix Interval Inspection (American Petroleum Institute, 1998) Kategori Kekritisan Confidence Level (tahun) 4 3 2 1 5E High Risk 1 2 6 N/A 4E,5D 2 4 6 N/A 3E,4D,5C 3 4 6 N/A 2E,3D,4C,5B Medium Risk 4 4 8 8 1E,2D,3C,4B,5A 4 6 8 8 1D,2C,3B,4A 6 6 8 10 1C,2B,3A Low Risk 6 8 10 10 1B,2A 8 10 10 15 1A 8 10 15 15 Berdasarkan perhitungan, laju korosi yang mempunyai nilai tertinggi pada pipa penyangga jembatan PCK-03-B adalah pipa penyangga dengan kode PL 2 segmen C yaitu sebesar 0,151 mm/tahun. Nilai pipa penyangga jembatan PCK- 03-B tersebut dinyatakan sebagai korosi sedang berdasarkan standard NACE RP 0169-83 untuk menentukan besaran nilai laju korosi. Tabel 9. Uji Ketebalan Profil Pipa Penyangga Jembatan PCK-03-B No Kode & Segmen Posisi Pengujian Ketebalan (mm) ta min 12 3 6 9 1. PL 2 A 5,78 5,63 5,54 5,61 5,54 PL 2 B 4,37 4,12 4,25 4,39 4,12 PL 2 C 3,85 3,63 3,62 3,69 3,62 2. PL 3 A 5,41 5,53 5,34 5,56 5,34 PL 3 B 4,56 4,30 4,46 4,39 5,30 PL 3 C 4,03 3,98 3,85 3,72 3,72 3. 3. HASIL DAN PEMBAHASAN Tabel 9 menampilkan data yang digunakan yaitu hasil uji ketebalan profil pipa penyangga jembatan PCK-03-B yang meliputi ketebalan maksimum dan ketebalan minimum dari elemen struktur Steel Pile dengan Tipe Profil Steel Pipe dari tiap pipa yang diperiksa pada tahun 2019. tr= PD VyEj (4) tr= PD VyEj (4) Dimana tr: Ketebalan minimum yang dipersyarat- kan; P: Beban truk eksplorasi (104,90 Psi); D: Diameter luar pipa; Vy: Tegangan yang diizinkan (24.791,591 Psi) dan Ej; Joint Quality Factor sebesar 0,6 (American National Standards Institute, 2012). kan; P: Beban truk eksplorasi (104,90 Psi); D: Diameter luar pipa; Vy: Tegangan yang diizinkan (24.791,591 Psi) dan Ej; Joint Quality Factor sebesar 0,6 (American National Standards Institute, 2012). 2.1. Sisa Umur Layan PL 4 A 5,11 5,18 5,21 5,15 5,11 PL 4 B 4,79 4,66 4,64 4,76 4,64 PL 4 C 3,87 3,93 4,02 3,91 3,87 Tabel 8. Matrix Interval Inspection (American Petroleum Institute, 1998) Petroleum Institute, 1998) Kategori Kekritisan Confidence Level (tahun) 4 3 2 1 5E High Risk 1 2 6 N/A 4E,5D 2 4 6 N/A 3E,4D,5C 3 4 6 N/A 2E,3D,4C,5B Medium Risk 4 4 8 8 1E,2D,3C,4B,5A 4 6 8 8 1D,2C,3B,4A 6 6 8 10 1C,2B,3A Low Risk 6 8 10 10 1B,2A 8 10 10 15 1A 8 10 15 15 Sebelum melakukan perhitungan perkiraan sisa umur layan konstruksi dihitung terlebih dahulu ketebalan minimum yang dipersyaratkan (tr), berdasarkan safety factor dari prediksi pembebanan struktur jembatan 2.1. Sisa Umur Layan Financial Model adalah dampak dari keuangan yang berkaitan dengan terjadinya kerusakan pada suatu material untuk memperbaiki jika terdapat kerusakan. Tabel 5 menunjukkan biaya yang sebenarnya dikeluarkan oleh perusahaan dengan kurun waktu tertentu guna memperbaiki jika menggunakan Criticality Matrix (Tabel 7). Criticality Matrix merupakan alat umum untuk penilaian dan komunikasi kekritisan suatu material yang digunakan untuk menentukan tingkat risiko. Tabel 8 menunjukkan matriks untuk peren g g g Tabel 8 menunjukkan matriks untuk peren j p canaan penjadwalan perbaikan yang disebut Matriks Interval Inspeksi (MII/Matrix Interval Inspection). Hasil perencanaan MII tersebut http://dx.doi.org/10.30656/intech.v9i1.6098 89 Jurnal INTECH Teknik Industri Universitas Serang Raya Vol 9 No 1 Juni 2023, 86-94 dipakai adalah diameter 12 inch schedule 80S dengan ketebalan (tebal initial) 12,7 mm. ditentukan dari kategori kekritisan yang telah didapat berdasarkan Criticality Matrix dengan indeks angka keyakinan (confidence factor) untuk mendapatkan rentang perencanaan jadwal per- baikan pipa penyangga jembatan PCK-03-B selanjutnya. Penentuan penjadwalan perbaikan berdasarkan hasil dari Matriks Interval Inspeksi selanjutnya diolah menggunakan Microsoft Excel yang nantinya menghasilkan Gantt Chart. g ( ) , 1. Perhitungan laju korosi pada pipa penyangga 1. Perhitungan laju korosi pada pipa penyangga PL 2 C CR= 12,7-3,62 60 = 0,151 mm/tahun 2. Perhitungan laju korosi pada pipa penyangga PL 3 pada segmen C CR= 12,7-3,72 60 = 0,150 mm/tahun p g CR= 12,7-3,72 60 = 0,150 mm/tahun 60 3. Perhitungan laju korosi pada pipa penyangga PL 4 pada segmen C CR= 12,7-3,82 60 = 0,147 mm/tahun B d k hit l j k i 60 3. Perhitungan laju korosi pada pipa penyangga PL 4 pada segmen C CR= 12,7-3,82 60 = 0,147 mm/tahun 60 3. Perhitungan laju korosi pada pipa penyangga PL 4 pada segmen C CR= 12,7-3,82 60 = 0,147 mm/tahun Tabel 7. Criticality Matrix (American Petroleum Institute, 2002) POF Consequence of Failure (CoF) A B C D E 5 5A 5B 5C 5D 5E 4 4A 4B 4C 4D 4E 3 3A 3B 3C 3D 3E 2 2A 2B 2C 2D 2E 1 1A 1B 1C 1D 1E 5 Catastrophic (Tinggi) 4 Significant (Sedang Tinggi) 3 Moderate (Sedang) 2 Minor (Rendah) 1 Insignificant (Sangat Rendah) Tabel 8. http://dx.doi.org/10.30656/intech.v9i1.6098 Tabel 13. Financial Model (CoF 2) Kode & Segmen Financial Model Waktu Perbaikan (hari) Rating 2 C 1-3 2 3 C 1-3 2 4 C 1-3 2 Tabel 12. Stand by Availability (CoF 1) Tabel 12. Stand by Availability (CoF 1) Tabel 12. Stand by Availability (CoF 1) Kode & Segmen Stand by Availability Rating 2 C Derating Max 20% 4 3 C Derating Max 20% 4 4 C Derating Max 10% 3 Tabel 12. Stand by Availability (CoF 1) 2. Perkiraan sisa umur layan pada pipa penyangga PL 3 C RL= 3,72-2,159 0,150 = 10 tahun , 3. Perkiraan sisa umur layan pada pipa penyangga PL 4 C RL = 3,87−2,159 0,147 = 11 tahun Tiap-tiap pipa penyangga jembatan PCK-03- B membutuhkan waktu 1-3 hari untuk memper- baiki pipa penyangga jembatan yang rusak apa- bila terjadi kerusakan, serta berdasarkan data perusahaan membutuhkan biaya sebesar Rp.100.000.000-Rp.300.000.000 untuk setiap melakukan perbaikan. Berdasarkan hal tersebut, financial model pada pipa penyangga jembatan memiliki kategori sedang dengan rating 2 (Tabel 13). Hasil kalkulasi prediksi sisa umur jembatan PCK-03-B ditampilkan pada Tabel 10. Perkiraan sisa umur pakai jembatan PCK-03-B yang paling kritis adalah 9 tahun yaitu pada pipa penyangga dengan kode PL 2 segmen C. Berdasarkan hasil tersebut dapat dinyatakan bahwa prediksi sisa umur layan konstruksi adalah 9 tahun kedepan sehingga jembatan tersebut saat ini masih aman untuk dilewati. Tabel 10. Prediksi Sisa Umur Layan Tabel 10. Prediksi Sisa Umur Layan Kode & Segmen Laju Korosi (CR) Sisa Umur Layan (tahun) Kategori Korosi 2 C 0,151 9 Sedang 3 C 0,150 10 Sedang 4 C 0,147 11 Sedang Tabel 13. Financial Model (CoF 2) 3.1. Sisa Umur Layan Dalam perhitungan sisa umur layan dihitung terlebih dahulu laju korosi maksimal mengguna- kan hasil pengukuran ketebalan masing-masing pipa penyangga. Laju korosi menggunakan Rumus 1 dengan data ketebalan minimum dari masing-masing kode pipa penyangga (Tabel 9) dan ketebalan maksimum awal jembatan dibangun yang menggunakan data prediksi bahwa profil awal dari pipa penyangga yang Berdasarkan rumus (4) maka tr= 104,90 x 12 24.791,591 x 0,6 = 0,085 inch =2,159 mm tr= 104,90 x 12 24.791,591 x 0,6 = 0,085 inch =2,159 mm Berdasarkan hasil tersebut dapat ditentukan dari masing-masing pipa penyangga dengan kode PL http://dx.doi.org/10.30656/intech.v9i1.6098 90 Jurnal INTECH Teknik Industri Universitas Serang Raya Vol 9 No 1 Juni 2023, 86-94 2, PL 3, dan PL 4, untuk perkiraan sisa umur layan konstruksinya, dengan menggunakan rumus (2): beban kendaraan s/d maksimum 10% yang dikategorikan sebagai Stand By Availability rendah dengan rating 3 karena RL pada pipa tersebut lebih dari 11 tahun (Tabel 12). 1. Perkiraan sisa umur layan pada pipa penyangga PL 2 C RL= 3,62-2,159 0,151 = 9 tahun 3.2. Risk Based Maintenance Tabel 14. Location Model (CoF 3) Tabel 14. Location Model (CoF 3) Penentuan PoF dengan mengacu pada Tabel 2. Pipa penyangga jembatan dengan kode PL 2C RL (Remaining Life)-nya berada di range 8- 10 tahun dikategorikan sebagai PoF dengan kemungkinan kecil akan terjadi kegagalan dengan rating 2 (dua), sedangkan pada kode PL 3C dan PL 4C didapatkan RL lebih dari 10 tahun yang dikategorikan sebagai PoF dengan sangat jarang untuk terjadinya kegagalan dengan rating 1 (satu) (Tabel 11). Kode & Segmen Location Model Lokasi Rating 2 C Area publik jauh dari pemukiman 4 3 C Area publik jauh dari pemukiman 4 4 C Area publik jauh dari pemukiman 4 Lokasi pipa penyangga jembatan PCK-03- B memiliki rating 4 yaitu Area Publik Jauh dari Pemukiman (lalu lintas tidak terlalu padat), dikarenakan struktur dari setiap komponen memiliki letak lokasi yang sama (Tabel 14). Berdasarkan nilai CoF1-CoF3 tersebut, ranking konsekuensi kegagalan (CoF) dapat direkapitulasi menjadi satu (Tabel 15). Hasil rekapitulasi CoF dimana dapat disimpulkan bahwa dari ketiga kategori tersebut didapatkan rata-rata sebesar 10 dengan hasil Criticality Matrix menurut API 580 untuk pipa penyangga jembatan dengan kode PL 2 C dan 3 C adalah D. Sedangkan pada pipa penyangga jembatan dengan kode PL 4 C sebesar 9 dengan hasil Criticality Matrix adalah C. Tabel 11. Probability of Failure Ranking Tabel 11. Probability of Failure Ranking Kode & Segmen Probability of Failure (POF) Remaining Life Rating 2 C 9 Tahun 2 3 C 10 Tahun 1 4 C 11 Tahun 1 Pipa penyangga jembatan pada kode PL 2C dan PL 3C ditetapkan jika penurunan beban kendaraan s/d maksimum 20% yang dikategori- kan sebagai Stand By Availability sedang dengan rating 4 karena RL pada pipa tersebut kurang dari 10 tahun. Sedangkan pipa penyangga jembatan pada kode PL 4C ditetapkan jika penurunan http://dx.doi.org/10.30656/intech.v9i1.6098 91 Jurnal INTECH Teknik Industri Universitas Serang Raya Vol 9 No 1 Juni 2023, 86-94 Tabel 15. Consequence of Failure dari tiap elemen Steel Pile Kode & Segmen Consequence of Failure ∑CoF CoF CoF1 CoF2 CoF3 2 C 4 2 4 10 D 3 C 4 2 4 10 D 4 C 3 2 4 9 C Tabel 15. Consequence of Failure dari tiap elemen Steel Pile tahun kedepan sebelum waktu Remaining Life yang telah ditentukan yaitu 9 tahun pada pipa PL 2 C dan 10 tahun pada pipa PL 3 C. 3.2. Risk Based Maintenance Sedangkan, pada pipa penyangga jembatan dengan kode PL 4 C memiliki rentang waktu perbaikan selama 10 tahun kedepan sebelum waktu Remaining Life yang telah ditentukan yaitu 11 tahun. Penjadwalan menggunakan Gantt Chart yang berguna untuk mengetahui jadwal perbaik- an, dimana Gantt Chart ini merupakan hasil dari penjadwalan ketiga pipa dengan kode PL 2 C, PL 3 C, dan PL 4 C yang dimana hasilnya berupa tahun untuk dilakukannya perbaikan pada pipa penyangga jembatan PCK-03-B. Pipa jembatan PCK-03-B akan dilakukan perbaikan pada tahun 2027 per-2019 berdasarkan pipa penyangga yang memiliki laju korosi tertinggi dan sisa umur layan terpendek yaitu pipa penyangga PL 2 C (Gambar 2). Penentuan tingkat risiko pada Analisis Risk Based Maintenance menggunakan Criticality Matrix untuk mengetahui risiko pipa pada setiap bagian jembatan PCK-03-B yang akan diukur dalam terminologi Probability of Failure dan Consequence of Failure dengan menggunakan rumus 3 (Tabel 16). Tabel 16. Rangking Risiko dari tiap Steel Pile Tabel 16. Rangking Risiko dari tiap Steel Pile Kode & Segmen PoF CoF Rating 2 C 2 D Sedang 3 C 1 D Rendah 4 C 1 C Sangat Rendah Gambar 2. Gantt Chart Penjadwalan Perbaikan Pipa Penyangga Jembatan PCK-03-B Berdasarkan Tabel 7 maka Criticality Matrix pada pipa penyangga PL 2 C didapatkan hasil yang berada pada kolom kuning dengan kategori tingkat kerusakan pipa yang sedang (3/moderate). Sementara itu untuk pipa penyangga PL 3 C didapatkan hasil yang berada pada kolom hijau muda dengan kategori tingkat kerusakan pipa yang rendah (2/minor). Dan untuk pipa penyangga PL 4 C didapatkan hasil yang berada pada kolom hijau tua dengan kategori tingkat kerusakan pipa yang sangat rendah (1/Insignificant). Gambar 2. Gantt Chart Penjadwalan Perbaikan Pipa Penyangga Jembatan PCK-03-B 3.3. Pembahasan tersebut dianalisis sehingga dapat ditentukan tingkat risiko pada pipa penyangga jembatan menggunakan Criticality Matrix (tingkat kekritisan) berdasarkan API 580 : 2002, dimana diperoleh hasil pada pipa penyangga dengan kode PL 2 C sebagai acuan kondisi keseluruhan jembatan PCK-03-B dengan memiliki tingkat risiko kerusakan pipa rangking 2D, yang berarti termasuk rating sedang (moderate), dengan nilai PoF sebesar 2 (dua) dan nilai COF sebesar 10 (sepuluh) atau kategori D (berdasarkan klasifikasi nilai CoF). Peningkatan kegiatan maintenance dilakukan dengan penjadwalan yang tepat sebelum waktu sisa umur layan (Remaining Life). Perencanaan penjadwalan perbaikan menggunakan Matrix Interval Inspection dimana untuk rangking 2D (kategori kekritisan medium risk) dan confidence factor sebesar 2 diperoleh rentang waktu perbaikan selama 8 tahun kedepan sebelum sisa umur layan tiba. Kemudian hasil tersebut diolah menggunakan Microsoft Excel dengan modul Gantt Chart dapat dilihat pada Gambar 2., dimana didapatkan jadwal perbaikan pada pipa penyangga jembatan pada tahun 2027 mendatang dari tahun inspeksi yang dilakukan pada tahun 2019. American National Standards Institute. (2012). B31.1: Power Piping, ASME Code for Pressure Piping. New York: The American Society of Mechanical Engineers. https://www.littlepeng.com/single- post/asme-b-31-1-asme-b-31-3-code- comparison#:~:text=Power Piping Code%3F-,ASME B31.,district heating and cooling systems. American Petroleum Institute. (1998). API 570: Piping Inspection Code. https://www.nrc.gov/docs/ML1233/ML12339 A557.pdf Perencanaan penjadwalan perbaikan menggunakan Matrix Interval Inspection dimana untuk rangking 2D (kategori kekritisan medium risk) dan confidence factor sebesar 2 diperoleh rentang waktu perbaikan selama 8 tahun kedepan sebelum sisa umur layan tiba. Kemudian hasil tersebut diolah menggunakan Microsoft Excel dengan modul Gantt Chart dapat dilihat pada Gambar 2., dimana didapatkan jadwal perbaikan pada pipa penyangga jembatan pada tahun 2027 mendatang dari tahun inspeksi yang dilakukan pada tahun 2019. American Petroleum Institute. (2002). API 580: Risk Based Inspection. https://www.api.org/products-and- services/individual-certification- programs/certifications/api580 Arunraj, N. S., & Maiti, J. (2007). Risk-based maintenance—Techniques and applications. Journal of Hazardous Materials, 142(3), 653–661. https://doi.org/10.1016/j.jhazmat.2006.06.06 9 3.3. Pembahasan Berdasarkan hasil penelitian ini, didapatkan hasil sisa umur layan (Remaining Life) terpendek yaitu 9 tahun mendatang pada pipa penyangga jembatan dengan kode PL 2 segmen C. Remaining Life tersebut dijadikan acuan karena pipa penyangga jembatan dengan kode PL 2 segmen C ini memiliki ketebalan pipa tertipis, sehingga mendapatkan hasil model risiko korosi tertinggi yang dapat dikatakan sebagai corrosion specific area, dengan nilai laju korosi (Corrosion Rate) sebesar 0,151 mm/tahun dengan kategori sedang, dimana nilai laju korosi tersebut lebih besar jika dibandingkan dengan pipa penyangga PL 3 C dan PL 4 C. Semakin tinggi nilai laju korosi, semakin rendah Remaining Life pada jembatan, dan sebaliknya. y g g g Penentuan jadwal perbaikan pada pipa penyangga jembatan PCK-03-B penghubung jalur pengeboran minyak melalui dua tahap, yaitu perencanaan penjadwalan perbaikan dan penjadwalan perbaikan. Hasil perencanaan matriks interval inspeksi ditentukan dari kategori kekritisan yang telah didapat berdasarkan Criticality Matrix dengan indeks angka keyakinan (confidence factor) untuk mendapatkan rentang perencanaan jadwal perbaikan pipa penyangga jembatan PCK-03-B selanjutnya. Setiap pipa penyangga memiliki tingkat risiko yang berbeda, dengan indeks angka keyakinan (confidence factor), dimana dari semua pipa penyangga jembatan memiliki confidence factor yang sama yaitu kode 2 dengan deteriorasi dapat diprediksi, data cacat diterima standar, dan data lengkap. Berdasarkan Tabel 8 maka dapat disimpulkan jika perencanaan penjadwalan perbaikan pada pipa penyangga jembatan dengan kode PL 2 C dan PL 3 C memiliki rentang waktu perbaikan selama 8 Analisis menggunakan metode Risk Based Maintenance bertujuan untuk mengurangi risiko yang akan timbul akibat kegagalan yang terjadi pada pipa penyangga jembatan PCK-03-B, sehingga dapat menjaga fungsi selama jembatan tersebut beroperasi. Risiko yang diperoleh berdasarkan dari kemungkinan kegagalan (Probability of Failure) dan konsekuensi kegagal- an (Consequence of Failure). Kedua risiko http://dx.doi.org/10.30656/intech.v9i1.6098 92 Jurnal INTECH Teknik Industri Universitas Serang Raya Vol 9 No 1 Juni 2023, 86-94 Teknik ITS, 4(1), G1–G5. http://ejurnal.its.ac.id/index.php/teknik/articl e/view/8931 tersebut dianalisis sehingga dapat ditentukan tingkat risiko pada pipa penyangga jembatan menggunakan Criticality Matrix (tingkat kekritisan) berdasarkan API 580 : 2002, dimana diperoleh hasil pada pipa penyangga dengan kode PL 2 C sebagai acuan kondisi keseluruhan jembatan PCK-03-B dengan memiliki tingkat risiko kerusakan pipa rangking 2D, yang berarti termasuk rating sedang (moderate), dengan nilai PoF sebesar 2 (dua) dan nilai COF sebesar 10 (sepuluh) atau kategori D (berdasarkan klasifikasi nilai CoF). Peningkatan kegiatan maintenance dilakukan dengan penjadwalan yang tepat sebelum waktu sisa umur layan (Remaining Life). 4. KESIMPULAN Fontana, M. G. (1986). Corrosion Engineering. McGraw-Hill. https://books.google.co.id/books?id=N95EA AAACAAJ Berdasarkan pembahasan hasil penelitian diatas, maka dapat disimpulkan bahwa tujuan penelitian telah tercapai yaitu untuk memi- nimalisasi tingkat risiko yang ditimbulkan akibat kerusakan material pada pipa penyangga jembatan PCK-03-B. Kondisi pipa penyangga, sisa umur layan (remaining life) dan tingkat risiko telah dihitung dan dianalisis, sehingga dapat ditentukan penjadwalan perbaikan yang dibutuhkan. Hasil perhitungan dan analisis tingkat risiko pada pipa penyangga dengan kode PL 2 C (terkritis) dijadikan sebagai acuan kondisi keseluruhan jembatan PCK-03-B sehingga diperoleh rentang penjadwalan perbaikan jembatan PCK-03-B ditetapkan pada 8 tahun (per-2019) mendatang yaitu di tahun 2027, sebelum masa umur pakai habis, sehingga dapat menghindari risiko terjadinya kecelakaan yang tidak diharapkan. Jaderi, F., Ibrahim, Z. Z., & Zahiri, M. R. (2019). Criticality analysis of petrochemical assets using risk based maintenance and the fuzzy inference system. Process Safety and Environmental Protection, 121, 312–325. https://doi.org/10.1016/j.psep.2018.11.005 Khan, F. I., & Haddara, M. (2004). Risk-based maintenance (RBM): A new approach for process plant inspection and maintenance. Process Safety Progress, 23(4), 252–265. https://doi.org/10.1002/prs.10010 Knöfel, D. (1978). Corrosion of Building Materials. Van Nostrand Reinhold. https://books.google.co.id/books?id=l5mwA AAAIAAJ Penelitian selanjutnya perlu berfokus pada metode dan teknik pemeliharaan yang efektif, baik yang bersifat pencegahan maupun penanggu- langan yang dapat meminimalisasi risiko korosi pada pipa penyangga sekaligus risiko terjadinya kecelakaan kerja seperti runtuhnya jembatan sebelum masa umur pakai digunakan. Limantoro, D. (2013). Total Productive Maintenance di PT. X. Jurnal Titra, 1(1), 13– 20. https://publication.petra.ac.id/index.php/tekn ik-industri/article/view/131 Mahardhika, M. M., & Pramudyo, C. S. (2023). Pengendalian Risiko Kecelakaan Kerja Menggunakan Metode HIRA dan HAZOP (Studi Kasus: WL Alumunium, Yogyakarta). Jurnal Serambi Engineering, 8(2), 5066– https://doi.org/10.36055/jiss.v4i1.4089 https://doi.org/10.36055/jiss.v4i1.4089 DAFTAR PUSTAKA DAFTAR PUSTAKA Afandi, Y. K., Arief, I. S., & Amiadji, A. (2015). Analisa Laju Korosi pada pelat baja Karbon dengan Variasi ketebalan coating. Jurnal http://dx.doi.org/10.30656/intech.v9i1.6098 93 Jurnal INTECH Teknik Industri Universitas Serang Raya Vol 9 No 1 Juni 2023, 86-94 http://dx.doi.org/10.30656/intech.v9i1.6098 5073. https://doi.org/10.32672/jse.v8i2.5450 5073. https://doi.org/10.32672/jse.v8i2.5450 Saputra, S. P., & Rahayu, G. H. N. N. (2019). Analisis Perhitungan Overall Equipment Effectiveness (OEE) Pada Mesin Pengemasan Susu Kental Manis Sachet PT Frisian Flag Indonesia [Universitas Pancasila]. https://perpus.univpancasila.ac.id/index.php /index.php?p=show_detail&id=123593 Mentari, D., & Lie, D. (2017). Analisis Pelaksanaan Kegiatan Pemeliharaan (Maintenance) Terhadap Kualitas Produk Pada Cv Green Perkasa Pematangsiantar. Maker: Jurnal Manajemen, 3(1), 40–48. https://www.maker.ac.id/index.php/maker/ar ticle/view/55 National Association of Engineer. (2002). Control of external corrosion on underground or submerged metallic piping systems. http://zinoglobal.com/wp- content/uploads/2019/12/NACE-RP-0169- 2002.pdf Siregar, T., Sitorus, E., Priastomo, Y., Bachtiar, E., Siagian, P., Mohamad, E., Gurning, K., Hasibuan, F. A., Destiarti, L., & Marzuki, I. (2021). Korosi dan Pencegahannya. Yayasan Kita Menulis. http://repository.uhn.ac.id/handle/12345678 9/8497 Rahayu, G. H. N. N. (2023). Tragedi Plumpang dan Urgensi Manajemen Risiko Bisnis. https://kumparan.com/gama-harta/tragedi- plumpang-dan-urgensi-manajemen-risiko- bisnis-1zy28slhriD Tulloh, R., Sodikin, I., & Khasanah, R. (2019). Usulan Perawatan Buoy Tsunami Dengan Menggunakan Metode Risk Based Maintenance (Rbm). Jurnal Rekavasi, 7(1), 51–61. https://journal.akprind.ac.id/index.php/rekav asi/article/view/1328 Rahayu, G. H. N. N., & Fitri, F. A. (2020). Penentuan alternatif pilihan strategi mitigasi riisiko kecelakaan kerja dengan metode ANP di PT XYZ. Jurnal Rekayasa Dan Optimasi Sistem Industri, 2(2), 44–50. https://journal.univpancasila.ac.id/index.php /jrosi/article/view/2476 Wijaya, S., & Sholihin, M. Y. M. (2022). Analisa Risiko Material Pipa Penyangga Jembatan PCK-03-A Pada Pengeboran Minyak di PT. X Menggunakan Metoda Risk Based Maintenance. Jurnal Teknik Industri, 3(02), 21–28. https://jurnal.pelitabangsa.ac.id/index.php/J UTIN/article/view/1518 Rahayu, G. H. N., Riyanto, R., & Ismail, A. H. (2018). Analisis Risiko Biaya Operasi Distal Medial Pada Departemen Orthopedi Dan Traumatologi Di Rumah Sakit XYZ. Journal Industrial Servicess, 4(1), 57–63. 94 http://dx.doi.org/10.30656/intech.v9i1.6098
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Gender-specific contribution of cardiometabolic index and lipid accumulation product to left ventricular geometry change in general population of rural China
BMC cardiovascular disorders
2,018
cc-by
9,682
Wang et al. BMC Cardiovascular Disorders (2018) 18:62 https://doi.org/10.1186/s12872-018-0798-0 Wang et al. BMC Cardiovascular Disorders (2018) 18:62 https://doi.org/10.1186/s12872-018-0798-0 Abstract Background: Despite current interest in the unfavorable impact of cardiometabolic index (CMI) and lipid accumulation product (LAP) on diabetes and cardiovascular risk, information regarding the relation of CMI and LAP to left ventricular (LV) geometry has not been specifically addressed. We aimed to examine the hypothesis: (1) CMI and LAP represent an independent determinant of LV remodeling in general population of rural China; (2) there are gender differences in obesity-related alterations in terms of LV morphology. Methods: The sample for this cross-sectional analysis included 11,258 participants (mean age 53.9 years; 54.0% females) who underwent assessment of basic metabolic and anthropometric parameters in rural areas of northeast China. Comprehensive echocardiography-defined LV geometric pattern was determined according to left ventricular mass index and relative wall thickness. Results: The prevalence rate of eccentric and concentric LV hypertrophy (LVH) presented a proportional increase with elevated quartiles of CMI and LAP in a dose-response manner (all P < 0.005). When CMI and LAP were entered as a continuous variable in multivariable adjusted model, we observed the independent effect of 1 SD increment in CMI and LAP with the probability of eccentric and concentric LVH, while this relationship was more pronounced in females than in males. Likewise, the odds ratio comparing the top versus bottom quartiles of CMI were 2.105 (95%CI:1.600–2. 768) for eccentric LVH and 2.236 (95%CI:1.419–3.522) for concentric LVH in females. Males in the highest CMI quartile exhibited a nearly doubled (OR:1.724, 95%CI:1.287–2.311) and 1.523-fold (95%CI:1.003–2.313) greater risk of eccentric and concentric LVH, respectively. Increasing LAP entailed a higher possibility of eccentric LVH by a factor of 3.552 and 1.768 in females and males, respectively. In contrast to females, where LAP fourth quartile and concentric LVH were positively associated (OR:2.544, 95%CI:1.537–4.209), higher LAP did not correlate with concentric LVH in males (OR:1. 234, 95%CI:0.824–1.849). Conclusions: CMI and LAP give rise to a new paradigm of accounting for gender difference in obesity-related abnormal LV geometry, an effect that was substantially greater in females. These two indices, acting in concert, may also be advantageous prognostically for refining cardiovascular risk stratification in individuals with LV remodeling. Gender-specific contribution of cardiometabolic index and lipid accumulation product to left ventricular geometry change in general population of rural China Haoyu Wang1, Yingxian Sun1, Zhao Li1, Xiaofan Guo1, Shuang Chen1, Ning Ye1, Yichen Tian1 and Lijun Zhang2* Haoyu Wang1, Yingxian Sun1, Zhao Li1, Xiaofan Guo1, Shuang Chen1, Ning Ye1, Yichen Tian1 a * Correspondence: zljcmu1h@163.com 2Department of Hematology, The First Hospital of China Medical University, 155 Nanjing North Street, Heping District, Shenyang 110001, Liaoning, People’s Republic of China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Keywords: Left ventricular geometry, Left ventricular remodeling, Cardiometabolic index, Lipid accumulation product, Obesity, Adipose tissue, Gender-specific, Epidemiology * Correspondence: zljcmu1h@163.com 2 * Correspondence: zljcmu1h@163.com p j @ 2Department of Hematology, The First Hospital of China Medical University, 155 Nanjing North Street, Heping District, Shenyang 110001, Liaoning, People’s Republic of China Full list of author information is available at the end of the article Background and all-cause mortality [19, 20]. Lately, a novel param- eter, named cardiometabolic index (CMI), has been put forward by Ichiro Wakabayashi to adequately discrimin- ate the presence of diabetes and atherosclerotic progres- sion [21–23]. It represents a combination of TG/ high- density lipoprotein cholesterol (HDL-C) ratio and WHtR, highlighting the product of them integrates the blood lipid and abdominal obesity into a clinically ac- cessible and conceptually appealing marker. Of note, CMI could be clinically relevant with respect to the LV geometry, with insulin resistance and carotid intima- media thickness (IMT) having a substantial effect on cardiac remodeling [24, 25]. However, none of the stud- ies that address the impact of measures of adiposity (CMI and LAP) on the pathogenesis of abnormal LV geometry has been specifically established. In effect, the gender difference in fat quantity, anatomic storage and consequent hemodynamic influence has been proposed to drive a better understanding of the link between obes- ity and LV remodeling. Accordingly, the aim of this study was to provide further insight into exploring CMI and LAP as a reliable biomarker for gender-specific identifying those subjects at higher risk for LV geometry in general population of rural China. Obesity is recognized as an important contributor to heart failure and cardiovascular disease (CVD) inde- pendent of comorbid illnesses [1–3]. The increase in body weight and composition was thought to be associ- ated with the changes in left ventricular (LV) structure, attributable to increased hemodynamic load and a clus- ter of altered inflammatory and metabolic state [4–6]. It has been well documented that chronic volume load in response to obesity exhibited increased LV mass and cavity size, resulting in eccentric left ventricular hyper- trophy (LVH) [7, 8]. Likewise, concentric LVH character- ized by an increase in LV wall thickness (LVWT) greater than the chamber radius and elevated mass-to-volume ratio, was largely related to the impact of excess adipos- ity [4, 8–10]. Given that being the precursor for worsen- ing cardiac function, LV structural abnormalities impose an elevated risk for progression of symptomatic heart fail- ure and are considered as a powerful, integrated predictor of cardiovascular morbidity and mortality [11–13]. On this basis, it is now becoming evident that the effect of obesity on LV remodeling offers a conceivable pathophysi- ologic explanation for the relationship of excess body fat with adverse cardiovascular outcomes. Page 2 of 11 Wang et al. BMC Cardiovascular Disorders (2018) 18:62 Study population y The data originated from a large population-based epi- demiological cross-sectional study of 11,956 permanent residents (≥35 years of age) in rural areas of China. The full details regarding the study design and definitions were extensively described elsewhere [26–28]. After 698 of subjects were excluded because of missing biochem- ical and clinical covariates, our complete dataset (adipos- ity indices and echocardiographic data) consisted of 11,258 participants in the current analysis. The Ethics Committee of China Medical University (Shenyang, China) approved the study protocol. We obtained writ- ten informed consent from each participant before enrollment, and the whole data and procedures con- formed to the principles of ethical standards. There is a diverse geometric pattern of left ventricle adaptations to fat distribution, in view of adipose depots inherently involve disparate metabolic process, structural composition, and functional manifestation [14]. In this context, a sample of 1073 young individuals from Boga- lusa Heart Study revealed that increases in body mass index (BMI), waist circumference (WC), and waist-to- height ratio (WHtR) were demonstrated to have inde- pendent influences on the development of eccentric LVH but not concentric LVH [15]. Interestingly, in a multiethnic cohort of 1262 adults from the Dallas Heart Study, the predominant impact of general or central adi- posity on LV phenotype was concentric remodeling without additional ejection fraction change [16]. The no- tion that visceral adipose tissue, an index of central adi- posity, was a key independent determinant of LV concentricity and unfavorable hemodynamics has been recently arisen from two observations [17, 18]. These findings placed particular emphasis on the potential role for adiposity and its distribution in LV morphology, accounting for the pathogenic correlates of future heart failure. Background Determining which measure of adiposity and obesity contributes to the changes in LV geometry could promote early identification and prevention of cardiovascular events. Echocardiography Information on the echocardiography examination was conducted according to standardized procedures, as pre- viously described [28, 29]. Two-dimensional, M-mode, spectral and color Doppler transthoracic echocardiog- raphy were performed with a commercially available Doppler machine (Vivid, GE Healthcare, United States) and were interpreted by three skilled echocardiographers. p y g p Measurements of LV end-diastolic internal dimension (LVIDD), interventricular septal thickness (IVST), and posterior wall thickness (PWT) were determined by the optimized parasternal long-axis view on the basis of the recommendation from the American Society of Echocar- diography [30]. Using the Teichholz equations [31], we assessed the LV end-diastolic volume (LVEDV), LV end- systolic volume (LVESV) to generate LV ejection fraction. Left ventricular mass (LVM) was derived from a necropsy validated formula: LVM = 0.8× [1.04{(IVST + PWT + LVIDD)3–LVIDD3}] + 0.6 g [32]. Normalization of LVM for height to the power of 2.7 was regarded as left ventricular mass index (LVMI). LVWT = (IVST + PWT) / 2. The relative wall thickness (RWT) was calculated as 2 × PWT/ LVIDD. The presence of LV hypertrophy was categorized by gender-specific cutoffs of LVMI > 46.7 g/ m2.7 for females and > 49.2 g/m2.7 for males. Concentric LV geometry was defined as a partition value of 0.42 for RWT in both females and males [33]. Four different pattern of LV geometry were defined: (1) normal geometry Statistical analyses Analyses were conducted separately for each gender. Baseline characteristics of all individuals are summarized as mean ± standard deviation or median (interquartile range) and numbers (percentage) as appropriate. Loga- rithmic transformation was performed for CMI and LAP because of skewness. Characteristics were assessed females and males using Student’s t or Mann-Whitney test to examine differences in means, while χ2 test for independence was utilized to compare differences of cat- egorical variables in proportions. Baseline demographic, clinical, biochemical, and anthropometric measures and echocardiogram parameters were compared across four categories of LV geometry using one-way ANOVA or Kruskal-Wallis test for continuous variables and χ2 test for categorical data. The strength of linear association between left ventricular geometry parameters and anthropometric indices was tested by Spearman correl- ation coefficients. CMI and LAP were stratified into quartiles in accordance with the distribution of each an- thropometric measure. We obtained gender-specific esti- mation of the OR for 1 SD increment in CMI and LAP to predict the risk of LV geometric abnormalities. Add- itionally, stratified by gender, multivariable logistic re- gression analysis was applied to explore an independent association of quartiles of CMI and LAP on the risk of eccentric LVH and concentric LVH, where three models were evaluated when potential confounding risk factors were progressively established. The results are expressed as odds ratios (ORs) and 95% confidence intervals (CIs). All of the statistical analyses involved the application of SPSS 22.0 software (IBM Corp) and a two-tailed P < 0.05 was adopted to be statistically significant. Fasting antecubital vein blood specimens were de- signed to be obtained by each participant in the morning after an overnight fasting with 12 h for assessing fasting plasma glucose (FPG), TG, and HDL-C. Comprehensive storage process and laboratory measurement methods were available in the previous reports [26–28]. The TG/ HDL-C ratio was calculated using available TG and HDL-C values. Definitions CMI was calculated according to the formula: CMI = TG/HDL-C × WHtR. Lipid accumulation product (LAP) is determined by using the following equation: LAP = TG (mmol/l) × (WC (cm) −58) for women and LAP = TG (mmol/l) × (WC (cm) −65) for men. Data collection and measurements The detailed process about data collection and methods selection of this sample has been fully reported in our prior publications [26–28]. Cardiologists and trained nurses administered a structured questionnaire to document specified data on demographic, health- related behaviors, anthropometric parameters, history of CVD (coronary heart disease, arrhythmia and heart failure), use of antihypertensive drug (at least one type versus no), use of antidiabetic drug (at least one type versus no), and use of lipid-lowering drug (at least one type versus no). Lipid accumulation product (LAP) is an emerging cen- tral lipid accumulation parameter in the light of taking both waist circumference (WC) and triglyceride (TG) levels into account, with prior studies reporting its strong predictive of clinical cardiovascular consequences Page 3 of 11 Wang et al. BMC Cardiovascular Disorders (2018) 18:62 Wang et al. BMC Cardiovascular Disorders (2018) 18:62 was confirmed when the values of LVMI and RWT were within normal range; (2) concentric LV remodeling with normal LVMI and increased RWT; (3) increased LVMI and normal RWT was classified as eccentric LVH; and (4) concentric LVH with increases in both LVMI and RWT. Study participants waited for at least 5 min in a relaxed and sitting position. Then blood pressure was measured by two randomly trained observers. The three consecutive readings were collected on the right arm at 1–2 min inter- vals and the average value was recorded for analysis. Anthropometric indices were conducted when the subjects wore in light clothing without shoes. Their weight was quantified to the nearest 0.1 kg in the utility of a calibrated digital scale. A portable stadiometer was used for height measurement (rounded to nearest 0. 1 cm) in a standing position. After full expiration, WC was measured using a steel measuring tape from the horizontal line at 1 cm above the belly button. An- thropometric indices were measured twice and then av- eraged. BMI was calculated as weight per height squared (kg/m2). WHtR was defined as WC divided by height in meters squared. Results Demographic, clinical, and echocardiographic characteristics of study participants stratified by gender The comparison between females (n = 6079) and males (n = 5179) with regard to the demographic, clinical, and echocardiographic characteristics were shown in Table 1. The mean age of the study subjects was 53.86 ± 10. Demographic, clinical, and echocardiographic characteristics of study participants stratified b Wang et al. Odds ratio (OR) and 95% confidence intervals for abnormal left ventricular geometry according to gender- specific continuous or quartiles of CMI and LAP Table 2 Spearman’s correlation analysis of CMI and LAP with left ventricular geometry parameters by gender LVM LVMI LVIDD LVEDVI LVESVI LVWT RWT Female CMI 0.276a 0.282a 0.154a 0.181a 0.139a 0.204a 0.130a LAP 0.382a 0.341a 0.238a 0.217a 0.167a 0.300a 0.141a Male CMI 0.195a 0.159a 0.118a 0.080a 0.039b 0.150a 0.074a LAP 0.289a 0.207a 0.207a 0.117a 0.064a 0.243a 0.058a Abbreviations: LVM left ventricular mass, LVMI left ventricular mass index, LVIDD left ventricular end-diastolic internal dimension, LVEDVI left ventricular end-diastolic volume index, LVESVI left ventricular end-systolic volume index, LVWT, LV wall thickness, RWT relative wall thickness, CMI cardiometabolic index, LAP lipid accumulation product a P < 0.01; b P < 0.05; Demographic, clinical, and echocardiographic characteristics of study participants stratified b BMC Cardiovascular Disorders (2018) 18:62 Page 4 of 11 Table 1 Demographic, clinical, and echocardiographic characteristics of study participants stratified by gender Variable Total (N = 11,258) Female (N = 6079) Male (N = 5179) P value* Age (years) 53.86 ± 10.54 53.39 ± 10.32 54.42 ± 10.78 < 0.001 Race (Han) (%) 10,681 (94.9) 5769 (94.9) 4912 (94.8) 0.893 Primary school or below (%) 5629 (50.0) 3459 (56.9) 2710 (41.9) < 0.001 Family income > 20,000 CNY/year (%) 3709 (32.9) 2014 (33.1) 1695 (32.7) 0.008 Low physical activity (%) 3336 (29.6) 2171 (35.7) 1165 (22.5) < 0.001 Diet score 2.32 ± 1.13 2.13 ± 1.11 2.54 ± 1.10 < 0.001 Current smoker (%) 3959 (35.2) 996 (16.4) 2963 (57.2) < 0.001 Current drinker (%) 2523 (22.4) 177 (2.9) 2346 (45.3) < 0.001 Systolic blood pressure (mmHg) 141.69 ± 23.40 140.09 ± 24.02 143.57 ± 22.51 < 0.001 Diastolic blood pressure (mmHg) 82.04 ± 11.75 80.57 ± 11.53 83.76 ± 11.78 < 0.001 Fasting plasma glucose (mmol/L) 5.91 ± 1.63 5.87 ± 1.61 5.95 ± 1.67 0.007 TG (mmol/L) 1.24 (0.88–1.89) 1.26 (0.90–1.91) 1.23 (0.86–1.88) 0.017 HDL-C (mmol/L) 1.41 ± 0.38 1.41 ± 0.34 1.41 ± 0.42 0.717 TG/HDL-C ratio 0.92 (0.59–1.54) 0.93 (0.60–1.53) 0.91 (0.58–1.57) 0.566 WC (cm) 82.43 ± 9.82 81.29 ± 9.73 83.76 ± 9.76 < 0.001 Height (m) 1.61 ± 0.08 1.56 ± 0.06 1.66 ± 0.06 < 0.001 WHtR 0.51 ± 0.06 0.50 ± 0.06 0.52 ± 0.06 < 0.001 Hypertension (%) 5738 (51.0) 2949 (48.5) 2789 (53.9) < 0.001 Diabetes (%) 1175 (10.4) 662 (10.9) 513 (9.9) 0.089 Antihypertensive drug (%)a 1701 (15.1) 1034 (17.0) 667 (12.9) < 0.001 Antidiabetic drug (%)a 449 (4.0) 294 (4.8) 155 (3.0) < 0.001 Lipid-lowering drug (%)a 371 (3.3) 211 (3.5) 160 (3.1) 0.258 History of CVD Coronary heart disease (%) 580 (5.2) 363 (6.0) 217 (4.2) < 0.001 Arrhythmia (%) 617 (5.5) 421 (6.9) 196 (3.8) < 0.001 Heart failure (%) 104 (0.9) 58 (1.0) 46 (0.9) 0.716 CMI 0.47 (0.29–0.82) 0.48 (0.30–0.83) 0.46 (0.27–0.82) 0.002 LAP (cm·mmol/L) 25.44 (13.39–47.38) 28.31 (16.17–50.95) 21.95 (10.71–43.60) < 0.001 ECG measures LVIDD (cm) 4.74 ± 0.46 4.54 ± 0.41 4.89 ± 0.45 < 0.001 IVST (cm) 0.89 ± 0.28 0.86 ± 0.26 0.92 ± 0.30 < 0.001 PWT (cm) 0.87 ± 0.31 0.85 ± 0.33 0.90 ± 0.29 < 0.001 LVWT (cm) 2.79 ± 0.28 2.69 ± 0.25 2.90 ± 0.27 < 0.001 RWT 0.38 ± 0.33 0.39 ± 0.41 0.37 ± 0.20 0.050 LVM (g) 132.32 (113.63–158.21) 122.26 (105.33–137.72) 147.78 (128.02–174.52) < 0.001 LVMI (g/m2.7) 36.99 (31.98–43.43) 36.69 (31.52–42.84) 37.40 (32.46–44.13) < 0.001 LVEDV (ml) 104.17 ± 22.95 95.87 ± 19.17 113.94 ± 23.18 < 0.001 LVESV (ml) 39.45 ± 13.60 36.16 ± 11.68 43.34 ± 14.64 < 0.001 LVEDVI (ml/m2.7) 29.07 ± 6.35 29.18 ± 6.29 28.93 ± 6.42 0.037 LVESVI (ml/m2.7) 11.01 ± 3.76 11.00 ± 3.65 11.01 ± 3.88 0.902 LV ejection fraction (%) 62.05 ± 10.02 62.22 ± 9.76 61.85 ± 10.31 0.048 Normal geometry 8780 (78.0) 4656 (76.6) 4124 (79.6) < 0.001 Concentric LV remodeling 792 (7.0) 466 (7.7) 326 (6.3) 0.005 Eccentric LVH 1185 (10.5) 689 (11.3) 496 (9.6) 0.002 Page 5 of 11 Wang et al. Demographic, clinical, and echocardiographic characteristics of study participants stratified b BMC Cardiovascular Disorders (2018) 18:62 Table 1 Demographic, clinical, and echocardiographic characteristics of study participants stratified by gender (Continued) Variable Total (N = 11,258) Female (N = 6079) Male (N = 5179) P value* Concentric LVH 501 (4.5) 268 (4.4) 233 (4.5) 0.817 Data are expressed as mean ± standard deviation or median (interquartile range) and numbers (percentage) as appropriate. CNY China Yuan (1CNY = 0.158 USD), TG triglyceride, HDL-C high-density lipoprotein cholesterol, WHtR waist-to-height ratio, CVD cardiovascular disease, CMI cardiometabolic index, LAP lipid accumula- tion product, LV left ventricular, IVST interventricular septal thickness, LVIDD left ventricular end-diastolic internal dimension, PWT posterior wall thickness, LVWT, LV wall thickness, RWT, relative wall thickness, LVM left ventricular mass, LVMI left ventricular mass index, LVEDV left ventricular end-diastolic volume, LVESV left ventricular end-systolic volume LVEDVI, left ventricular end-diastolic volume index, LVESVI left ventricular end-systolic volume index, ECG echocardiogram, LVH left ventricular hypertrophy * P values are for a Student’s t or Mann-Whitney test (continuous) and chi-square test (categorical) comparison across left ventricular geometry a At least one, versus no LVMI, LVIDD, LVEDVI, LVESVI, LVWT and RWT for both sexes, and the strength of the correlations was more pronounced among females (all P < 0.05). Put to- gether, subjects showed a greater LV geometry parameter response to ascending LAP, a pattern that was more prominent than CMI. LVMI, LVIDD, LVEDVI, LVESVI, LVWT and RWT for both sexes, and the strength of the correlations was more pronounced among females (all P < 0.05). Put to- gether, subjects showed a greater LV geometry parameter response to ascending LAP, a pattern that was more prominent than CMI. 54 years, and the average age of males was slightly higher than females. Overall, the metabolic parameters like systolic blood pressure (SBP), diastolic blood pres- sure (DBP), FPG, WHtR, and WC are prone to higher in males than females (all P < 0.05). No significant differ- ences were presented between the groups for HDL-C, TG/HDL-C and diabetes. Relative to males, females had greater TG, CMI, and LAP, with a more frequency in use of antihypertensive drug, antidiabetic drug, and his- tory of coronary heart disease and arrhythmia. For de- scriptive results of echocardiographic indices related to LV structure, females were associated with lower LVIDD, IVST, PWT, LVWT, LVM, LVMI, LVEDV, LVESV in comparison to males, without a significant distinction in LV end-systolic volume index (LVESVI). Odds ratio (OR) and 95% confidence intervals for abnormal left ventricular geometry according to gender- specific continuous or quartiles of CMI and LAP Odds ratio (OR) and 95% confidence intervals for abnormal left ventricular geometry according to gender- specific continuous or quartiles of CMI and LAP The multivariable logistic regression analysis was carried out to evaluate the gender-specific association of CMI and LAP with eccentric and concentric LVH (Table 3). In analyses modeling CMI and LAP as a continuous variable, we revealed a 32% and 50% higher risk for ec- centric LVH with each SD increment in CMI and LAP levels in females, respectively. Similar results were ac- quired for the effect of 1 SD increase in CMI and LAP with eccentric LVH in males (OR, 1.187; 95% CI, 1.075– 1.311; OR, 1.251; 95% CI, 1.123–1.394, respectively; model 3), while this association was particularly pro- nounced among females. Likewise, CMI carried concen- tric LVH odds (95% CIs) of 1.280 (1.122 to 1.461) and 1. 172 (1.019 to 1.347) in females and males, respectively, per SD increase. The relationship between LAP and con- centric LVH was significant with higher risks in the former (Females, OR, 1.384; 95% CI, 1.191–1.609; Males, OR, 1.233; 95% CI, 1.056–1.441). In models Characteristics of study population according to the left ventricular geometry were listed in Additional file 1: Table S1. Of 11,258 adults, 8780 (78.0%) presented with normal LV geometry, 792 (7.0%) presented with concen- tric remodeling, 1185 (10.5%) presented with eccentric LVH, and 501 (4.5%) presented with concentric LVH. The level of SBP, DBP, FPG, TG, and TG/HDL-C tended to increase in proportion to LV structure from normal LV geometry to concentric LVH. In anthropometric measures of CMI and LAP, participants with either ec- centric or concentric LVH showed the more unfavorable mean value than normal LV geometry. Especially, use of antihypertensive drug, and antidiabetic drug, lipid- lowering drug and history of CVD (coronary heart dis- ease, arrhythmia and heart failure) were more prevalent in subjects with LVH, regardless of the geometric pat- tern, than in adults with normal LV geometry. Demographic, clinical, and echocardiographic characteristics of study participants stratified b As expected, in- creased RWT, LV end-diastolic volume index (LVEDVI), and LV ejection fraction were found in females. The pro- portion of concentric remodeling and eccentric LVH were somewhat higher in females apart from concentric LVH which was quite comparable in both genders. 54 years, and the average age of males was slightly higher than females. Overall, the metabolic parameters like systolic blood pressure (SBP), diastolic blood pres- sure (DBP), FPG, WHtR, and WC are prone to higher in males than females (all P < 0.05). No significant differ- ences were presented between the groups for HDL-C, TG/HDL-C and diabetes. Relative to males, females had greater TG, CMI, and LAP, with a more frequency in use of antihypertensive drug, antidiabetic drug, and his- tory of coronary heart disease and arrhythmia. For de- scriptive results of echocardiographic indices related to LV structure, females were associated with lower LVIDD, IVST, PWT, LVWT, LVM, LVMI, LVEDV, LVESV in comparison to males, without a significant distinction in LV end-systolic volume index (LVESVI). As expected, in- creased RWT, LV end-diastolic volume index (LVEDVI), and LV ejection fraction were found in females. The pro- portion of concentric remodeling and eccentric LVH were somewhat higher in females apart from concentric LVH which was quite comparable in both genders. Spearman’s correlation analysis of CMI and LAP with left ventricular geometry parameters by gender Abbreviations: LVM left ventricular mass, LVMI left ventricular mass index, LVIDD left ventricular end-diastolic internal dimension, LVEDVI left ventricular end-diastolic volume index, LVESVI left ventricular end-systolic volume index, LVWT, LV wall thickness, RWT relative wall thickness, CMI cardiometabolic index, LAP lipid accumulation product a P < 0.01; b P < 0.05; Table 2 indicated the unadjusted Spearman’s correlation coefficients regarding the association between measures of obesity and LV geometry by different genders. In- creasing of CMI and LAP were highly related to LVM, Wang et al. Spearman’s correlation analysis of CMI and LAP with left ventricular geometry parameters by gender As a result of this, it was consistent in females that higher CMI and LAP levels were independently and positively related to the probability of eccentric or con- centric LVH in a dose-response pattern (all P for trend < 0.001). In contrast to females, when the highest and lowest categories were compared, a high LAP level dem- onstrated a 1.77-fold increase in the odds of eccentric LVH in males (95% CI, 1.305 to 2.396; P for trend< 0. 001), whereas there was no relationship between con- centric LVH and increasing LAP (P for trend = 0.120). Additionally, ORs of eccentric and concentric LVH in- creased by a factor of 1.72 and 1.52 for males with rising levels of CMI, respectively. In general, the associations of CMI and LAP with eccentric LVH or concentric LVH were more prominent in females. Confirming CMI and LAP as a key independent deter- minant of LV hypertrophy and geometry had crucial im- plications for exploring potential areas of future investigations targeting CMI and LAP gender-specifically to prevent or weaken the effects of LV remodeling on heart disease. Early data pointed out that due to a state of chronic volume overload, the prime effect of obesity on LV struc- ture has been considered as eccentric LVH, with a resulting parallel increases in LV cavity size and LVWT and no alteration in RWT [7, 8, 34]. Instead, the results of recent studies consistently advocated a concentric pattern to be linked with increased weight, in nature with a greater extent increases in LVWT as compared to LVIDD accompanied by an increased RWT [4, 10, 35]. The majority of prior works evaluating the adverse im- pact of adiposity on LV geometry have mainly focused on parameters of general obesity such as BMI. The im- plication of BMI on eccentric LVH, represented by higher LVEDV, and concentric LVH geometric pattern, expressed by increased LVM/volume ratio, has been pre- viously demonstrated in a cross-sectional fashion [4, 36]. A growing body of evidence supports the prognostic value of long-term change in BMI on certain aspects of LV geometry. Two researches from the Framingham Heart Study, performed in middle-aged adults, suggested that longitudinal tracking of LVWT, RWT and LV di- mensions increased progressively with BMI over a 16 years period [37, 38]. Spearman’s correlation analysis of CMI and LAP with left ventricular geometry parameters by gender BMC Cardiovascular Disorders (2018) 18:62 Table 3 Odds ratio (OR) and 95% confidence intervals for abnormal left ventricular geometry according to gender-specific continu- ous or quartiles of CMI and LAP (Continued) Variables Eccentric LVH Concentric LVH OR (95% CI) OR (95% CI) Case (%) Model 1 Model 2 Model 3 Case (%) Model 1 Model 2 Model 3 165 (33.5) 2.215 (1.670– 2.938) 2.489 (1.864– 3.324) 1.768 (1.305– 2.396) 89 (38.2) 2.063 (1.421– 2.995) 2.345 (1.606– 3.423) 1.234 (0.824– 1.849) P value for trend < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.091 Model 1: unadjusted; Model 2: adjusted for age, race, education level, family income, diet score, current smoking and drinking status, physical activity; Model 3: adjusted for all the factors in Model 2 and hypertension, diabetes, antihypertensive drug, and antidiabetic drug, lipid-lowering drug, and history of cardiovascular disease (coronary heart disease, arrhythmia and heart failure). Abbreviations: OR odd ratio; 95% CI 95% confidence interval, CMI cardiometabolic index, LAP lipid accumulation product, LVH left ventricular hypertrophy Model 1: unadjusted; Model 2: adjusted for age, race, education level, family income, diet score, current smoking and drinking status, physical activity; Model 3: adjusted for all the factors in Model 2 and hypertension, diabetes, antihypertensive drug, and antidiabetic drug, lipid-lowering drug, and history of cardiovascular disease (coronary heart disease, arrhythmia and heart failure). Abbreviations: OR odd ratio; 95% CI 95% confidence interval, CMI cardiometabolic index, LAP lipid accumulation product, LVH left ventricular hypertrophy incorporating CMI and LAP quartiles, after taking into account the effect of confounding factors (model 2), par- ticipants in the highest quartile of CMI and LAP exhib- ited a 2.74-fold (95%CI, 2.13 to 3.61) and 4.99-fold (95% CI, 3.68 to 6.77) elevated risk of eccentric LVH com- pared with the lowest quartile in females. Similarly, the adjusted ORs of concentric LVH were statistically signifi- cant in the top quartile of CMI (OR, 3.610; 95% CI, 2. 330 to 3.593) and LAP (OR, 4.904; 95% CI, 3.206 to 7. 949). Adding SBP, DBP, FPG, use of antihypertensive drug, and antidiabetic drug, lipid-lowering drug and his- tory of CVD (coronary heart disease, arrhythmia and heart failure) to the model 3 did not materially alter the results and the associations maintained statistical signifi- cance. Spearman’s correlation analysis of CMI and LAP with left ventricular geometry parameters by gender Notably, in a Coronary Artery Risk Development in Young Adults (CARDIA) study, in- creasing BMI longitudinally over 25 years contributes to LV structural remodeling assessed by larger LV volume, greater LVM/height, and LVM/LVEDV ratio [39]. Spearman’s correlation analysis of CMI and LAP with left ventricular geometry parameters by gender BMC Cardiovascular Disorders (2018) 18:62 Page 6 of 11 Table 3 Odds ratio (OR) and 95% confidence intervals for abnormal left ventricular geometry according to gender-specific continu- ous or quartiles of CMI and LAP Variables Eccentric LVH Concentric LVH OR (95% CI) OR (95% CI) Case (%) Model 1 Model 2 Model 3 Case (%) Model 1 Model 2 Model 3 Females CMI (per 1 SD increase) 1.601 (1.482– 1.730) 1.453 (1.339– 1.576) 1.319 (1.209– 1.438) 1.683 (1.500– 1.888) 1.527 (1.352– 1.723) 1.280 (1.122– 1.461) Quartiles of CMI Q1 (≤0.30) 84 (12.2) 1.000 (reference) 1.000 (reference) 1.000 (reference) 26 (9.7) 1.000 (reference) 1.000 (reference) 1.000 (reference) Q2 (0.30–0.48) 117 (17.0) 1.491 (1.116– 1.992) 1.341 (0.998– 1.802) 1.258 (0.934– 1.695) 45 (16.8) 1.829 (1.123– 2.981) 1.609 (0.983– 2.634) 1.451 (0.881– 2.389) Q3 (0.48–0.83) 222 (32.2) 2.938 (2.261– 3.817) 2.407 (1.841– 3.146) 2.063 (1.572– 2.709) 77 (28.7) 3.083 (1.965– 4.837) 2.443 (1.548– 3.854) 1.806 (1.135– 2.874) Q4 (> 0.83) 266 (38.6) 3.736 (2.890– 4.829) 2.742 (2.106– 3.572) 2.105 (1.600– 2.768) 120 (44.8) 5.065 (3.294– 7.787) 3.610 (2.330– 5.593) 2.236 (1.419– 3.522) P value for trend < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 LAP (per 1 SD increase) 1.874 (1.716– 2.047) 1.668 (1.541– 1.849) 1.496 (1.357– 1.650) 1.955 (1.713– 2.232) 1.742 (1.518– 1.998) 1.384 (1.191– 1.609) Quartiles of LAP Q1 (≤16.17) 54 (7.9) 1.000 (reference) 1.000 (reference) 1.000 (reference) 20 (7.5) 1.000 (reference) 1.000 (reference) 1.000 (reference) Q2 (16.17–28.31) 134 (19.5) 2.587 (1.870– 3.579) 2.356 (1.695– 3.274) 2.112 (1.515– 2.946) 40 (15.0) 1.999 (1.163– 3.435) 1.756 (1.017– 3.031) 1.395 (0.802– 2.427) Q3 (28.31–50.95) 199 (29.0) 4.034 (2.959– 5.499) 3.329 (2.429– 4.563) 2.713 (1.970– 3.735) 77 (28.9) 3.949 (2.402– 6.492) 3.095 (1.873– 5.114) 2.108 (1.263– 3.517) Q4 (> 50.95) 300 (43.7) 6.580 (4.877– 8.879) 4.957 (3.653– 6.727) 3.552 (2.587– 4.876) 129 (48.5) 6.858 (4.258– 11.046) 4.904 (3.206– 7.949) 2.544 (1.537– 4.209) P value for trend < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 Males CMI (per 1 SD increase) 1.264 (1.156– 1.383) 1.327 (1.208– 1.458) 1.187 (1.075– 1.311) 1.309 (1.155– 1.483) 1.389 (1.221– 1.579) 1.172 (1.019– 1.347) Quartiles of CMI Q1 (≤0.27) 85 (17.1) 1.000 (reference) 1.000 (reference) 1.000 (reference) 39 (16.7) 1.000 (reference) 1.000 (reference) 1.000 (reference) Q2 (0.27–0.46) 108 (21.8) 1.219 (0.908– 1.638) 1.167 (0.865– 1.575) 1.158 (0.855– 1.567) 55 (23.6) 1.348 (0.888– 2.047) 1.356 (0.890– 2.065) 1.270 (0.826– 1.954) Q3 (0.46–0.82) 138 (27.8) 1.719 (1.296– 2.280) 1.722 (1.292– 2.295) 1.553 (1.160– 2.080) 58 (24.9) 1.527 (1.010– 2.309) 1.613 (1.063– 2.448) 1.300 (0.846– 1.997) Q4 (> 0.82) 165 (33.3) 2.018 (1.534– 2.654) 2.216 (1.672– 2.935) 1.724 (1.287– 2.311) 81 (34.8) 2.089 (1.414– 3.085) 2.395 (1.611– 3.562) 1.523 (1.003– 2.313) P value for trend < 0.001 < 0.001 < 0.001 < 0.001 0.001 < 0.001 < 0.001 0.057 LAP (per 1 SD increase) 1.353 (1.228– 1.490) 1.437 (1.297– 1.591) 1.251 (1.123– 1.394) 1.461 (1.273– 1.677) 1.559 (1.351– 1.793) 1.233 (1.056– 1.441) Quartiles of LAP Q1 (≤10.71) 77 (15.6) 1.000 (reference) 1.000 (reference) 1.000 (reference) 43 (18.5) 1.000 (reference) 1.000 (reference) 1.000 (reference) Q2 (10.71–21.85) 98 (19.9) 1.242 (0.912– 1.692) 1.200 (0.877– 1.642) 1.092 (0.795– 1.499) 41 (17.6) 0.914 (0.591– 1.412) 0.900 (0.581– 1.395) 0.711 (0.454– 1.113) Q3 (21.85–43.60) 153 (31.0) 2.031 (1.526– 2.702) 2.035 (1.521– 2.723) 1.682 (1.250– 2.264) 60 (25.8) 1.357 (0.910– 2.023) 1.396 (0.933– 2.090) 0.940 (0.619– 1.427) Q4 (> 43.60) Table 3 Odds ratio (OR) and 95% confidence intervals for abnormal left ventricular geometry according to gender-specific continu- ous or quartiles of CMI and LAP Page 7 of 11 Page 7 of 11 Wang et al. Discussion Novel findings in our study include an outstanding gen- der difference in the positive correlation of adiposity in- dices (CMI and LAP) with elevated likelihood for LV structural abnormality among the general Chinese popu- lation. The greater degree of eccentric and concentric LVH seen in obesity in response to increasing CMI and LAP is not only irrespective of conventional cardiovas- cular risk factors but is also pronounced in females than males, suggesting that excess adiposity per se influence abnormal LV geometry substantially more in females. BMI has been the subject of numerous previous stud- ies to interpret the influences of obesity on cardiovascu- lar risk, while it was reported to be insusceptible to make a distinction between the excess adipose tissue and heavy muscle mass. It is now clear that body fat dis- tribution, especially central adiposity, is believed to pro- vide additional information than sheet quantity of body fat [40, 41]. The prospective cohort Atherosclerosis Risk Page 8 of 11 Wang et al. BMC Cardiovascular Disorders (2018) 18:62 There was a greater detrimental influence of CMI on ab- normal LV morphology in females compared with males. Our findings were coherent with those of previous clin- ical studies in that obesity was proved to elicit a cluster of LV geometric abnormalities, which is especially evi- dent in females. A community-based sample of the MONICA Augsburg cohort described that the rise of LVH prevalence in response to obesity and hypertension was generally higher in females [44]. In keeping with this, a large, biracial cohort of elderly participants specu- lated that obese females, but not males, were signifi- cantly accompanied by abnormal LV geometry [42]. In the 2919 members of the Strong Heart Study cohort, in- creased LV mass was a function of ascending waist-to- hip ratio, a typical index reported in the context of cen- tral obesity [45]. There was a further demonstration that females, in whom adipose tissue was much more abun- dant, denoted excess LV mass substantially greater than males. In our study, the statistical effect of visceral dis- tribution on the variability of LV mass in females was in- dicated by the significant impact of CMI and LAP. At each BMI-level, the greater degree of adipose tissue in females provides an explanation for this differential re- sponse [46]. Discussion A considerable proportion of females pos- sess a higher percentage of body fat than males, which may in turn expose them to central obesity [47]. In addition, as the average age of females in our study was 53.39 years where sex hormone levels have declined, the favorable cardiovascular effects of estrogen were inevit- ably disappeared. Owing to the fact that estrogen is be- lieved to inhibit cardiac hypertrophy and testosterone promotes LV hypertrophy, endogenous sex hormone dif- ferences might account for gender difference in obesity- related LV remodeling [48]. On the other hand, central obesity can cause dramatic changes in the release of in- flammatory markers [49], which is the major driving force for regulating cardiac energy metabolism [50]. It is well known that as for normotensive obese females, pro- inflammatory cytokines have been implicated in both echocardiographic abnormalities and the amount of vis- ceral adipose tissue [51]. Obesity and female gender interacted in determining myocardial glucose uptake and insulin sensitivity, which demonstrated gender-related differences in the myocardial substrate metabolism due to obesity [52]. in Communities study (ARIC) noted that greater metric of abdominal obesity, defined by WC, constituted an in- dependent risk factor for subclinical abnormalities in LV structure [42]. In contrast to a recent study from Boga- lusa Heart Study in which an eccentric LVH but not concentric LVH was predominant for WC [15], the Dal- las Heart Study and a report from South African com- munity proposed that WC raised considerably the possibility of concentric LV remodeling phenotype, which is prognostically worse due to a high risk for car- diovascular death [10, 16]. Furthermore, there have dis- crepant conclusions concerning the malefic health consequences of visceral adiposity index (VAI) as a valid estimate for the prediction of LV remodeling and geom- etry. Current investigations of LV morphology have elu- cidated that individuals with more visceral adiposity predisposed to greater LVM/volume ratio and LVWT concurrent with a decrease in LVEDV, which repre- sented the summation of multiple diverse aspects of concentric LV remodeling [17, 18]. Conversely, Hu T and colleagues failed to support long-term increases, in both magnitude and velocity of VAI, as a marker associ- ated with abnormal LV geometry [15]. Discussion Hence, it would be useful to embark on a novel and easily assessed marker of abdominal adiposity in clinical practice to stratify the risk of divergent cardiovascular structural and hemodynamic phenotypes given the paucity of effi- cient diastolic heart failure therapies. It is noteworthy that LAP, which is computed from WC and fasting TG, has offered mechanistic insight into worse cardiometabolic profile given its independent rela- tionship with incident cardiovascular events [19, 20, 43], yet is not quantified in the clinical setting of abnormal LV geometry. At present, CMI, a product of TG/HDL-C ratio and WHtR, has the advantage of being applicable in the assessment of diabetes and atherosclerotic pro- gression [21–23]. Ichiro Wakabayashi et al. reflected that contribution of elevated levels of CMI to the prevalent diabetes risk was somewhat stronger in females than in males [22]. A relevant study further expanded current knowledge by confirming the importance of central adi- posity (by CMI) as a potential etiology of atherosclerotic progression (by IMT) in subjects with peripheral arterial disease [21]. Given the fact that diabetes and athero- sclerosis have been the primary mediators of subclinical LV remodeling, it is likely that CMI could explain the adverse cardiovascular effects of central obesity [24, 25]. In this sense, this middle-aged, cross-sectional, population-based design is initiated to examine and val- idate the practicality of CMI and LAP as the key corre- lates of LV geometric abnormalities in rural China. There has been a great interest in exploring the under- lying mechanism regarding the potential adverse impact of CMI and LAP on morphologic LV abnormalities. The cardiac workload in visceral obesity is constantly in- creased, probably as a consequence of supplying the high energy demands of the adipose tissue [53, 54]. An in- crease in LVM might be the result of chronic volume overload and insufficient adaptation of peripheral resist- ance to the increased cardiac output, characterizing a Our data underlined the clinical value of new central adiposity measures such as LAP and CMI in assessing the risk of LVH which differed somewhat by gender. Wang et al. BMC Cardiovascular Disorders (2018) 18:62 Page 9 of 11 Page 9 of 11 state of increased stroke work [54]. Acknowledgements Here, I would like to express my sincere thanks to all the authors. The authors would like to thank Dr. Yingxian Sun who was responsible for the project completion. Abbreviations BMI: Body mass index; CMI: Cardiometabolic index; CVD: Cardiovascular disease; DBP: Diastolic blood pressure; ECG: Echocardiogram; FPG: Fasting plasma glucose; HDL-C: High-density lipoprotein cholesterol; IVST: Interventricular septal thickness; LAP: Lipid accumulation product; LV: Left ventricular; LVEDV: Left ventricular end-diastolic volume; LVEDVI: Left ventricular end-diastolic volume index; LVESV: Left ventricular end-systolic volume; LVESVI: Left ventricular end-systolic volume index; LVH: Left ventricular hypertrophy; LVIDD: Left ventricular end-diastolic internal dimension; LVM: Left ventricular mass; LVMI: Left ventricular mass index; LVWT: LV wall thickness; PWT: Posterior wall thickness; RWT: Relative wall thickness; SBP: Systolic blood pressure; TG: Triglyceride; VAI: Visceral adiposity index; WC: Waist circumference; WHtR: Waist-to-height ratio g Our results from this study are subject to some limita- tions. First, the cross-sectional design allows for only de- termining an association of CMI and LAP with abnormal LV geometry, but no potential cause-effect re- lationships can be drawn. Further confirmations in pro- spective studies to assess the prognostic role of CMI and LAP in LV morphology are warranted. Secondly, more detailed and precise imaging phenotypes of adiposity tis- sue distribution such as visceral or abdominal subcuta- neous fat are not available in our study. Thirdly, our sample is comprised of only Chinese adults, and it is un- known whether our findings are also applicable to other racial or ethnic populations. Notwithstanding these limi- tations, the potential public health implications for the prevention and treatment of LVH and heart failure also merit comment. Our population-based design (which permits extensive multivariable adjustment for several confounders) for the first time acknowledges that CMI and LAP have a fulfilling supplementary beneficial effect on predicting LV geometric pattern in a large sample of community members of Northeast China. Within the scope of the epidemiological study, CMI and LAP are much more cost-effective and clinically feasible parameter to evaluate obesity-related pathologic cardiac remodeling when compared with quantitative measurements of vis- ceral fat from advanced imaging. Under this scenario, car- diovascular risk stratification might be improved by adoption of simple central adiposity measures (defined by CMI and LAP). Funding h d This study was supported by grants from ‘Thirteenth Five-Year’ program funds (The National Key Research and Development Program of China, Grant #2017YFC1307600) and ‘Twelfth Five-Year’ project funds (The National Sci- ence and Technology Support Program of China, Grant #2012BAJ18B02). Discussion Secondly, central fat distribution, which has been assumed to be the metabol- ically active compartment of fat deposits, could mediate increased LVM through the effects of expression of circu- lating inflammatory cytokines, elevated availability of angiotensinogen, and increased myocardial fibrosis [53, 55]. Pro-inflammatory visceral adipocytes generate a cascade of neuro-hormonal signals, which play a critical role in insulin resistance and cardiac remodeling [56, 57]. Also, the crucial importance of hyperinsulinemia and insulin resistance in favoring myocardial hypertrophy is well recognized in the context of growth-stimulating effect of insulin or expansion of blood volume [58]. Thirdly, exposure of the heart to de- position of fat tissue and the presence of high fatty acid and TG levels in the myocardium impair cardiac structure and induce an increase in LVM [58, 59]. Moreover, super- imposing higher systolic blood pressures to obesity com- bines hemodynamic (pressure, volume overload) and non- hemodynamic stimuli (fat infiltration, inflammation), which together has an additive effect on concentric myocardial re- modeling [60]. decrement in their associations among males. Given the gender differences in LV adaptation to adiposity exists, our results add further impetus to translating CMI and LAP into a clinical and public health recommendations for reducing the burden of LVH. Efforts to target individ- uals at higher cardiometabolic risk on a gender-specific basis, integrating assessments of CMI and LAP may pre- vent abnormal LV geometry and incident heart failure. Additional file Additional file 1: Table S1. Characteristics of study population according to the left ventricular geometry. (DOCX 18 kb) Additional file 1: Table S1. Characteristics of study population according to the left ventricular geometry. (DOCX 18 kb) Authors’ contributions HYW participated in the design, coordination and execution of the study, analyzed data and drafted the manuscript. ZL and XFG contributed to the echocardiographic measurements. SC, NY, and YCT took responsible for acquisition of data, revising it critically for important intellectual content. YXS and LJZ were involved in supervisory role in the project and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work. All authors have read and approved the final manuscript. Availability of data and materials h d d d l The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate The study was approved by the Ethics Committee of China Medical University. All procedures were performed in accordance with ethical standards. Informed consent was signed by all the participants. Consent for publication Consent for publication was obtained in all participants. Conclusion Females with greater CMI and LAP were more likely to lead in eccentric and concentric LVH with a concurrent Consent for publication C f bl Consent for publication Consent for publication was obtained in all participants. References Aijaz B, Ammar KA, Lopez-Jimenez F, Redfield MM, Jacobsen SJ, Rodeheffer RJ. Abnormal cardiac structure and function in the metabolic syndrome: a population-based study. Mayo Clin Proc. 2008;83(12):1350–7. 28. Zhang N, Chen Y, Guo X, Sun G, Dai D, Sun Y. Metabolic abnormalities, but not metabolically healthy obesity, are associated with left ventricular hypertrophy. Heart Lung Circ. 2017;26(3):251–7. 7. 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Accurate and Precise DNA Quantification in the Presence of Different Amplification Efficiencies Using an Improved Cy0 Method
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Introduction Real-time quantitative polymerase chain reaction (real-time PCR) is the method of choice for absolute or relative quantifica- tion of nucleic acids (DNA and RNA if preceded by the reverse transcription step) because of its rapidity, accuracy and sensitivity [1–3]. Although real-time PCR is well accepted as the gold standard in nucleic acid quantification and is widely used for validating the results of large-scale microarray experiments [4], two key issues, namely data quality assurance and proper data analysis, may compromise the acquisition of reliable biological results [5–7]. Fn~F0(1zE) n ð1Þ ð1Þ where n is the cycle number, F0 is the base fluorescence value and efficiency (E = 1) is constant and maximal over the entire reaction. However, empirical evidence shows that E is not always 1 and constant, conversely it progressively declines at the building up of products and consuming of reagents [10,11]. Hence, we can distinguish E in Emax defined as the maximal initial value of PCR efficiency, En is the efficiency value at the n cycle and the rate of loss in E after each cycle is defined as DE. To provide a complete picture we will discuss overall efficiency when describing the global efficiency of the amplification system calculated using the standard curve method [8]: where n is the cycle number, F0 is the base fluorescence value and efficiency (E = 1) is constant and maximal over the entire reaction. However, empirical evidence shows that E is not always 1 and constant, conversely it progressively declines at the building up of products and consuming of reagents [10,11]. Hence, we can distinguish E in Emax defined as the maximal initial value of PCR efficiency, En is the efficiency value at the n cycle and the rate of loss in E after each cycle is defined as DE. To provide a complete picture we will discuss overall efficiency when describing the global efficiency of the amplification system calculated using the standard curve method [8]: The widely accepted quantification method for determining the quantification cycle (Cq) in real-time PCR is the Ct (threshold cycle) method. The Ct value is defined as the fractional cycle number in the log-linear region of PCR amplification in which the reaction reaches fixed amounts of amplicon DNA [8]. This method requires generating serial dilutions of a given sample and performing multiple PCR reactions on each dilution. Accurate and Precise DNA Quantification in the Presence of Different Amplification Efficiencies Using an Improved Cy0 Method Michele Guescini*., Davide Sisti., Marco B. L. Rocchi, Renato Panebianco, Pasquale Tibollo, Vilberto Stocchi Guescini*., Davide Sisti., Marco B. L. Rocchi, Renato Panebianco, Pasquale Tibollo Stocchi Vilberto Stocchi Department of Biomolecular Sciences, University of Urbino ‘‘Carlo Bo’’ Via I Maggetti, Urbino, Italy Abstract Quantitative real-time PCR represents a highly sensitive and powerful technology for the quantification of DNA. Although real-time PCR is well accepted as the gold standard in nucleic acid quantification, there is a largely unexplored area of experimental conditions that limit the application of the Ct method. As an alternative, our research team has recently proposed the Cy0 method, which can compensate for small amplification variations among the samples being compared. However, when there is a marked decrease in amplification efficiency, the Cy0 is impaired, hence determining reaction efficiency is essential to achieve a reliable quantification. The proposed improvement in Cy0 is based on the use of the kinetic parameters calculated in the curve inflection point to compensate for efficiency variations. Three experimental models were used: inhibition of primer extension, non-optimal primer annealing and a very small biological sample. In all these models, the improved Cy0 method increased quantification accuracy up to about 500% without affecting precision. Furthermore, the stability of this procedure was enhanced integrating it with the SOD method. In short, the improved Cy0 method represents a simple yet powerful approach for reliable DNA quantification even in the presence of marked efficiency variations. Editor: Giovanni Maga, Institute of Molecular Genetics IMG-CNR, Italy Received March 26, 2013; Accepted June 5, 2013; Published July 8, 2013 pyright:  2013 Guescini et al. This is an open-access article distributed under the terms of the Creative Commons Attribution L restricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Funding: The authors have no support or funding to report. erests: The authors have declared that no competing interests exis Competing Interests: The authors have declared that no competing interests exist. * E-mail: michele.guescini@uniurb.it . These authors contributed equally to this work. DNA template should double at each cycle according to the equation: DNA template should double at each cycle according to the equation: July 2013 | Volume 8 | Issue 7 | e68481 Introduction The threshold-cycle values are then plotted versus the Log of the dilution and a linear regression, from which the mean efficiency can be derived, is performed [9]. This approach involves the comparison of identical efficiency among amplifications. PCR efficiency can be defined as the fold change in the amount of amplified DNA after each cycle of PCR. In an ideal PCR, the Eoverall~10 { 1 slope{1 ð2Þ ð2Þ where the slope is calculated from the linear regression between the Log of the initial DNA template and the Cq. Small amplification variations may occur in any PCR quanti- fication potentially leading to estimate uncertainty. Among the PLOS ONE | www.plosone.org 1 July 2013 | Volume 8 | Issue 7 | e68481 Accurate and Precise Real-Time PCR Quantification manufacturer’s instructions. The final concentration of the standard plasmid was estimated spectophotometrically by averag- ing three replicate A260 absorbance determinations. numerous factors that affect the sensitivity, accuracy, and reliability of real-time PCR assay, are many substances found in biological samples such as co-extracted contaminants, which can inhibit PCR, confounding template amplification and analysis [12–15]. This DNA fragment was amplified for quantification by the high amplification efficiency (HE) primer pair (forward ND1F2:59- ACGCCATAAAACTCTTCACCAAAG-39 and reverse ND2:59- TAGTAGAAGAGCGATGGTGAGAGCTA-39) and low ampli- fication efficiency (LE) primer pair (forward ND1F5:59- ATAAAACTCTTCACCAAAGAG-39 and reverse ND2:59- TAGTAGAAGAGCGATGGTGAGAGCTA-39). Furthermore, poor quality primers or difficult samples may lead to sub-optimal amplification. Imperfect amplification primers may be the result not only of non-optimal primer design but, sometimes, primer sequence and/or position are constrained, as in the case of detection of sequence variants, intron spanning primers etc. [16]. Sub-optimal amplification may also be the result of the inhibiting agents used during nucleic acid extraction or co- purified components from the biological sample such as bile salts, urea, haeme, heparin, and immunoglobulin G. The presence of inhibitors results in deviations of the amplification kinetic from the optimal amplifications obtained using standard samples [17]. Real-time PCR amplifications were conducted using Light- CyclerH 480 SYBR Green I Master (Roche) according to the manufacturer’s instructions, with 500 nM primers and a variable amount of DNA standard in a 20 ml final reaction volume. Thermocycling was conducted using a LightCyclerH 480 (Roche) initiated by a 10 min incubation at 95uC, followed by 40 cycles (95uC for 5 s; 60uC for 5 s; 72uC for 20 s) with a single fluorescent reading taken at the end of each cycle. Muscle Fine Needle Aspiration (FNA) The most common procedure used to account for any differences in PCR efficiencies among samples is to amplify a reference gene in parallel with the reporter gene and to relate their expression levels. However, this approach assumes that the two assays are inhibited to the same degree, which is not always the case. Such variations in assay inhibition are a particular problem in absolute quantification, where an external calibration curve is used to calculate the number of transcripts in the test samples, an approach that is commonly adopted for quantification of pathogens [22]. Students affiliated with the University of Urbino took part in this study. Individuals were considered eligible if they were .18 years old. Written informed consent was obtained from all participants. Ethical approval for this study conformed to the standards of the Declaration of Helsinki. The Urbino University Ethical Committee (Approval Number 28507) approved the protocol before study initiation. p y Skeletal muscle was obtained by fine needle aspiration (FNA) from the vastus lateralis muscle. Muscle FNA was performed with a 22-G spinal needle (Becton Dickinson, Madrid) under ultrasound guidance as previously described by Guescini et al. [30]. Muscle tissue was rapidly thawed and incubated with 0.2 mg/ml of Proteinase K at 55uC for 10 min. Subsequently, genomic DNA and total RNA were co-purified using silica micro columns by the RNeasy Micro Kit (Qiagen) according to the manufacturer’s instructions. Real-time PCR amplifications were conducted using Specific primers for MT-CO1 (forward: 59-GTGCTATAGTG- GAGGCCGGA-39 and reverse: 59-GGGTGGGAG- TAGTTCCCTGC-39). and ATP6 (forward: 59-ATGAGCGGG- CACAGTGATTA-39 and reverse: 59- AGGGAAGGTTAATGGTTGATA-39) in a Light-Cycler 480 SYBR Green I Master (Roche, Basilea, Switzerland) according to the manufacturer’s instructions, with 300 nM primers and 2 ml of purified FNA sample in 20 ml of final reaction volume. Thermo- cycling was performed using a LightCycler 480 (Roche) initiated by a 10 min incubation at 95uC, followed by 40 cycles (95uC for 5 sec; 60uC for 5 sec; 72uC for 10 sec) with a single fluorescent reading taken at the end of each cycle. Each reaction was conducted in duplicate and completed with a melt curve analysis to confirm the specificity of amplification and lack of primer dimers. In the last few years, a number of studies have attempted to address these problems determining reaction amplification effi- ciency through the application of different mathematical models [17,23–28]. Muscle Fine Needle Aspiration (FNA) However, the key issue is that the exact equation of the PCR amplification kinetics is still elusive, and the proposed models are only good approximations of single portions of the amplification kinetic. Bearing in mind these concerns, our group proposed an alternative method (Cy0 method), in which a new quantitative entity, Cy0, is defined. The Cy0 method is similar to Ct, but it offers the important advantage of taking into account the kinetic parameters of the amplification curve and may compensate for small variations among the samples being compared [29]. Conversely, with a marked decrease in amplification efficiency the Cy0 method underestimates. Our proposed improvement in the Cy0 method is based on the use of kinetic parameters calculated in the curve inflection point to compensate for efficiency variations. In addition, the enhanced Cy0 method was integrated with SOD analysis to quantify starting DNA quantity in the presence of different amplification kinetics due to inhibition of primer extension and imperfect primer design. The advantages of the improved Cy0 method were then evaluated in mtDNA quantifications from skeletal muscle samples obtained from fine needle aspiration [30]. Cy0 Method The Cy0 value [29] is the intersection point between the abscissa axis and tangent of the inflection point of the Richards curve obtained by the nonlinear regression of raw data. The Richards equation is an extension of the symmetric logistic-type growth curve; specifically, when d coefficient is equal to 1, the symmetric logistic-type and Richards (asymmetric logistic-type) curves are the same. Shape of amplification curves in real-time PCR ranges from a perfect symmetric to a strongly asymmetric shape, for example in Introduction Each reaction combination, was performed in 4–6 replicates and all the runs were completed with a melt curve analysis to confirm the specificity of amplification and lack of primer dimers. For these reasons, many tools, based on amplification efficiency, have been developed to detect outlier amplifications [18–20]. A quality test tool, called SOD, has also recently been developed. SOD is not based on direct amplification efficiency estimation in order to detect outliers, but relies on monitoring the shape of the amplification curve [21]. Accurate and Precise Real-Time PCR Quantification Accurate and Precise Real-Time PCR Quantification Accurate and Precise Real-Time PCR Quantification subsequently, using this equation it is possible to calculate the value of the starting reaction efficiency: subsequently, using this equation it is possible to calculate the value of the starting reaction efficiency: presence of inhibitors [21]. The Cy0 method was performed by nonlinear regression fitting of the Richards function [31], in order to fit fluorescence readings to the 5-parameter Richards function. ln Emaxz1 ð Þ~ 2m yf ð7Þ y~ Fmax 1ze {1 b x{c ð Þ   " #d zFb ð3Þ ð7Þ ð3Þ This relation shows that the slope of the inflection point, scaled for its ordinate, is the natural logarithmic value of starting efficiency (Emax+1). Where y is the reaction fluorescence at cycle x, Fmax is the maximal reaction fluorescence, b can be considered a slope parameter, c is the transition parameter and d represents the Richards coefficient, and Fb is the background reaction fluorescence. The five parameters that characterized each run were used to calculate the Cy0 value by the following equation, corrected in order to eliminate the influence of baseline correction (Fb). Cy0 values were obtained as follows: Starting from two amplification reactions (A and B) showing the same starting DNA template (y0), but different Emax, we can consider the following equivalence: yfA 1z EmaxAz1  xfA ~ yfB 1z EmaxBz1  xfB ð8Þ ð8Þ from which, introducing some useful approximations and trans- formations (see Data File S1), it is possible to formulate the following equation: Cy0~c{b(1z 1 d { ln d) ð4Þ ð4Þ xfA~ xfB 2mB yfB { ln yfB yfA ! yfA 2mA ð9Þ When the flex ordinate of the raw data curve is lower than Fmax/e (where e = 2.718281…), the Richards curve does not yield a good fit because it is not possible to estimate the parameter d; in this case, the upper points are progressively removed, until an acceptable fitting is achieved [32]. Cy0 was calculated using the following web interface: http://www.cy0method.org/, specifically developed by the authors of this article. When the flex ordinate of the raw data curve is lower than Fmax/e (where e = 2.718281…), the Richards curve does not yield a good fit because it is not possible to estimate the parameter d; in this case, the upper points are progressively removed, until an acceptable fitting is achieved [32]. Accurate and Precise Real-Time PCR Quantification Cy0 was calculated using the following web interface: http://www.cy0method.org/, specifically developed by the authors of this article. ð9Þ where xf and yf are the x- and y-coordinates of the inflection point of the amplification curve and m is the slope of the curve in inflection point. In this equation the xf values represent the Cq of the two amplification reactions while yf and m account for the kinetic parameters of the amplification (Emax and DE). In particular, in the presence of different amplification kinetics for curves A and B, the Cq of curve B (xfB) can be overlapped to the Cq of curve A (xfA) applying Eq. 9. Notably, to perform this procedure the only parameters needed are xf, yf and m of the two curves. SOD Calculation Shape based kinetic outlier detection (SOD) was based on the shapes of the amplification curves. In order to fit fluorescence raw data, nonlinear regression fitting of 5-parameter Richards function was performed. The following shape parameters were used: the plateau value of the amplification curve (Fmax), the tangent straight line slope in the inflection point (m) and the y-coordinate of the inflection point (Yf). For further details, see Sisti et al. [21]. The proposed procedure refers to the comparison of two amplification curves; however, this formula can be easily generalized to the standard curve quantification method. In fact, we propose using yf and m as reference values calculated as the arithmetic mean of each yfi and mi, estimated from all standard amplification curves, and as Cq, we replaced xf with Cy0. After profile correction, the two curves become parallel justifying the use of the Cy0 value as Cq. Hence, in the presence of unknown amplification profiles significantly different in shape from that of the standard curve the obtained Cy0 value should be corrected using the following formula: f All other statistical elaborations and graphics were obtained using specific VBA macro developed in the MS Excel and Statistical Packages for Social Sciences (SPSS 13.0). Mathematical Model for Cy0 Correction Regarding the kinetic of PCR amplification we considered the logistic model as previously reported by Chervoneva et al. and Rutledge & Stewart [33,34]. Cy0corrected~ Cy0 2m yf { ln yf yf ! yf 2m ð10Þ ð10Þ y~ Fmax 1z Fmax y0 {1   Emaxz1 ð Þ{x ð5Þ ð5Þ Quantitative Real-Time PCR Quantitative Real-Time PCR The DNA standard consisted of a pGEM-T (Promega) plasmid containing a 104 bp fragment of the human mitochondrial gene NADH dehydrogenase 1 (MT-ND1) as insert. This plasmid was purified using the Plasmid Midi Kit (Qiagen) according to the July 2013 | Volume 8 | Issue 7 | e68481 PLOS ONE | www.plosone.org 2 Assessing the Cy0 Correction Model in Theoretical Logistic Curves Assessing the Cy0 Correction Model in Theoretical Logistic Curves where Fmax is the height of amplification profile, Emax is the starting maximal efficiency value of curve growth rate and y0 is the initial amount of DNA template. By a simple mathematical elaboration (reported in Data File S1), it is possible to determine the curve slope of the inflection point as: The proposed Cy0 correction provides for: a) curve shift of a factor equal to [ln(yfB/yfA)]/(yfB/2mA)] and b) scaling on the abscissa axis using the ratio (mB/yfB*yfA/mA). It should be noted that all these procedures were not necessarily applied simultaneously. Their application depends on the characteristics of the curves to be compared and consequently Eq. 10 progressively becomes simpler when one of the standard curve parameters (m or yf) matches with one of the corresponding parameters of the unknown curve. m~Fmax ln Emaxz1 ð Þ 4 ð6Þ ð6Þ PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org July 2013 | Volume 8 | Issue 7 | e68481 3 Accurate and Precise Real-Time PCR Quantification In an attempt to demonstrate the performance of the proposed C 0 ti i E 5 t d t lifi ti fil resulting profiles allowed us to show how the Cy0 correction k d d t l i th hifti d li f t d i E Figure 1. Simulation of amplification curves with different kinetics using logistic equation. Left plots show the starting fluorescence profiles A and B, in which A represents the optimal amplification while B refers to the non-optimal amplification profile. Right plots report the amplification profiles after application of proposed correction. In these analyses, we used profile A as a reference, hence the A profiles always remain the same while the B profiles were corrected using the formula reported near the plots. All the reported formulas derive from Eq. 10. In an attempt to explain the components of this equation, we deleted the terms that nullify according to the differences and overlappings in the amplification profiles. The inner tables show the kinetic parameters of the fluorescence profiles and the Cq calculated as Ct, Cy0 and Cy0corr and differences among them are also shown. doi:10.1371/journal.pone.0068481.g001 Figure 1. Simulation of amplification curves with different kinetics using logistic equation. Left plots show the starting fluorescence profiles A and B, in which A represents the optimal amplification while B refers to the non-optimal amplification profile. Assessing the Cy0 Correction Model in Theoretical Logistic Curves Right plots report the amplification profiles after application of proposed correction. In these analyses, we used profile A as a reference, hence the A profiles always remain the same while the B profiles were corrected using the formula reported near the plots. All the reported formulas derive from Eq. 10. In an attempt to explain the components of this equation, we deleted the terms that nullify according to the differences and overlappings in the amplification profiles. The inner tables show the kinetic parameters of the fluorescence profiles and the Cq calculated as Ct, Cy0 and Cy0corr and differences among them are also shown. doi:10 1371/journal pone 0068481 g001 doi:10.1371/journal.pone.0068481.g001 resulting profiles allowed us to show how the Cy0 correction worked and to explain the shifting and scaling factors used in Eq. 10. In these analyses we showed two fluorescence profiles: A and B; curve A represented the optimal condition, whilst curve B In an attempt to demonstrate the performance of the proposed Cy0 correction, using Eq. 5, we created two amplification profiles characterized by different Fmax and Emax and hence different DE [11], but the same initial DNA template (y0). The analysis of the PLOS ONE | www.plosone.org July 2013 | Volume 8 | Issue 7 | e68481 July 2013 | Volume 8 | Issue 7 | e68481 4 Accurate and Precise Real-Time PCR Quantification Figure 2. Description of the High Efficiency (HE) and Low Efficiency (LE) systems. The HE and LE systems were obtained amplifying the mitochondrial MT-ND1 gene with the same reverse primer ND1R2 but using ND1F2 or ND1F5 as forward primer, respectively. Panel (A) shows the primer annealing locations in the mitochondrial sequence; panel (B) shows the secondary structure of the obtained amplification products calculated using http://mfold.rna.albany.edu/. This analysis shows that ND1F5 primer overlaps the stem-loop at 59 terminus, The overlapping coupled with its reduced length impairs the primer’s ability to bind the DNA template. Panel (C) shows the overall efficiency analysis of the two systems (HE and LE). The slopes of the two regressions are significantly different, with slopeHE = 23.488; slopeLE = 23.942; consequently, the overall efficiencies, calculated using the equation Eoverall~10{ 1 slope{1, were Eoverall-HE = 0.93; Eoverall-LE = 0.79. doi:10.1371/journal.pone.0068481.g002 Figure 2. Description of the High Efficiency (HE) and Low Efficiency (LE) systems. Assessing the Cy0 Correction Model in Theoretical Logistic Curves The HE and LE systems were obtained amplifying the mitochondrial MT-ND1 gene with the same reverse primer ND1R2 but using ND1F2 or ND1F5 as forward primer, respectively. Panel (A) shows the primer annealing locations in the mitochondrial sequence; panel (B) shows the secondary structure of the obtained amplification products calculated using http://mfold.rna.albany.edu/. This analysis shows that ND1F5 primer overlaps the stem-loop at 59 terminus, The overlapping coupled with its reduced length impairs the primer’s ability to bind the DNA template. Panel (C) shows the overall efficiency analysis of the two systems (HE and LE). The slopes of the two regressions are significantly different, with slopeHE = 23.488; slopeLE = 23.942; consequently, the overall efficiencies, calculated using the equation Eoverall~10{ 1 slope{1, were Eoverall-HE = 0.93; Eoverall-LE = 0.79. doi:10.1371/journal.pone.0068481.g002 represented a non-optimal fluorescence profile (Fig. 1 left graphs). After correction, the fluorescence profile of curve B was modified following the proposed procedure using the parameters of curve A as a reference (Fig. 1 right graphs) and Cy0 was recalculated (Cy0corr). two previous cases, in this elaboration two profiles differing in both Emax and Fmax were created, and in this case as well the proposed correction allowed us to compensate for these differences (Fig. 1 lower panels). Hence, we can conclude that Cy0 correction as reported in Eq. 10 can be applied in all conditions. In the first case, the two curves were characterized by equal Emax (Emax = 1) and different Fmax corresponding to 1 and 0.7, respectively (Fig. 1 upper panels). In these conditions, profile B was less steep and slightly shifted towards the right compared to profile A. The Cy0 values obtained from the two curves differed, and in particular, the Cy0 value calculated from the curve with lower Fmax slightly overestimated the input DNA quantity, whilst the Ct underestimated the starting quantity. As we can observe in the inner table, after Cy0 correction, the error became very small (Cy0A-Cy0corr-B ,1025). Then, we created two profiles where Fmax was equal but m differed, as shown in Figure 1 (middle panels) Curve B (Emax = 0.88) was significantly more delayed than curve A producing a marked underestimation using both Cy0 and Ct methods. After the application of the proposed correction, curves A and B overlapped resulting in a full correction of the initial DNA estimate. July 2013 | Volume 8 | Issue 7 | e68481 Low and High Amplification Efficiency Systems g p y y To examine the usefulness of the developed quantification method in compensating for PCR efficiency, two amplification systems were used. The first is characterized by a high amplification efficiency (HE; Fig. 2C black symbols), while the second showed a low amplification efficiency (LE; Fig. 2C white symbols) (Fig. 2). The two systems were obtained amplifying the same DNA sequence with the same reverse primer ND1R2 but using ND1F2 or ND1F5 as forward primer for the HE and LE systems, respectively. Specifically, the annealing region of the primer ND1F5 is shifted only 5 nucleotides downstream from the ND1F2 primer location and shortened by three nucleotides (Fig. 2A). In this region, the DNA template shows a stem-loop secondary structure (evaluated using http://mfold.rna.albany. edu/results/10/12Aug30-10-05-01/) that competes with the forward primers for annealing (Fig. 2B). The primer ND1F5 is probably less effective than ND1F2 in template binding. Using this strategy, we were able to amplify the same DNA input template with two amplification systems that work with marked efficiency differences. The amplification efficiencies of the HE and LE systems were determined by the standard curve method. The obtained standard curves and the corresponding regression equations are shown in Fig. 2C and the obtained overall efficiencies of HE and LE were Eoverall-HE = 0.93 and Eoverall- LE = 0.79. Hence, we investigated the possible application of this procedure to experimental amplification plots obtained by the HE and LE systems (Fig. 2). The LE system is clearly far from a logistic profile, hence this allowed us to evaluate the robustness of the proposed model in asymmetric amplifications that deviate from a logistic shape. Correction of the Cy0 value estimated from an amplification reaction is necessary only in the presence of a marked decrease in the amplification rate compared to that of standard curve samples. In fact, as reported in our previous work, slight efficiency variations are well compensated by the Cy0 method alone [29]. Hence, we developed a flow chart that integrates the proposed new Cy0 correction with the previously developed SOD method [21]. As depicted in Figure 3, the first step involves the comparison of the shape parameters (Fmax, yf and m) of the unknown amplification with the mean shape parameters (Fmax,yf and m) estimated from the standard samples using the procedure reported in Sisti et al [21]. Assessing the Cy0 Correction Model in Theoretical Logistic Curves Finally, we provided an example that summarizes the The presented data showed that theoretically, it is possible to account for differences in kinetic parameters between two amplification profiles estimating xf, yf and m. The improved Cy0 method re-proposes two previously intro- duced concepts: curve normalization on Fmax [35] and Cq correction based on the value of the slope in the maximal of the second derivative [36]. These aspects were incorporated in the proposed method and further developed starting from the logistic curve. Specifically, the significant differences consisted in the use of yf for normalization to avoid bias due to asymmetric amplifications and the weighting of the slope of the curve on the yf value. Furthermore, to the best of our knowledge, the introduced shift factor [ln(yfB/yfA)]/(yfB/2mA)] has never been reported in literature. July 2013 | Volume 8 | Issue 7 | e68481 PLOS ONE | www.plosone.org 5 Accurate and Precise Real-Time PCR Quantification Figure 3. Flowchart of the integration between Cy0 and SOD methods and application of the Cy0 correction. Fmax, yf and m are the shape parameters corresponding to each unknown amplification curve. These parameters are compared with mean shape parameters (marked above) calculated as the mean of the single parameters of all the standard curves. doi:10.1371/journal.pone.0068481.g003 Figure 3. Flowchart of the integration between Cy0 and SOD methods and application of the Cy0 correction. Fmax, yf and m are the shape parameters corresponding to each unknown amplification curve. These parameters are compared with mean shape parameters (marked above) calculated as the mean of the single parameters of all the standard curves. doi:10.1371/journal.pone.0068481.g003 Low and High Amplification Efficiency Systems Fmax,yf and m were calculated as the arithmetic mean of all Fmax, yf and m values corresponding to each of the amplification curves that make up the standard curve. For SOD negative runs the flow chart leads to the original Cy0 value whilst the SOD positive values require the application of the proposed Cy0 correction (Eq. 10) in order to compensate for the difference in amplification efficiency. To test this protocol we used an inhibition PLOS ONE | www.plosone.org July 2013 | Volume 8 | Issue 7 | e68481 July 2013 | Volume 8 | Issue 7 | e68481 6 Accurate and Precise Real-Time PCR Quantification Figure 4. Effect of increasing PCR inhibition on the accuracy of the HE and LE amplification systems. PCR quantifications were performed using HE and LE systems in the presence of an equal starting number of template molecules and increasing blocked primer concentrations. Panels (A) and (B) show the obtained amplification plots for each system. Panels (C) and (D) show the relative errors obtained using Cy0 and Cy0 corrected following Eq. 10. The relative error was reported as Log(DNAexp)–Log(DNAobs) where Log(DNAexp) is the number of expected molecules and Log(DNAobs) is the number of calculated molecules using the Cy0 (Black dots) or Cy0corrected (White dots). Each symbol represents a mean 6 runs. SOD detected almost all the runs in which the blocked primer was added as outliers. doi:10.1371/journal.pone.0068481.g004 Figure 4. Effect of increasing PCR inhibition on the accuracy of the HE and LE amplification systems. PCR quantifications were performed using HE and LE systems in the presence of an equal starting number of template molecules and increasing blocked primer concentrations. Panels (A) and (B) show the obtained amplification plots for each system. Panels (C) and (D) show the relative errors obtained using Cy0 and Cy0 corrected following Eq. 10. The relative error was reported as Log(DNAexp)–Log(DNAobs) where Log(DNAexp) is the number of expected molecules and Log(DNAobs) is the number of calculated molecules using the Cy0 (Black dots) or Cy0corrected (White dots). Each symbol represents a mean 6 runs. SOD detected almost all the runs in which the blocked primer was added as outliers. doi:10.1371/journal.pone.0068481.g004 and 20.62 using Cy0 and Cy0corr, respectively. This means that, under these conditions, Cy0 underestimated the true value by about 1000 fold, whereas Cy0corr underestimated the value by only 4 fold. Low and High Amplification Efficiency Systems Likewise, for the LE system we obtained Log [DNAexp]– Log [DNAobs] = 23.94 and 20.45 using Cy0 and Cy0corr, respectively, corresponding to an underestimation of about 10,000 and 2.5 fold when using Cy0 or Cy0corr, respectively. However, one might doubt that such extreme conditions occur often in real-time PCR quantifications. To address this concern, we set up an additional real-time PCR quantification experiment in which the forward primers, ND1F2 and ND1F5, were mixed in different ratios while maintaining the total amount of forward primers constant. This set up mimics a condition in which two primers compete to bind the same template sequence and after primer annealing the polymerization can occur. Hence, using this system we reduced the efficiency of the annealing phase. This setting also offered the advantage of a known amplification model that involved the addition of a blocked primer to the PCR mix. We estimated the inflection point parameters of the amplification curves fitting the Richards equation [29]. We used the Richards curve because it fits very well [28] allowing us to better estimate the inflection point; moreover, it has the advantage of becoming a symmetric logistic-type curve when the Richards parameter d = 1 (Data File S2). The blocked reverse primer was progressively added to HE and LE quantification systems resulting in the shift towards the right of the amplification curves (Fig. 4 A and B; efficiencies ranged from 1.93 to 1.67 and from 1.79 to 1.56 for HE and LE systems, respectively) that led to a marked underestimation of input DNA molecules (Fig. 4 C and D). The SOD method efficiently detected almost all the inhibited reactions in which the blocked reverse primer was added. The proposed correction allowed us to decrease the bias in quantification considerably. For example, for the HE system in extreme conditions we obtained Log [DNAexp]–Log [DNAobs] = 23.01 model that involved the addition of a blocked primer to the PCR mix. We estimated the inflection point parameters of the amplification curves fitting the Richards equation [29]. We used the Richards curve because it fits very well [28] allowing us to better estimate the inflection point; moreover, it has the advantage of becoming a symmetric logistic-type curve when the Richards parameter d = 1 (Data File S2). The blocked reverse primer was progressively added to HE and LE quantification systems resulting in the shift towards the right of the amplification curves (Fig. Low and High Amplification Efficiency Systems PCRs were performed in the presence of an equal starting number of template molecules by using 500 nM of ND1R2 as reverse primers and different combinations of ND1F2 and ND1F5 as forward primers. Specifically, the PCRs were set as follows: 1) 500 nM ND1F2; 2) 375 nM ND1F2 and 125 nM ND1F5; 3) 250 nM ND1F2 and 250 nM ND1F5; 4) 200 nM ND1F2 and 300 nM ND1F5; 5) 150 nM ND1F2 and 350 nM ND1F5; 6) 125 nM ND1F2 and 375 nM ND1F5; 7) 100 nM ND1F2 and 400 nM ND1F5; 8) 50 nM ND1F2 and 450 nM ND1F5; and 9) 500 nM ND1F5. This experimental model offers the advantage of known PCR efficiencies of extreme conditions (condition 1 corresponds to HE while condition 9 corresponds to LE); hence, we were able to reproduce a gradient of mild decreasing efficiency ranging from 0.93 to 0.79. Black dots represent Cy0 values while white dots correspond to Cy0corrected values. Each symbol represents a mean of 4 runs. SOD detected all the runs obtained using [ND1F5]$250 nM as outliers. Figure 5. Real-time PCR quantifications in the presence of primers with different annealing abilities. Panel (A) shows amplification plots obtained for each condition; (B) shows the relative errors Log(DNAexp)–Log(DNAobs) obtained using Cy0 and Cy0 corrected following Eq. 10. PCRs were performed in the presence of an equal starting number of template molecules by using 500 nM of ND1R2 as reverse primers and different combinations of ND1F2 and ND1F5 as forward primers. Specifically, the PCRs were set as follows: 1) 500 nM ND1F2; 2) 375 nM ND1F2 and 125 nM ND1F5; 3) 250 nM ND1F2 and 250 nM ND1F5; 4) 200 nM ND1F2 and 300 nM ND1F5; 5) 150 nM ND1F2 and 350 nM ND1F5; 6) 125 nM ND1F2 and 375 nM ND1F5; 7) 100 nM ND1F2 and 400 nM ND1F5; 8) 50 nM ND1F2 and 450 nM ND1F5; and 9) 500 nM ND1F5. This experimental model offers the advantage of known PCR efficiencies of extreme conditions (condition 1 corresponds to HE while condition 9 corresponds to LE); hence, we were able to reproduce a gradient of mild decreasing efficiency ranging from 0.93 to 0.79. Black dots represent Cy0 values while white dots correspond to Cy0corrected values. Each symbol represents a mean of 4 runs. SOD detected all the runs obtained using [ND1F5]$250 nM as outliers. Low and High Amplification Efficiency Systems The coefficient of variation was calculated among the corrected Cy0 values of the replicates corresponding to HE and LE amplifications where blocked primers were added and HE/LE competition. doi:10.1371/journal.pone.0068481.g006 Figure 6. Analysis of the variability among replicates intro- duced after Cy0 correction in the studied models. The coefficient of variation was calculated among the corrected Cy0 values of the replicates corresponding to HE and LE amplifications where blocked primers were added and HE/LE competition. doi:10.1371/journal.pone.0068481.g006 Figure 6. Analysis of the variability among replicates intro- duced after Cy0 correction in the studied models. The coefficient of variation was calculated among the corrected Cy0 values of the replicates corresponding to HE and LE amplifications where blocked primers were added and HE/LE competition. doi:10.1371/journal.pone.0068481.g006 Figure 6. Analysis of the variability among replicates intro- duced after Cy0 correction in the studied models. The coefficient of variation was calculated among the corrected Cy0 values of the replicates corresponding to HE and LE amplifications where blocked primers were added and HE/LE competition. doi:10.1371/journal.pone.0068481.g006 conditions, where in the presence of a difference in overall efficiency of 14.18%, we were able to decrease the relative error from 5.89-fold to 0.65-fold using Cy0corr (Fig. 5 B). Finally, but no less important, we evaluated the variability of the Cy0corr values among replicates in the efficiency models used. As expected, these values proved to be very stable with a CV among replicates ranging from 0.002 to 0.02 (Fig. 6). To further substantiate the presented results related to Cy0 method in presence of decreasing amplification efficiency, the Cy0 correction was applied to the freely available data set named ‘‘Competimer’’, reported in Ruijter et al. [32]. This data set is independent and obtained with completely different chemistry and PCR hardware (Data File S4). The proposed Cy0 correction allowed us to efficiently quantify the inhibited amplifications as shown by the low Cq shift in presence of increasing % Competimer (Fig. 7A) and by the marked decrease in standard deviation of Cy0 corrected values compared with uncorrected Cy0 values (Fig. 7B) in all the conditions tested. These results further demonstrate the effectiveness of the proposed Cy0 correction and support the notion that it is generally applicable. Figure 5. Real-time PCR quantifications in the presence of primers with different annealing abilities. Panel (A) shows amplification plots obtained for each condition; (B) shows the relative errors Log(DNAexp)–Log(DNAobs) obtained using Cy0 and Cy0 corrected following Eq. 10. Low and High Amplification Efficiency Systems 4 A and B; efficiencies ranged from 1.93 to 1.67 and from 1.79 to 1.56 for HE and LE systems, respectively) that led to a marked underestimation of input DNA molecules (Fig. 4 C and D). The SOD method efficiently detected almost all the inhibited reactions in which the blocked reverse primer was added. The proposed correction allowed us to decrease the bias in quantification considerably. For example, for the HE system in extreme conditions we obtained Log [DNAexp]–Log [DNAobs] = 23.01 July 2013 | Volume 8 | Issue 7 | e68481 7 July 2013 | Volume 8 | Issue 7 | e68481 PLOS ONE | www.plosone.org Accurate and Precise Real-Time PCR Quantification efficiency of the extreme conditions (in the presence of only ND1F2 or ND1F5 primers). In these experiments the curve shift was present but it was less pronounced than in the previous experiments. Moreover, as the efficiency of the curves decreased Figure 5. Real-time PCR quantifications in the presence of primers with different annealing abilities. Panel (A) shows amplification plots obtained for each condition; (B) shows the relative errors Log(DNAexp)–Log(DNAobs) obtained using Cy0 and Cy0 corrected following Eq. 10. PCRs were performed in the presence of an equal starting number of template molecules by using 500 nM of ND1R2 as reverse primers and different combinations of ND1F2 and ND1F5 as forward primers. Specifically, the PCRs were set as follows: 1) 500 nM ND1F2; 2) 375 nM ND1F2 and 125 nM ND1F5; 3) 250 nM ND1F2 and 250 nM ND1F5; 4) 200 nM ND1F2 and 300 nM ND1F5; 5) 150 nM ND1F2 and 350 nM ND1F5; 6) 125 nM ND1F2 and 375 nM ND1F5; 7) 100 nM ND1F2 and 400 nM ND1F5; 8) 50 nM ND1F2 and 450 nM ND1F5; and 9) 500 nM ND1F5. This experimental model offers the advantage of known PCR efficiencies of extreme conditions (condition 1 corresponds to HE while condition 9 corresponds to LE); hence, we were able to reproduce a gradient of mild decreasing efficiency ranging from 0.93 to 0.79. Black dots represent Cy0 values while white dots correspond to Cy0corrected values. Each symbol represents a mean of 4 runs. SOD detected all the runs obtained using [ND1F5]$250 nM as outliers. doi:10.1371/journal.pone.0068481.g005 Figure 6. Analysis of the variability among replicates intro- duced after Cy0 correction in the studied models. Evaluation of the Cy0 Correction in Muscle Samples Obtained using Fine Needle Aspiration g p In order to evaluate the output of the improved Cy0 method in biological samples, we quantified mtDNA from skeletal muscle fine needle aspiration [30]. This technique allowed us to recover only small quantities of biological materials, hence it was very difficult, if not impossible, to establish PCR efficiency using the standard curve method. To assess the accuracy of the proposed method we estimated the mtDNA quantity by using two amplification systems, one targeted to the MT-CO1 gene and the other to ATP6 gene. Because the mtDNA is a circular DNA molecule, the correlation analysis between MT-CO1 and ATP6 estimated from several specimens should tend to one. The overall MT-CO1 and ATP6 efficiencies, calculated with the standard curve method, using genomic DNA extracted from U87MG cells, were slightly different (Eoverall = 0.89; Eoverall = 0.94 for ATP6 and MT-CO1, respectively). We analyzed 40 aspirates and for each one MT-CO1 and ATP6 amplifications were performed in duplicate for a total of 160 runs. SOD analysis detected 14 and 5 runs as outliers for ATP6 and MT-CO1, respectively. Interestingly, the amplification system doi:10.1371/journal.pone.0068481.g005 doi:10.1371/journal.pone.0068481.g005 doi:10.1371/journal.pone.0068481.g005 efficiency of the extreme conditions (in the presence of only ND1F2 or ND1F5 primers). In these experiments the curve shift was present but it was less pronounced than in the previous experiments. Moreover, as the efficiency of the curves decreased (demonstrated using the standard curve method), the fluorescence profiles showed decreased Fmax and steepness, and increased asymmetry (Fig. 5 A; and Data file S3). We used the amplification in the presence of 500 nM ND1F2 and 500 nM ND1R2 as reference (HE system). Once again, SOD correctly identified the runs obtained with ND1F5.250 nM as outliers. The most promising finding showing the effectiveness of the proposed method came to light from the comparison of the extreme PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org July 2013 | Volume 8 | Issue 7 | e68481 8 Accurate and Precise Real-Time PCR Quantification Figure 7. Real-time PCR quantifications in the presence of ‘‘Competimer.’’ The Cy0 and Cy0corrected values were calculated from the independent freely available data set named ‘‘Competimer’’, reported in Ruijter et al. [32]. Briefly, the amplification curves were obtained using decreasing input gDNA amounts (64, 16, 4, 1, 0.25 and 0.0625 ng/ml) and in optimal (% Competimer = 0–5) and inhibited (increasing % Competimer from 10 to 50%) amplification conditions. Evaluation of the Cy0 Correction in Muscle Samples Obtained using Fine Needle Aspiration The yf and m parameters were estimated from optimal amplification conditions. Panel (A) shows Cy0 and Cy0corrected values obtained for each condition; Panel (B) shows the standard deviation calculated from the Cqs (Cy0 and Cy0corrected) corresponding to the same input cDNA quantity. doi:10.1371/journal.pone.0068481.g007 Figure 7. Real-time PCR quantifications in the presence of ‘‘Competimer.’’ The Cy0 and Cy0corrected values were calculated from the independent freely available data set named ‘‘Competimer’’, reported in Ruijter et al. [32]. Briefly, the amplification curves were obtained using decreasing input gDNA amounts (64, 16, 4, 1, 0.25 and 0.0625 ng/ml) and in optimal (% Competimer = 0–5) and inhibited (increasing % Competimer from 10 to 50%) amplification conditions. The yf and m parameters were estimated from optimal amplification conditions. Panel (A) shows Cy0 and Cy0corrected values obtained for each condition; Panel (B) shows the standard deviation calculated from the Cqs (Cy0 and Cy0corrected) corresponding to the same input cDNA quantity. doi:10.1371/journal.pone.0068481.g007 higher R2 value (Fig. 8A). In order to evaluate the agreement between Cy0 and Cy0corr outcomes in fine needle aspiration the Bland Altman plot was performed. Using this approach it is possible to show that the quantification differences were propor- tional to input DNA and that the quantification errors were lower after correction (p,0.005; permutation test for paired data). Overall bias in Cy0 quantification was 225.8% whereas in Cy0corr the bias was reduced to 210.2%. Specifically, in Figure 8B we can observe that, in outlier runs, uncorrected Cy0 tended to showing the lowest efficiency presented more outlier curves. Subsequently, the correction of the Cy0 values using the efficiency parameters estimated in the inflection point (Eq. 10) were performed only in the outlier runs. Figure 8A shows the descriptive correlation plot between the MT-CO1 and ATP6 estimates before and after Cy0 correction. The graph shows that after the proposed correction the slope and the intercept of the correlation straight- line were closer to 1 and 0, respectively; hence, the proposed method enhanced the accuracy of the quantification. The increased precision obtained after correction was shown by the Figure 8. MtDNA real-time PCR quantifications in muscle samples obtained from Fine Needle Aspiration. Mt-CO1 and ATP6 mitochondrial genes were quantified from 40 samples. Only SOD positive samples were included in this analysis. A) shows the descriptive correlation analysis between Mt-CO1 and ATP6 quantification while panel B) shows the Bland-Altman plot. doi:10.1371/journal.pone.0068481.g008 Figure 8. Acknowledgments The authors would like to thank Dr. Michela Mantuano for technical assistance. Data File S1 Word file that describes the algebra used in Cy0 correction. (XLS) Data File S4 Fluorescence data, fitting elaborations and Cy0 calculations obtained using the freely available data set named ‘‘Competimer’’ reported in Ruijter et al. [32]. (XLS) Conclusion Currently, the analysis of real-time PCR data is based almost exclusively on the determination of a quantitative fractional cycle (Cq) value of each sample using the Ct method. This value is subsequently referred to a standard curve whose slope estimates overall PCR efficiency. Author Contributions Conceived and designed the experiments: MG DS. Performed the experiments: MG PT. Analyzed the data: MG DS MBLR VS. Contributed reagents/materials/analysis tools: VS RP. Wrote the paper: MG DS. Critically revised the manuscript: VS MBLR. Conceived and designed the experiments: MG DS. Performed the experiments: MG PT. Analyzed the data: MG DS MBLR VS. Contributed reagents/materials/analysis tools: VS RP. Wrote the paper: MG DS. Critically revised the manuscript: VS MBLR. Accurate and Precise Real-Time PCR Quantification Accurate and Precise Real-Time PCR Quantification underestimate the starting DNA and the proposed correction based on kinetic parameters allowed us to account for this. shown by the low coefficient of inter-run variation. Moreover, the improved Cy0 method was very accurate even when overall efficiency dropped to about 60–80%. These results show the effectiveness of estimating amplification efficiency using only the parameters of the curve inflection point. Data File S1 Word file that describes the algebra used in Cy0 correction. Data File S1 Word file that describes the algebra used in Cy0 correction. (DOC) The Cy0 method represents a reliable alternative method to calculate a Cq. The advantage of this method stems from the fact that it works very well even in sub-optimal amplification conditions where amplification efficiency may have slight varia- tions among runs [20,29,32]. However, in the presence of significant amplification efficiency decrease (in the range 1.60– 1.93) the Cy0 is impaired; hence, determination of amplification efficiency is essential to achieve a reliable quantification. Data File S2 Fluorescence data and fitting elaborations of standard sample amplifications (standard curve) and amplifications obtained in the presence reverse blocked primer. (XLS) Data File S3 Analysis of the variation in curve asymme- try for each amplification curve obtained using the HE and LE primer assays in presence or absence of blocked and finally in HE/LE competition setup. (TIF) In low efficiency reactions, ideally, each single quantitative analysis should yield three parameters: a Cy0 value, as a measure of the number of initial target quantities, an initial efficiency value of the amplification system and an estimate of the rate at which initial amplification efficiency decreases. The improved Cy0 method represents a simple but powerful approach to achieve reliable DNA quantification even in the presence of a marked decrease in Emax and DE. The main advantage of the proposed method is that it uses the kinetic parameters of the amplification curve using the information estimated in the inflection point of the amplification curve where the fluorescence readings are very reliable and minimally affected by noise. This is noteworthy, as the methods based on the analysis of fluorescence in the first cycles of curve growth are very sensitive to baseline evaluation and variation [37]. Moreover, the proposed method relies on the fitting of the whole fluorescence reading avoiding the serious problem of point selection by the user. Finally, the stability of this procedure is further ensured by its integration with the SOD method allowing us to detect and correct only those amplifications which showed marked different efficiencies. Taken together, these features make the proposed method very precise, as Data File S4 Fluorescence data, fitting elaborations and Cy0 calculations obtained using the freely available data set named ‘‘Competimer’’ reported in Ruijter et al. [32]. Evaluation of the Cy0 Correction in Muscle Samples Obtained using Fine Needle Aspiration MtDNA real-time PCR quantifications in muscle samples obtained from Fine Needle Aspiration. Mt-CO1 and ATP6 mitochondrial genes were quantified from 40 samples. Only SOD positive samples were included in this analysis. A) shows the descriptive correlation analysis between Mt-CO1 and ATP6 quantification while panel B) shows the Bland-Altman plot. doi:10.1371/journal.pone.0068481.g008 July 2013 | Volume 8 | Issue 7 | e68481 PLOS ONE | www.plosone.org 9 9 References (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification. J Forensic Sci 39: 362–372. 1. Gingeras TR, Higuchi R, Kricka LJ, Lo YM, Wittwer CT (2005) Fifty years of molecular (DNA/RNA) diagnostics. Clin Chem 51: 661–671. 13. Tichopad A, Didier A, Pfaffl MW (2004) Inhibition of real-time RT-PCR quantification due to tissue-specific contaminants. Mol Cell Probes 18: 45–50. 2. Nolan T, Hands RE, Bustin SA (2006) Quantification of mRNA using real-time RT-PCR. Nature Protocols 1: 1559–1582. 14. Wilson IG (1997) Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 63: 3741–3751. 3. 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Nucleic Acids Res 31: e93. application of standard curves. Accurate and Precise Real-Time PCR Quantification Accurate and Precise Real-Time PCR Quantification 24. Liu W, Saint DA (2002) A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics. Anal Biochem 302: 52–59. 30. Guescini M, Fatone C, Stocchi L, Guidi C, Potenza L, et al. (2007) Fine needle aspiration coupled with real-time PCR: a painless methodology to study adaptive functional changes in skeletal muscle. Nutr Metab Cardiovasc Dis 17: 383–393. g 31. Richards F (1959) A flexible growth function for empirical use. Journal of experimental Botany 10: 290–300. 25. Peirson SN, Butler JN, Foster RG (2003) Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis. Nucleic Acids Res 31: e73. 32. Ruijter JM, Pfaffl MW, Zhao S, Spiess AN, Boggy G, et al. 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Spiess AN, Feig C, Ritz C (2008) Highly accurate sigmoidal fitting of real-time PCR data by introducing a parameter for asymmetry. BMC Bioinformatics 9: 221. 35. Larionov A, Krause A, Miller W (2005) A standard curve based method for relative real time PCR data processing. BMC Bioinformatics 6: 62. 36. Shain EB, Clemens JM (2008) A new method for robust quantitative and qualitative analysis of real-time PCR. Nucleic Acids Res 36: e91. 36. Shain EB, Clemens JM (2008) A new method for robust qua 29. Guescini M, Sisti D, Rocchi MB, Stocchi L, Stocchi V (2008) A new real-time PCR method to overcome significant quantitative inaccuracy due to slight amplification inhibition. BMC Bioinformatics 9: 326. qualitative analysis of real-time PCR. Nucleic Acids Res 36: e91. 37. Ruijter JM, Ramakers C, Hoogaars WM, Karlen Y, Bakker O, et al. References Nucleic Acids Res 31: e93. 20. Bar T, Kubista M, Tichopad A (2012) Validation of kinetics similarity in qPCR. Nucleic Acids Res 40: 1395–1406. 9. 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Akane A, Matsubara K, Nakamura H, Takahashi S, Kimura K (1994) Identification of the heme compound copurified with deoxyribonucleic acid July 2013 | Volume 8 | Issue 7 | e68481 PLOS ONE | www.plosone.org 10 31. Richards F (1959) A flexible growth function for empirical use. Journal of experimental Botany 10: 290–300. 32. Ruijter JM, Pfaffl MW, Zhao S, Spiess AN, Boggy G, et al. (2013) Evaluation of qPCR curve analysis methods for reliable biomarker discovery: Bias, resolution, precision, and implications. Methods 59: 32–46. Accurate and Precise Real-Time PCR Quantification (2009) Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res 37: e45. PLOS ONE | www.plosone.org July 2013 | Volume 8 | Issue 7 | e68481 PLOS ONE | www.plosone.org 11
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STN1 OB Fold Mutation Alters DNA Binding and Affects Selective Aspects of CST Function
PLOS genetics
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STN1 OB Fold Mutation Alters DNA Binding and Affects Selective Aspects of CST Function * jason.stewart@sc.edu (JS); carolyn.price@uc.edu (CMP) * jason.stewart@sc.edu (JS); carolyn.price@uc.edu (CMP) a1111 OPEN ACCESS Citation: Bhattacharjee A, Stewart J, Chaiken M, Price CM (2016) STN1 OB Fold Mutation Alters DNA Binding and Affects Selective Aspects of CST Function. PLoS Genet 12(9): e1006342. doi:10.1371/journal.pgen.1006342 Editor: Karel Riha, Masaruk University, CZECH REPUBLIC Editor: Karel Riha, Masaruk University, CZECH REPUBLIC Received: May 16, 2016 Accepted: September 6, 2016 Published: September 30, 2016 Copyright: © 2016 Bhattacharjee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by grants NIH RO1 GM041803, NIH K99 GM1104409 and NIH R00 GM1104409. https://www.nigms.nih.gov/ Pages/default.aspx. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. RESEARCH ARTICLE a11111 Abstract Mammalian CST (CTC1-STN1-TEN1) participates in multiple aspects of telomere replica- tion and genome-wide recovery from replication stress. CST resembles Replication Protein A (RPA) in that it binds ssDNA and STN1 and TEN1 are structurally similar to RPA2 and RPA3. Conservation between CTC1 and RPA1 is less apparent. Currently the mechanism underlying CST action is largely unknown. Here we address CST mechanism by using a DNA-binding mutant, (STN1 OB-fold mutant, STN1-OBM) to examine the relationship between DNA binding and CST function. In vivo, STN1-OBM affects resolution of endoge- nous replication stress and telomere duplex replication but telomeric C-strand fill-in and new origin firing after exogenous replication stress are unaffected. These selective effects indicate mechanistic differences in CST action during resolution of different replication problems. In vitro binding studies show that STN1 directly engages both short and long ssDNA oligonucleotides, however STN1-OBM preferentially destabilizes binding to short substrates. The finding that STN1-OBM affects binding to only certain substrates starts to explain the in vivo separation of function observed in STN1-OBM expressing cells. CST is expected to engage DNA substrates of varied length and structure as it acts to resolve dif- ferent replication problems. Since STN1-OBM will alter CST binding to only some of these substrates, the mutant should affect resolution of only a subset of replication problems, as was observed in the STN1-OBM cells. The in vitro studies also provide insight into CST binding mechanism. Like RPA, CST likely contacts DNA via multiple OB folds. However, the importance of STN1 for binding short substrates indicates differences in the architec- ture of CST and RPA DNA-protein complexes. Based on our results, we propose a dynamic DNA binding model that provides a general mechanism for CST action at diverse forms of replication stress. * jason.stewart@sc.edu (JS); carolyn.price@uc.edu (CMP) RESEARCH ARTICLE Introduction Although DNA replication must occur rapidly and with high fidelity, the replisome frequently encounters obstacles such as DNA damage or repetitive sequence that cause the replication fork to stall. Since stalled forks can lead to double strand breaks and genomic instability, multiple pathways exist to ensure their resolution [1,2]. Telomeres pose a particularchallenge to the repli- cation machinery due to their repetitive G-rich sequence and the inability of DNA polymerase to completely replicate the DNA 5’ terminus [3–5]. To ensure telomeres are duplicated effi- ciently, the replication process occurs in several distinct steps [3,6,7] and involves a number of ancillary proteins [8–11]. First, the repetitive dsDNA is duplicated by the replisome with assis- tance from various accessory factors. Next, the chromosome ends are processed by nucleases to form a single-strandedoverhang on the 3’ G-rich strand (termed the G-overhang). In telomerase positive cells, the G-overhang is then extended by telomerase. Finally, much of the elongated overhang is converted to duplex DNA by DNA polymerase alpha (pol α) in a process known as C-strand fill-in. This leaves a short G-overhang that is then bound by telomere proteins. g y CST is a protein complex that binds ssDNA and promotes telomere replication in a wide range of eukaryotes [12–16]. Budding yeast CST (Cdc13-Stn1-Ten1) binds the G-overhang where it protects the telomere, recruits telomerase and mediates C-strand fill-in [17–20]. Mam- malian CST (CTC1-STN1-TEN1) is less important for telomere-end protection but it functions both in telomere duplex replication and C-strand fill-in [21–25]. It is also proposed to limit telo- merase action, perhaps by competing for binding to the telomere protein TPP1 [26,27]. CST has additional genome-wide roles that are just starting to be appreciated [13,24,28–31]. In humans, CST facilitates recovery from various forms of replication stress throughout the genome. It promotes activation of dormant or late firing origins in response to replication fork stalling [24] and enhances viability when cells are treated with drugs that block replication fork progression [30]. Mutations in CTC1 cause the diseases Coats plus and dyskeratosis congenita [32–34]. The telomeric and non-telomeric roles of CST are likely to underlie the severity of these diseases. Although the mechanism of CST action is still unclear, multiple studies indicate a link to pol α. Mammalian CTC1 and STN1 were originally identified as Alpha Accessory Factor (AAF), a factor that co-purifiedwith pol α and enhanced its processivity and affinity for ssDNA templates [35,36]. Effect of STN1 Mutation on CST Function and defects in chromosome segregation. Currently, we do not understand how CST acts to ensure the resolution of very diverse types of replication problem. We set out to address this question by studying a mutant form of CST that was predicted to alter DNA binding. The mutations are in the STN1 subunit. In vivo, the STN1 mutant (STN1-OBM) affects some aspects of CST function while others are normal. The effects of STN1-OBM do not align with the telomeric versus non-telomeric roles of CST but instead separate out differ- ent aspects of CST function at telomeres and genome-wide. In vitro binding studies indi- cate that STN1-OBM disrupts binding to only short DNA substrates. Since CST is likely to encounter DNA substrates of varied length and structure in vivo as it helps resolve differ- ent replication problems, this finding starts to explain why STN1-OBM affects only certain aspects of CST function. Our in vitro binding studies also shed light on how CST actually binds to DNA and they suggest a novel “dynamic binding model” that provides a mecha- nistic explanation for how CST helps resolve a diverse array of replication problems to preserve genome stability. Author Summary Mammalian CST (CTC1/STN1/TEN1) is a three protein complex that aids in several steps during telomere replication and has genome-wide roles during recovery from replication fork stalling. Loss of CST leads to abnormalities in telomere structure, genomic instability Competing Interests: The authors have declared that no competing interests exist. 1 / 24 PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 Effect of STN1 Mutation on CST Function mammals [20,21,37,38]. Xenopus CST stimulates DNA priming by pol α on ssDNA [39] while Candida CST enhances primase activity and primase to polymerase switching [40]. CST exhibits notable structural similarities to Replication Protein A (RPA) the eukaryotic ssDNA binding protein that directs the assembly of multi-protein complexes needed for DNA replication, recombination and repair (Fig 1A) [23]. RPA has three subunits (RPA1, RPA2 and RPA3) that together harbor six OB (oligonucleotide-oligosaccharide binding) folds (Fig 1A) [41,42]. Four of the OB folds participate in DNA binding. Because RPA has multiple DNA binding sites, individual OB folds can undergo rapid dissociation and re-association without causing the protein to fall off the DNA [43,44]. This dissociation and re-association of OB folds underlies RPA function as it makes binding dynamic and enables RPA to diffuse along DNA to melt DNA structure or load and unload proteins needed for replication, recombina- tion or repair [45]. Like RPA, CST appears to harbor OB folds in all three subunits and X-ray crystallography indicates striking structural similarity between STN1-TEN1 and RPA2-RPA3 dimers [46–48]. The structural conservation encompasses the OB-fold and winged helix domains and the dimerization interface. The large subunits of RPA and CST appear less well conserved. Although RPA1 and Cdc13 from budding yeast each harbor 4 OB folds, Cdc13 needs only one OB fold for high affinity binding [42,49]. Moreover, Cdc13 dimerizes through its N-terminal OB fold to form a DNA pol α binding surface, whereas RPA1 does not self-associate [50]. In mammalian cells, Cdc13 is replaced by CTC1 but the two proteins share little sequence identity and the extent of structural or functional conservation is unclear [37]. Protein threading pro- grams (Phyr2 and HHpred) predict 5–6 OB folds in human CTC1 with the three most C-ter- minal folds resembling those of RPA1 (Fig 1A). In vitro studies have revealed an additional parallel between RPA and human CST as in each case high affinity DNA binding requires for- mation of the three protein complex [23,26,41,42]. The structural similarities between RPA and CST raise the possibility that dynamic DNA binding through multiple OB folds may also contribute to CST function. Since so little is known about CST mechanism of action and the relationship between DNA binding and CST function, we set out to analyze how reduced DNA binding affects CST activ- ity at telomeres and elsewhere in the genome. We describe a STN1 OB fold mutant that prefer- entially affects in vitro binding to short DNA substrates. In vivo, the mutant can substitute for wild type STN1 in some aspects of CST function but other aspects are impaired. DNA binding studies indicate that, like RPA, CST appears to contact DNA via multiple OB folds and to have distinct modes of binding. However, we also provide evidence that the organization of OB-fold engagement by CST is quite different. PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 Introduction CST and pol α have since been shown to interact in yeast, plants and PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 2 / 24 Fig 1. STN1 OB fold mutation affects DNA binding but not CST complex formation. (A) Cartoon depicting known and predicted OB fold domains in RPA and CST, white boxes indicate OB folds. Black bars mark OB folds in CST (known for STN1, predicted for CTC1, see Materials and Methods) where mutations alter DNA binding [15,36]. (B) Alignment of human STN1 and RPA32 OB folds showing identical and similar (+) residues [52]. Arrows indicate mutations in the STN1 OB fold mutant (STN1-OBM), arrowheads mark corresponding RPA residues that contact DNA directly in the U. maydis crystal structure [41]. (C) Western blots showing co-immunoprecipitation of CTC1 and TEN1 with STN1 or STN1-OBM using extracts from TEN1 overexpressing HeLa cells transiently transfected with HA-CTC1 and FLAG-STN1 or FLAG-STN1-OBM. (D) Silver-stained gels Effect of STN1 Mutation on CST Function OS G i | DOI 10 13 1/j l 1006342 S b 30 2016 4 / 24 Effect of STN1 Mutation on CST Function Fig 1. STN1 OB fold mutation affects DNA binding but not CST complex formation. (A) Cartoon depicting known and predicted OB fold domains in RPA and CST, white boxes indicate OB folds. Black bars mark OB folds in CST (known for STN1, predicted for CTC1, see Materials and Methods) where mutations alter DNA binding [15,36]. (B) Alignment of human STN1 and RPA32 OB folds showing identical and similar (+) residues [52]. Arrows indicate mutations in the STN1 OB fold mutant (STN1-OBM), arrowheads mark corresponding RPA residues that contact DNA directly in the U. maydis crystal structure [41]. (C) Western blots showing co-immunoprecipitation of CTC1 and TEN1 with STN1 or STN1-OBM using extracts from TEN1 overexpressing HeLa cells transiently transfected with HA-CTC1 and FLAG-STN1 or FLAG-STN1-OBM. (D) Silver-stained gels Fig 1. STN1 OB fold mutation affects DNA binding but not CST complex formation. (A) Cartoon depicting known and predicted OB fold domains in RPA and CST, white boxes indicate OB folds. Black bars mark OB folds in CST (known for STN1, predicted for CTC1, see Materials and Methods) where mutations alter DNA binding [15,36]. (B) Alignment of human STN1 and RPA32 OB folds showing identical and similar (+) residues [52]. Arrows indicate mutations in the STN1 OB fold mutant (STN1-OBM), arrowheads mark corresponding RPA residues that contact DNA directly in the U. maydis crystal structure [41]. Alteration of DNA-binding by STN1 OB-fold mutation To generate the STN1 OB-fold mutant (STN1-OBM) we changed three residues (W89A, R139L, Y141A) that are conservedbetween STN1 and the OB fold of RPA2 and which either directly contact, or lie very close to DNA in RPA crystal structures (Fig 1B) [41,51]. The W89A and Y141A mutations were chosen because the equivalent mutations in mouse STN1/AAF-44 reduced DNA binding by ~60% in pull-down assays with biotin-labeled poly-dC [36] Co-immunoprecipitation and tandem affinity purification experiments verified that the STN1 mutant retained the ability to form a complex with CTC1 and TEN1 (Fig 1C and 1D). In initial experiments, we co-expressed FLAG-tagged STN1 or STN1-OBM with HA-CTC1 in a previously characterized HeLa cell line over-expressing TEN1 [30]. When STN1-OBM was immunoprecipitated from whole cell lysate, both CTC1 and TEN1 co-purified(Fig 1C). We To generate the STN1 OB-fold mutant (STN1-OBM) we changed three residues (W89A, R139L, Y141A) that are conservedbetween STN1 and the OB fold of RPA2 and which either directly contact, or lie very close to DNA in RPA crystal structures (Fig 1B) [41,51]. The W89A and Y141A mutations were chosen because the equivalent mutations in mouse STN1/AAF-44 reduced DNA binding by ~60% in pull-down assays with biotin-labeled poly-dC [36] Co-immunoprecipitation and tandem affinity purification experiments verified that the STN1 mutant retained the ability to form a complex with CTC1 and TEN1 (Fig 1C and 1D). In initial experiments, we co-expressed FLAG-tagged STN1 or STN1-OBM with HA-CTC1 in a previously characterized HeLa cell line over-expressing TEN1 [30]. When STN1-OBM was immunoprecipitated from whole cell lysate, both CTC1 and TEN1 co-purified(Fig 1C). We 3 / 24 PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 Effect of STN1 Mutation on CST Function showing co-purification of FLAG-CTC1 and TEN1 with His-STN1 or His-STN1-OBM expressed in insect cells. Left, a full 12% gel showing all three subunits. Right, portion of a more heavily stained 15% gel showing STN1 and TEN1. (E-F) EMSAs showing CST(WT) or CST(STN1-OBM) binding to non-telomeric (NonTel) or telomeric G-strand (TelG) oligonucleotides or dsDNA (DS) of the indicated lengths. Increasing concentrations of CST were incubated with 0.1 nM labeled DNA for 30 min prior to separation in agarose gels. (E) The same CST(WT) or CST(STN1-OBM) preparation was used in all the EMSAs with ssDNA. showing co-purification of FLAG-CTC1 and TEN1 with His-STN1 or His-STN1-OBM expressed in insect cells. Left, a full 12% gel showing all three subunits. Right, portion of a more heavily stained 15% gel showing STN1 and TEN1. (E-F) EMSAs showing CST(WT) or CST(STN1-OBM) binding to non-telomeric (NonTel) or telomeric G-strand (TelG) oligonucleotides or dsDNA (DS) of the indicated lengths. Increasing concentrations of CST were incubated with 0.1 nM labeled DNA for 30 min prior to separation in agarose gels. (E) The same CST(WT) or CST(STN1-OBM) preparation was used in all the EMSAs with ssDNA. doi:10.1371/journal.pgen.1006342.g001 doi:10.1371/journal.pgen.1006342.g001 also generated recombinant CST complexes containing wild type STN1 (CST(WT)) or STN1-OBM (CST(STN1-OBM)) by co-infectinginsect cells with baculovirusencoding FLAG- tagged CTC1, untagged TEN1 and His-tagged STN1 or STN1-OBM. Protein complexes were affinity purified on nickel resin followed by FLAG beads (Fig 1D) and again CTC1 and TEN1 co-purifiedwith STN1-OBM. We next examined how STN1-OBM affects the ability of CST to bind a range of DNA sub- strates. As the affinity of CST for short versus long substrates seems to depend on DNA sequence [26], we monitored binding of CST(WT) and CST(STN1-OBM) to telomeric and non-telomeric oligonucleotides of various lengths (Fig 1E and 1F and Table 1). When we used electrophoretic mobility shift assays (EMSA) to compare binding of CST(WT) and CST (STN1-OBM) to 48 nt substrates, the two complexes appeared to bind both non-telomeric (NonTel-48) and telomeric G-strand (TelG-48) DNA with similar affinity. However relative to CST(WT), the CST(STN1-OBM)) bound less efficiently to the 36 nt non-telomeric (NonTel- 36) and the 18 nt telomeric G-strand (TelG-18) substrates. Neither complex bound equivalent concentrations of the 18 nt non-telomeric oligonucleotide (NonTel-18) or dsDNA (Fig 1E) [23]. These results suggest that the STN1-OBM preferentially affects binding to short sub- strates. Note, we refer to TelG-18 as a short substrate because CST has very low affinity for DNA with fewer telomeric repeats, e.g. TelG-12 [26]. Our results also confirm that CST binds both telomeric and non-telomeric DNA but that telomeric DNA is preferred when substrate length is short [26]. (C) Western blots showing co-immunoprecipitation of CTC1 and TEN1 with STN1 or STN1-OBM using extracts from TEN1 overexpressing HeLa cells transiently transfected with HA-CTC1 and FLAG-STN1 or FLAG-STN1-OBM. (D) Silver-stained gels PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 4 / 24 Effect of STN1 Mutation on CST Function In initial experiments, we asked if STN1-OBM could rescue the increase in anaphase bridges that occurs after STN1 depletion [24]. STN1-OBM cells and a series of control cells (shSTN1, STN1 R d hNT t t hRNA t l) t d i it i ith d l Fig 2. In vivo expression of STN1-OBM causes anaphase bridges. (A) Western blot showing levels of STN1 in HeLa cells expressing non-target shRNA (shNT) or STN1 shRNA (shSTN1) and shSTN1 cells with sh-resistant mutant STN1 (STN1-OBM) or wild type STN1 (STN1-Res). Blot was probed with antibody to STN1 or to actinin for a loading control. (B) Left; representative images of DAPI-stained anaphase cells with/ without bridges. Anaphase cells with no bridge in shNT (top) and with bridges in shSTN1 and STN1-OBM cells (middle and bottom). Right; quantification of bridges (200 anaphases counted per cell line per experiment. n = 3 experiments, mean ± S.E.M, p-values are indicated above bars). doi:10.1371/journal.pgen.1006342.g002 Fig 2. In vivo expression of STN1-OBM causes anaphase bridges. (A) Western blot showing levels of STN1 in HeLa cells expressing non-target shRNA (shNT) or STN1 shRNA (shSTN1) and shSTN1 cells with sh-resistant mutant STN1 (STN1-OBM) or wild type STN1 (STN1-Res). Blot was probed with antibody to STN1 or to actinin for a loading control. (B) Left; representative images of DAPI-stained anaphase cells with/ without bridges. Anaphase cells with no bridge in shNT (top) and with bridges in shSTN1 and STN1-OBM cells (middle and bottom). Right; quantification of bridges (200 anaphases counted per cell line per experiment. n = 3 experiments, mean ± S.E.M, p-values are indicated above bars). doi:10.1371/journal.pgen.1006342.g002 Fig 2. In vivo expression of STN1-OBM causes anaphase bridges. (A) Western blot showing levels of STN1 in HeLa cells expressing non-target shRNA (shNT) or STN1 shRNA (shSTN1) and shSTN1 cells with sh-resistant mutant STN1 (STN1-OBM) or wild type STN1 (STN1-Res). Blot was probed with antibody to STN1 or to actinin for a loading control. (B) Left; representative images of DAPI-stained anaphase cells with/ without bridges. Anaphase cells with no bridge in shNT (top) and with bridges in shSTN1 and STN1-OBM cells (middle and bottom). Right; quantification of bridges (200 anaphases counted per cell line per experiment. n = 3 experiments, mean ± S.E.M, p-values are indicated above bars). PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 STN1-OBM fails to rescue to anaphase bridges after endogenous STN1 depletion To examine the in vivo effects of STN1 OB fold mutation, we generated HeLa cells that stably express FLAG-tagged STN1-OBM (Fig 2A and S1A Fig) by introducing an shRNA-resistant STN1-OBM cDNA into a previously characterized HeLa cell line expressing STN1 shRNA (shSTN1) [24,25]. A cell line expressing FLAG-tagged shRNA-resistant wild type STN1 (STN1-Res) was previously made in the same manner [24]. Table 1. Sequence of oligonucleotides used in DNA binding assays. Oligo Sequence (5’-3’) TelG-18 GGTTAGGGTTAGGGTTAG TelG-36 GGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAG TelG-48 GGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAG NonTel-18 AGCGTATCCGTTCAGTTG NonTel-36 AGCGTATCCGTTCAGTTGAGCGTATCCGTTCAGTTG NonTel-48 AGCGTATCCGTTCAGTTGAGCGTATCCGTTCAGTTGAGCGTATCCGTT ds 15 TTCGATCTACGTCAGCA 5’ T ............... TTGCTAGATGCAGTCGT 3’ ds 40 TTTACGTCAGCACGATCTACGTCAGCACGATCTACGTCAGCA 5’ T ........................................ TTATGCAGTCGTGCTAGATGCAGTCGTGCTAGATGCAGTCGT 3’ doi:10.1371/journal.pgen.1006342.t001 PLOS Genetics | DOI 10 1371/jo rnal pgen 1006342 September 30 2016 5 / 24 Table 1. Sequence of oligonucleotides used in DNA binding assays. 5 / 24 STN1-OBM affects telomere duplex replication but not C-strand fill-in Anaphase bridges can have a number of causes including telomere-to-telomere fusion and the presence of unresolved replication intermediates either at telomeres or elsewhere in the genome [53,54]. Thus, to ask more specificallywhether STN1-OBM affects the telomeric roles of CST, we looked for changes in telomere structure. Metaphase spreads from STN1-OBM and control cells were hybridized with telomere probe and examined for telomere loss, telomere fusions or other abnormal telomere signals. As previously reported, we did not observe an increase in telomere loss or telomere fusions in the shSTN1 and STN1-Res cells [24] (S1B Fig). This was also true for the STN1-OBM cells (S1B Fig), indicating that the anaphase bridges caused by STN1-OBM expression are unlikely to be caused by telomere fusions. However, rela- tive to the STN1-Res control, the STN1-OBM cells showed a large increase in individual chro- matids exhibiting Multiple Telomeric FISH Signals (MTS) (Fig 3A). As expected,the shSTN1 cells also showed an increase in MTS but it was lower than in the STN1-OBM cells. Again this may reflect the displacement of residual endogenous STN1 with STN1-OBM in CST com- plexes. Past studies have shown that MTS arise after fork stalling during replication of the telo- mere duplex [8] and they occur after depletion of the various factors needed for telomere replication, including CST [8,25]. In particular, STN1 depletion slows replication through the telomere duplex and causes the appearance of MTS [25]. We therefore conclude that STN1-OBM is unable to rescue the deficiencyin telomere duplex replication caused by STN1 depletion. We next asked if STN1-OBM affects telomere length or G-overhang structure. Genomic DNA was isolated from STN1-OBM or control cells and telomere restriction fragments were examined by Southern blotting or in-gel hybridization to monitor telomere length (S1C Fig). This analysis revealed that telomeres of shSTN1, STN1-Res and STN1-OBM cells were very similar in length, thus confirming our previous finding that telomere length in HeLa cells is largely unaffected by STN1 knockdown [25] and indicating that STN1-OBM has dominant negative effect. We then used an in-gel hybridization assay to ask if STN1-OBM affects G-over- hang structure. Restriction digested DNA was separated briefly in agarose gels and hybridized with a probe to the telomeric G-strand under non-denaturing conditions (Fig 3B). The DNA was then denatured and re-hybridized with the same probe. Effect of STN1 Mutation on CST Function doi:10.1371/journal.pgen.1006342.g002 doi:10.1371/journal.pgen.1006342.g002 doi:10.1371/journal.pgen.1006342.g002 In initial experiments, we asked if STN1-OBM could rescue the increase in anaphase bridges that occurs after STN1 depletion [24]. STN1-OBM cells and a series of control cells (shSTN1, STN1-Res and shNT, a non-target shRNA control) were arrested in mitosis with nocadozole, released for 45–60 min, fixed and scored for the number of anaphase cells with DAPI-stained bridges (Fig 2B). As previously described,depletion of STN1 caused an increase in bridges and this was rescued by expression of sh-resistant wild type STN1 [24]. In contrast, expression of sh-resistant STN1-OBM did not rescue bridge formation but instead further increased the frac- tion of anaphase cells with bridges. The reason for the higher level of bridges in the STN1-OBM cells relative to the shSTN1 cells is unclear but a possible cause is that STN1-OBM replaces residual endogenous STN1 in CST complexes. Overall, the inability of STN1-OBM to rescue the anaphase bridge phenotype indicates that STN1-OBM cannot substitute for wild type STN1 in some aspects of CST function. 6 / 24 PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 STN1-OBM affects telomere duplex replication but not C-strand fill-in Quantification of the overhang sig- nal relative to total telomeric DNA revealed the expected increase in overhang amount in the STN1-depleted cells (Fig 3C). This increase has previously been shown to result from ineffi- cient C-strand fill-in following telomerase extension [21,25]. Given that STN1-OBM affects binding to telomeric G-strand DNA in vitro (Fig 1E) and telomere duplex replication in vivo (Fig 2), we anticipated that the STN1-OBM cells would also have a deficiencyin C-strand fill- in. However, to our surprise we found that STN1-OBM cells had normal length overhangs (Fig 3C) implying that the mutant STN1 was able to rescue C-strand fill-in. G-overhang length is determined by a number of activities that occur at specific stages in the cell cycle. Overhangs are elongated in S-phase as a result of G-strand synthesis by telome- rase and C-strand resection by nuclease [6,7]. They are then returned to their original length in late S/G2 via C-strand fill-in by DNA pol α [25]. Given this balance between activities, it was possible that the normal length overhangs in the STN1-OBM cells result from decreased G- strand extension in S-phase in combination with decreased C-strand fill-in during late S/G2. To investigate this possibility, we examined G-overhang length dynamics during the cell cycle. Cells were synchronized in G1/S with a double thymidine block, released into S-phase and har- vested at intervals as they passed through mid S-phase, G2/M and back into G1 (Fig 4A and S2A Fig). Following DNA isolation, relative overhang length was examined by in-gel hybridiza- tion as describedabove (Fig 4B and 4C). Quantification of the overhang signal indicated that 7 / 24 PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 STN1-OBM does not disrupt interaction with TPP1 or DNA pol α Several studies have shown that STN1 can interact with the shelterin protein TPP1 [26,56], suggesting that this interaction might be important for recruiting CST or stabilizing CST bind- ing at the telomere. Given that OB folds can mediate protein-protein interactions as well as DNA binding [42], we considered the possibility that the in vivo effects of STN1-OBM expres- sion might reflect decreased binding of CST to TPP1. To test for a disruption in the TPP1-STN1 interaction, we transfected 293T cells with constructs encoding FLAG-tagged STN1 or STN1-OBM and HA-mCherry tagged TPP1 [57] and monitored co-immunoprecipi- tation of TPP1 with STN1. When TPP1 was precipitated with antibody to HA, Western blot analysis showed that the STN1 and STN1-OBM co-precipitated with equivalent efficiency(Fig 4D). We therefore conclude that STN1-OBM retains the ability to bind TPP1. We also tested whether STN1-OBM disrupts binding to DNA pol α, the only other known CST binding part- ner [21,58]. 293T cells were transfected with constructs encoding TEN1, FLAG or Myc-tagged CTC1 and FLAG-STN1 or FLAG-STN1-OBM, and CST was then precipitated from extracts with FLAG antibody. Western blot analysis of the immunoprecipitates showed that pol α co- precipitated with FLAG-STN1 only if both CTC1 and STN1 were overexpressed (S2B Fig). However the level of pol α precipitation was similar with CST(WT) and CST(STN1-OBM), indicating that STN1-OBM does not prevent CST from binding to pol α (Fig 4E and S2B Fig). Our finding that C-strand fill-in is unaffected by STN1-OBM (Figs 3B and 4C) provides further support for a functional interaction between pol α and CST(STN1-OBM), Effect of STN1 Mutation on CST Function monitored by in-gel hybridization with (A2TC3)4 probe. (B) Representative gels showing hybridization to genomic DNA from the indicated cells under native or denaturing conditions. (C) Quantification of G- overhang abundance in asynchronous cells (n = 3 experiments, mean ± S.E.M., p-values are indicated above bars). monitored by in-gel hybridization with (A2TC3)4 probe. (B) Representative gels showing hybridization to genomic DNA from the indicated cells under native or denaturing conditions. (C) Quantification of G- overhang abundance in asynchronous cells (n = 3 experiments, mean ± S.E.M., p-values are indicated above bars). doi:10.1371/journal.pgen.1006342.g003 doi:10.1371/journal.pgen.1006342.g003 the STN1-Res cells showed the expected increase in overhang abundance as they transitioned from G1 (0 hr) into mid S-phase (6 hrs) [6,7,25]. The overhang signal then declined due to C- strand fill-in as the cells transitioned into G2 (8 hrs) and G1 of the next cell cycle (10–12 hrs) [25,55]. Interestingly, the pattern of overhang elongation and shortening in the STN1-OBM cells was indistinguishable from that seen with the control STN1-Res cells indicating that STN1-OBM does not affect overhang elongation or C-strand fill-in. In contrast, the shSTN1 cells exhibited the expected delay in overhang shortening in late S/G2 reflecting the deficiency in C-strand fill-in [25]. Thus although STN1-OBM affects telomere duplex replication, it does not appear to affect C-strand fill-in by DNA pol α. Effect of STN1 Mutation on CST Function Fig 3. STN1-OBM causes multiple telomere signals (MTS) but does not affect G-overhang maintenance. (A) Telomere FISH of STN1-OBM or shSTN1 cells. Left; representative images of single metaphase chromosomes. White arrows, MTS; green, FITC-(C3TA2)3 probe; blue, DAPI. Right; Quantification of MTS (n = 4 experiments mean ± S.E.M.). Individual chromosomes were scored positive for MTS if they had MTS at one or more telomeres. (B-C) G-overhang abundance in asynchronous cells Fig 3. STN1-OBM causes multiple telomere signals (MTS) but does not affect G-overhang maintenance. (A) Telomere FISH of STN1-OBM or shSTN1 cells. Left; representative images of single metaphase chromosomes. White arrows, MTS; green, FITC-(C3TA2)3 probe; blue, DAPI. Right; Quantification of MTS (n = 4 experiments mean ± S.E.M.). Individual chromosomes were scored positive for MTS if they had MTS at one or more telomeres. (B-C) G-overhang abundance in asynchronous cells PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 8 / 24 STN1-OBM can function in replication rescue after genome wide replication fork stalling Since the above studies indicate that STN1-OBM has selective effects on CST function, we next examined whether the mutant affects the response to genome-wide replication fork stalling. In initial experiments, we asked if STN1-OBM could substitute for wild type STN1 to maintain cell viability after HU (hydroxyurea) treatment. STN1-OBM and control cells were treated with 2 mM HU for 0–24 hrs, allowed to recover for 24 hrs then cell viability was monitored by MTT assay (Fig 5A). As observedpreviously, STN1 depletion increased sensitivity to HU [30]. However, wild type STN1 (STN1-Res) and STN1-OBM rescued this sensitivity to an equal extent, indicating the mutant was sufficient to allow CST function in recovery from prolonged fork stalling. 9 / 24 PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 doi:10.1371/journal.pgen.1006342.g004 PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 Fig 5. STN1-OBM rescues viability and restores origin firing after HU treatment. (A) MTT assay showing viability afte HU treatment. Cells were treated with 2 mM HU for the indicated times and harvested for MTT assay 24 hrs later. Values a relative to untreated cells of the same cell type. Each time point was assayed in triplicate and the data are shown as the mean ± S.D from 3 independent experiments. For each cell line, the value of the untreated sample was set at 1. (B-D) DNA fiber analysis of origin firing following release from 2 mM HU. (B) Left: schematic showing timing of IdU and CldU labeling relative to HU treatment. Right: types of replication event scored. (C) Representative images of DNA tracks. Red, IdU; Green, CldU. (D) Graph indicating the percent of DNA tracks showing new origin firing (green-only tracks) (n = 7 experiments, mean ± S.E.M, p-values are indicated above bars). doi:10.1371/journal.pgen.1006342.g005 Effect of STN1 Mutation on CST Functio Effect of STN1 Mutation on CST Function Fig 5. STN1-OBM rescues viability and restores origin firing after HU treatment. (A) MTT assay showing viability aft HU treatment. Cells were treated with 2 mM HU for the indicated times and harvested for MTT assay 24 hrs later. Values relative to untreated cells of the same cell type. Each time point was assayed in triplicate and the data are shown as the mean ± S.D from 3 independent experiments. For each cell line, the value of the untreated sample was set at 1. (B-D) DN fiber analysis of origin firing following release from 2 mM HU (B) Left: schematic showing timing of IdU and CldU labeling Fig 5. STN1-OBM rescues viability and restores origin firing after HU treatment. (A) MTT assay showing viability after HU treatment. Cells were treated with 2 mM HU for the indicated times and harvested for MTT assay 24 hrs later. Values are relative to untreated cells of the same cell type. Each time point was assayed in triplicate and the data are shown as the mean ± S.D from 3 independent experiments. For each cell line, the value of the untreated sample was set at 1. (B-D) DNA fiber analysis of origin firing following release from 2 mM HU. (B) Left: schematic showing timing of IdU and CldU labeling relative to HU treatment. Right: types of replication event scored. (C) Representative images of DNA tracks. Effect of STN1 Mutation on CST Function Fig 4. STN1-OBM is competent for C-strand fill-in and TPP1 and pol α interaction. (A-C) Analysis of C-strand fill-in. (A) FACS analysis of DNA content showing synchrony of STN1-OBM cells used in (B). (B) Representative gels showing in-gel hybridization of (TA2C3)4 probe to DNA from cells harvested at the indicated times after release from G1/S block. (C) Quantification of G-overhang abundance. Cell types were analyzed in pairs, n = 3 experiments for shSTN1 + STN1-OBM, mean ± S.E.M.; n = 2 experiments for STN1-OBM + STN1-Res, error bars show min/max values). (D) Western blot showing co-immunoprecipitation of TPP1 with STN1 or STN1-OBM. Cells were transfected with FLAG-STN1 or FLAG-STN1-OBM plus HA-mCherry-TPP1 expression constructs. TPP1 was precipitated with antibody to HA. (E) Co- immunoprecipitation of DNA pol α with CST. Cells were transfected with FLAG-STN1 or FLAG-STN1-OBM, Myc-CTC1 and TEN1. CST was precipitated with FLAG beads. doi:10 1371/journal pgen 1006342 g004 Fig 4. STN1-OBM is competent for C-strand fill-in and TPP1 and pol α interaction. (A-C) Analysis of C-strand fill-in. (A) FACS analysis of DNA content showing synchrony of STN1-OBM cells used in (B). (B) Representative gels showing in-gel hybridization of (TA2C3)4 probe to DNA from cells harvested at the indicated times after release from G1/S block. (C) Quantification of G-overhang abundance. Cell types were analyzed in pairs, n = 3 experiments for shSTN1 + STN1-OBM, mean ± S.E.M.; n = 2 experiments for STN1-OBM + STN1-Res, error bars show min/max values). (D) Western blot showing co-immunoprecipitation of TPP1 with STN1 or STN1-OBM. Cells were transfected with FLAG-STN1 or FLAG-STN1-OBM plus HA-mCherry-TPP1 expression constructs. TPP1 was precipitated with antibody to HA. (E) Co- immunoprecipitation of DNA pol α with CST. Cells were transfected with FLAG-STN1 or FLAG-STN1-OBM, Myc-CTC1 and TEN1. CST was precipitated with FLAG beads. PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 10 / 24 Effects of STN1-OBM on binding affinity and stability Our finding that STN-OBM affects only specific aspects of CST function is analogous to what has been observedfor certain RPA OB-fold mutants, which support DNA replication but are defective for DNA repair [60,61]. These mutants cause only a small decrease in overall affinity of RPA for ssDNA and the deficit in repair is thought to result from a change in the dynamics of RPA binding through its multiple OB folds [45,61]. The structural similarities between CST and RPA suggest that CST function could also rely on dynamic binding using multiple OB folds. We therefore set out to explore the extent to which RPA binding can be used as a paradigm for understanding CST activity and the in vivo separation of function observedwith STN1-OBM. As a first step, we revisited the effect of STN1-OBM on DNA binding by using filter binding assays to quantify the affinity of CST(WT) and CST(STN1-OBM) for telomeric and non-telo- meric substrates of various lengths (Fig 6A, 6C and S4 Fig). CST purified from insect cells was incubated with 32P-labeled DNA then the DNA-protein complexes were separated from free DNA by filtration through a sandwich of nitrocellulose and HyBond membrane. The bound versus free DNA was quantified and used to calculate the apparent dissociation constant (Kd, app). Despite the different approach used to separate bound from free DNA in the filter bind- ing and the original gel shift assay (Fig 1E), the results of the two assays were qualitatively simi- lar. The filter binding indicated that CST(WT) and CST(STN1-OBM) bound the 48 nt telomeric and non-telomeric substrates with a similar Kd,app while binding to TelG-18 was decreased for CST(STN1-OBM) relative to CST(WT) (Fig 6A and 6C). Thus, the filter binding analysis again indicated that STN1-OBM preferentially affects binding to short DNA sub- strates. However the analysis also revealed that the overall decrease in Kd,app for CST (STN1-OBM) binding to TelG-18 was only 2–3 fold. Our finding that STN-OBM affects only specific aspects of CST function is analogous to what has been observedfor certain RPA OB-fold mutants, which support DNA replication but are defective for DNA repair [60,61]. These mutants cause only a small decrease in overall affinity of RPA for ssDNA and the deficit in repair is thought to result from a change in the dynamics of RPA binding through its multiple OB folds [45,61]. Effect of STN1 Mutation on CST Function silanized slides by hydrodynamic flow [59]. The fibers were stained with antibody to IdU and CldU then visualized by confocal microscopy to score the replication events (Fig 5B–5D and S3 Fig). As observedpreviously, the HU-treated shSTN1 cells exhibited fewer green-only (CldU- only) tracks than the shNT and STN1-Res control cells [24,30], indicating that STN1 depletion caused a decrease in new origin firing after HU release. In contrast, the HU-treated STN1-OBM cells exhibited a similar number of green-only tracks to the control cells. The fre- quency of other replication events was also similar (S3 Fig). We therefore conclude that the STN1-OBM can substitute for wild type STN1 to promote new origin firing. Overall, our results indicate that STN1-OBM does not affect the capacity of CST to aid in the restart of rep- lication following exogenous replication stress. This is in direct contrast to the inability of STN1-OBM to rescue the effects of endogenous stress as seen by the increase in anaphase brid- ges and MTS in unchallenged STN1-OBM cells. Effects of STN1-OBM on binding affinity and stability The structural similarities between CST and RPA suggest that CST function could also rely on dynamic binding using multiple OB folds. We therefore set out to explore the extent to which RPA binding can be used as a paradigm for understanding CST activity and the in vivo separation of function observedwith STN1-OBM. As a first step, we revisited the effect of STN1-OBM on DNA binding by using filter binding assays to quantify the affinity of CST(WT) and CST(STN1-OBM) for telomeric and non-telo- meric substrates of various lengths (Fig 6A, 6C and S4 Fig). CST purified from insect cells was incubated with 32P-labeled DNA then the DNA-protein complexes were separated from free DNA by filtration through a sandwich of nitrocellulose and HyBond membrane. The bound versus free DNA was quantified and used to calculate the apparent dissociation constant (Kd, app). Despite the different approach used to separate bound from free DNA in the filter bind- ing and the original gel shift assay (Fig 1E), the results of the two assays were qualitatively simi- lar. The filter binding indicated that CST(WT) and CST(STN1-OBM) bound the 48 nt telomeric and non-telomeric substrates with a similar Kd,app while binding to TelG-18 was decreased for CST(STN1-OBM) relative to CST(WT) (Fig 6A and 6C). Thus, the filter binding analysis again indicated that STN1-OBM preferentially affects binding to short DNA sub- strates. However the analysis also revealed that the overall decrease in Kd,app for CST (STN1-OBM) binding to TelG-18 was only 2–3 fold. When gel shift assays were used to examine CST(STN1-OBM) binding to the TelG-18 and NonTel-36 substrates a substantial amount of DNA was seen to migrate between the bands corresponding to free DNA and CST-bound DNA (Fig 1E). This observation suggested that the DNA-protein complexes were dissociating and hence the decrease in Kd,app for CST (STN1-OBM) might reflect less stable binding. To test this possibility, we measured the rate of CST(WT) and CST(STN1-OBM) dissociation (t½) from selected substrates. CST complexes were bound to 32P-labeled oligonucleotide, challenged with an excess of the corresponding cold oligonucleotide for various times and the remaining labeled DNA/protein complex was quantified by filter binding. This experiment revealed that CST(STN1-OBM) dissociated from the labeled TelG-18 and NonTel-36 1.6–2.6-fold faster than CST(WT) whereas dissociation from TelG-48 and NonTel-48 was essentially the same (Fig 6B and 6C). We therefore conclude that the STN1-OB fold acts to stabilize CST binding to short ssDNA substrates. PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 Red, IdU; Green, CldU. (D) Graph indicating the percent of DNA tracks showing new origin firing (green-only tracks) (n = 7 experiments, mean ± S.E.M, p-values are indicated above bars). doi:10.1371/journal.pgen.1006342.g005 doi:10.1371/journal.pgen.1006342.g005 To further explore the effect of STN1-OBM on recovery from fork stalling, we performed DNA fiber analysis to determine if the mutant can substitute for endogenous STN1 to promote origin firing after HU treatment. Cells were labeled with IdU (iododeoxyuridine)for 15 min- utes, treated with HU for two hours then released into media containing CldU (chlorodeoxyur- idine) for 60 min (Fig 5B). The cells were then collected, lysed and the DNA fibers spread on 11 / 24 PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 Effect of STN1 Mutation on CST Function Fig 6. Analysis of CST DNA binding parameters. (A) Binding isotherms used to determine apparent dissociation constants for CST(WT) or CST(STN1-OBM) and the indicated ssDNA substrates. Data were obtained by filter binding assay and fit to a one site specific binding model. Mean ± SEM, n = 3 independent experiments each with a different protein preparation. (B) Dissociation kinetics for CST bound to the indicated substrates The fraction of labeled DNA remaining bound was determined by filter binding at the indicated times. Data were fit to a one phase exponential decay equation to obtain the dissociation rate (t½)). Mean ± SEM, n = 3 independent experiments. (C) Table summarizing Kd(app) and t½ for CST(WT) or CST(STN1-OBM) and the indicated ssDNA substrates.—undetectable binding, ND: not determined. doi:10.1371/journal.pgen.1006342.g006 :10.1371/journal.pgen.1006342 September 30, 2016 13 / 24 Fig 6. Analysis of CST DNA binding parameters. (A) Binding isotherms used to determine apparent dissociation constants for CST(WT) or CST(STN1-OBM) and the indicated ssDNA substrates. Data were obtained by filter binding assay and fit to a one site specific binding model. Mean ± SEM, n = 3 independent experiments each with a different protein preparation. (B) Dissociation kinetics for CST bound to the indicated substrates The fraction of labeled DNA remaining bound was determined by filter binding at the indicated times. Data were fit to a one phase exponential decay equation to obtain the dissociation rate (t½)). Mean ± SEM, n = 3 independent experiments. (C) Table summarizing Kd(app) and t½ for CST(WT) or CST(STN1-OBM) and the indicated ssDNA substrates.—undetectable binding, ND: not determined. doi:10.1371/journal.pgen.1006342.g006 Fig 6. Analysis of CST DNA binding parameters. (A) Binding isotherms used to determine apparent dissociation constants for CST(WT) or CST(STN1-OBM) and the indicated ssDNA substrates. Data were obtained by filter binding assay and fit to a one site specific binding model. Mean ± SEM, n = 3 independent experiments each with a different protein preparation. (B) Dissociation kinetics for CST bound to the indicated substrates The fraction of labeled DNA remaining bound was determined by filter binding at the indicated times. Data were fit to a one phase exponential decay equation to obtain the dissociation rate (t½)). Mean ± SEM, n = 3 independent experiments. (C) Table summarizing Kd(app) and t½ for CST(WT) or CST(STN1-OBM) and the indicated ssDNA substrates.—undetectable binding, ND: not determined. doi:10 1371/journal pgen 1006342 g006 Fig 6. Effects of STN1-OBM on binding affinity and stability PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 12 / 24 CST subunit interactions with ssDNA substrates While CST appears to resemble RPA in terms of subunit composition and utilization of multi- ple OB folds for DNA binding, our finding that CST(STN1-OBM) destabilizes binding to short oligonucleotides suggested a significant difference in how the two complexes bind short DNA substrates (Fig 6C). RPA binds DNA in a 5’ to 3’ direction with the OB-folds of RPA1 contact- ing DNA towards the 5’ end and providing the highest affinity binding sites [41,62]. As a result, OB-A and OB-B of RPA1 provide the only contacts to an 8 nt substrate. OB-A, -B and–C of RPA1 contact substrates of 12–23 nt but RPA2 (the STN1 equivalent) only contacts longer sub- strates of ~30 nt [41,42]. Consequently, mutations in RPA2 OB-D affect binding to long rather than short ssDNA [60,62]. Our finding that STN1-OBM destabilizes binding to short (e.g. TelG-18) but not long (TelG-48 & NonTel-48) substrates (Fig 6C) suggested that, unlike RPA2, STN1 directly engages the DNA of short substrates to stabilize binding. To further explore this possibility, we used photo-crosslinking to explore the proximity of individual CST subunits to the 5’ or 3’ ends of 18 or 48 nt TelG oligonucleotides. CST(WT) and CST(STN1-OBM) were incubated with 32P-labeled TelG-18 or TelG-48 that had a photoactivata- ble 4-thiothymidine (s4T) at the third nucleotide from the 5’ or 3’ end (Fig 7A and S5 Fig). The DNA-protein complexes were cross-linked by irradiation with UVA and then separated in a SDS-polyacrylamidegel. The gel was scanned by phosphorimager to determine whether CTC1, STN1 or TEN1 had been cross-linked to the labeled DNA. Equivalent UV-irradiated samples sep- arated in the same gel were used for Western blot analysis to determine the positions of uncross- linked CTC1, STN1 and TEN1. The low level of cross-linking precluded detection of the cross- linked DNA-protein complexes by Western blot. Additional reactions that had not been subject to cross-linking were analyzed by EMSA to monitor DNA binding. As shown in Fig 7B, the s4T residues did not significantly alter CST(WT) or CST(STN1-OBM) binding to either substrate. Analysis of the crosslinking products obtained with CST(WT) and 5’- or 3’-s4T TelG-18 revealed labeled bands that migrated at positions expected for CTC1 (130 kD) and STN1 (>43 kD) (Fig 7C) indicating cross-linking to either substrate. However, cross-linking of STN1 relative to CTC1 was less efficient with the 5’-s4T TelG-18, suggesting that STN1 was posi- tioned closer to the DNA 3’ end. Effect of STN1 Mutation on CST Function The 2–3 fold decrease in affinity of CST(STN1-OBM) for TelG-18 resembles the modest decrease in RPA affinity for ssDNA that has been observedafter mutation of individual OB folds [60,62]. In the case of RPA, the small effect on overall binding affinity reflects the presence of multiple DNA binding domains within the complex such that disruption of one binding domain has a small effect on the macroscopic affinity constant. Thus, the observeddecrease in CST (STN1-OBM) binding fits with the model that CST also engages DNA via multiple DNA bind- ing domains. Given the six predicted OB folds in CTC1, we anticipate that these multiple DNA binding domains correspond to the STN1 OB fold plus some or all of the OB folds in CTC1. PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 Analysis of CST DNA binding parameters. (A) Binding isotherms used to determine apparent dissociation constants for CST(WT) or CST(STN1-OBM) and the indicated ssDNA substrates. Data were obtained by filter binding assay and fit to a one site specific binding model. Mean ± SEM, n = 3 independent experiments each with a different protein preparation. (B) Dissociation kinetics for CST bound to the indicated substrates The fraction of labeled DNA remaining bound was determined by filter binding at the indicated times. Data were fit to a one phase exponential decay equation to obtain the dissociation rate (t½)). Mean ± SEM, n = 3 independent experiments. (C) Table summarizing Kd(app) and t½ for CST(WT) or CST(STN1-OBM) and the indicated ssDNA substrates.—undetectable binding, ND: not determined. doi:10.1371/journal.pgen.1006342.g006 PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 13 / 24 CST subunit interactions with ssDNA substrates It was not possible to tell if TEN1 was cross-linked to either substrate because TEN1 migrated in the same region of the gel as the uncross-linked DNA. Thus, bands corresponding to TEN1-TelG-18 may be obscured by the heavy signal from the uncross-linked DNA. Overall, the results indicate that binding of CST to a short 18 nt substrate positions the DNA in close proximity to STN1. Comparison of the cross-linking products obtained with the 3’ modifiedTelG-18 and CST(WT) or CST(STN1-OBM) revealed that cross-linking to STN1-OBM was reduced relative to wild type STN1. This finding indicates that the contacts between STN1 and DNA are altered by STN1-OBM. Analysis of the products obtained with CST(WT) bound to TelG-48 revealed that only CTC1 was reproducibly cross-linked to the 5’-s4T substrate. In contrast, the 3’-s4T substrate crosslinked to all three CST subunits. CTC1 cross-linked more efficiently than STN1 or TEN1 PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 14 / 24 PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 Effect of STN1 Mutation on CST Function products observed only in some experiments. They may represent CTC1 or STN1 degradation products. Markers on the phosphorimager scans were obtained by laying the gels on nitrocellulose membrane and marking the positions of the marker bands with radioactive ink. For the Western blots, the membrane was cut into pieces, probed with antibody to CTC1, STN1 or TEN1, reassembled and exposed to film. The film was laid over the membrane and photographed to visualize both the markers and the CST bands. (E) Dynamic binding model of CST showing micro- dissociation of an individual OB fold (blue) to allow binding of an alternative protein (yellow). products observed only in some experiments. They may represent CTC1 or STN1 degradation products. Markers on the phosphorimager scans were obtained by laying the gels on nitrocellulose membrane and marking the positions of the marker bands with radioactive ink. For the Western blots, the membrane was cut into pieces, probed with antibody to CTC1, STN1 or TEN1, reassembled and exposed to film. The film was laid over the membrane and photographed to visualize both the markers and the CST bands. (E) Dynamic binding model of CST showing micro- dissociation of an individual OB fold (blue) to allow binding of an alternative protein (yellow). doi:10.1371/journal.pgen.1006342.g007 doi:10.1371/journal.pgen.1006342.g007 and the level of TEN1 cross-linking was somewhat variable (Fig 7D) (note: cross-linking of TEN1 to TelG-48 retards TEN1 migration enough for the band from the cross-linked product to become visible above the uncross-linked DNA). The cross-linking of STN1 and TEN1 to the 3’-s4T substrate but not the 5’-s4T substrate indicates that both subunits must be in close prox- imity to the DNA 3’ terminus but not the 5’ terminus. Examination of the photo-products obtained with of CST(STN1-OBM) bound to 3’-s4T TelG-48 revealed less cross-linking to STN1 and TEN1 relative to CST(WT) but CTC1 photoproducts were still formed, again indi- cating that STN1-OBM alters how STN1 contacts DNA. Taken together the above results demonstrate that CST(WT) binds long substrates with the DNA 3’ end positioned close to the CTC1-STN1-TEN1 interface while the 5’ end only contacts CTC1. We therefore infer that, CST binds DNA in a similar orientation to RPA: i.e. with the large subunits of each complex contacting DNA near the 5’ end and the two smaller subunits positioned at the 3’ end. However, our data indicate that the identity of the binding sites used to engage short DNA substrates differs between CST and RPA. For CST, the binding sites lie close to the interface between CTC1, STN1 and TEN1, they engage DNA toward the 3’ end, and STN1 plays an important role in stabilizing the interaction. For RPA, the primary binding sites for short substrates are OB-A and OB-B of RPA1 and these engage DNA at the 5’ end. Thus, despite sharing some common structural features CST and RPA engage DNA quite differently. In addition to addressing the architecture of CST-DNA complexes, the in vitro cross-linking studies combine with the analysis of DNA binding affinity start to explain the in vivo separation of function observedwith STN1-OBM cells. Our results show that the STN1 OB-fold mutation alters the interaction between STN1 and ssDNA and this translates into altered binding of CST to some but not all DNA substrates. In vivo, CST is likely to encounter DNA substrates of varied length and structure as the complex helps resolve a wide range of replication problems at telo- meres and genome-wide. Thus, similar to what has been observedfor RPA OB-fold mutants [60,61], the altered DNA binding caused by STN1-OBM is likely to impair the ability of CST to bind and mediate the resolution of only a subset of replication intermediates. PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 Effect of STN1 Mutation on CST Function Fig 7. Photo-crosslinking of CST subunits to thiothymidine substituted ssDNA. (A) Positions of s4T substitutions in TelG-18 and TelG-48 DNA substrates. (B) EMSA showing the s4T substitutions do not affect CST (WT) or CST(STN1-OBM) binding to TelG-18 or TelG-48. (C-D) Products obtained after photo-crosslinking. Left a right panels: Phosphorimager scans showing 32P-labeled cross-linking products. Central panels: Western blots fr the same gels showing positions of uncross-linked CTC1, STN1 and TEN1. (C) Products obtained with 3’ (left) or (right) modified TelG-18. (D) Products obtained with 3’ (left) or 5’ (right) modified TelG-48. * indicates cross-linkin Fig 7. Photo-crosslinking of CST subunits to thiothymidine substituted ssDNA. (A) Positions of s4T 4 Fig 7. Photo crosslinking of CST subunits to thiothymidine substituted ssDNA. (A) Positions of s T substitutions in TelG-18 and TelG-48 DNA substrates. (B) EMSA showing the s4T substitutions do not affect CST (WT) or CST(STN1-OBM) binding to TelG-18 or TelG-48. (C-D) Products obtained after photo-crosslinking. Left and right panels: Phosphorimager scans showing 32P-labeled cross-linking products. Central panels: Western blots from the same gels showing positions of uncross-linked CTC1, STN1 and TEN1. (C) Products obtained with 3’ (left) or 5’ (right) modified TelG-18. (D) Products obtained with 3’ (left) or 5’ (right) modified TelG-48. * indicates cross-linking PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 15 / 24 Effect of STN1 Mutation on CST Function telomeric loci [27,30]. Our findings underscore the importance of CST for multiple processes associated with telomere replication and genome-wide replication rescue. They also strongly suggest that different DNA binding transactions are needed for CST to resolve different forms of replication stress with a subset of these transactions being disrupted by STN1-OBM. While STN1-OBM did not inhibit interactions with TPP1 or pol α (Fig 4), it is possible that STN1-OBM disrupts CST interaction with as yet unidentified partner proteins. If this is the case, the interaction of STN1 with such proteins might provide an additional mechanism to target CST to its various sites of action within the genome. Our in vitro DNA binding studies using CST(WT) and CST(STN1-OBM) provide new insight into the mechanism of mammalian CST binding to ssDNA. Past studies provided con- flicting information concerning the sequence specificity of CST binding [23,26]. We now con- firm that human CST binds long (48 nt) substrates with little sequence specificity, however sequence identity is important for binding to short (18 nt) substrates as the telomeric G-strand substrate TelG-18 is bound with high affinity while binding to the non-telomeric substrate NonTel-18 is undetectable. We also provide evidence that human CST harbors multiple DNA binding domains. The STN1-OB fold comprises one of these domains and based on structure prediction, we suggest that OB folds in CTC1 comprise the others. Since CST only bound the 18 nt substrate that had the sequence of telomeric G-strand DNA (Fig 1), the domain(s) that bind short oligonucleotides (i.e. the STN1 OB fold or an adjacent OB fold in CTC1) must pro- vide important determinants for sequence-specificbinding. Given that long substrates (telo- meric and non-telomeric) are bound with higher affinity than short substrates and their binding is less affected by STN1-OBM, it seems likely that these substrates contact additional DNA-binding domains beyond those used to contact short substrates. The known structural similarity between STN1-TEN1 and RPA2-RPA3, together with the likely presence of multiple OB folds in CTC1, had previously suggested an RPA-like binding mechanism whereby mammalian CST contacts DNA via multiple OB folds. However, this was not a foregone conclusion because S. cerevisae CST binds DNA through one high affinity bind- ing site in Cdc13 [49]. Discussion Here we describe a series of in vivo and in vitro experiments that address the mechanism of CST action at telomeres and elsewhere in the genome. We show that a STN1-OB-fold mutant (STN1-OBM) which preferentially decreases affinity of CST for short ssDNA substrates is competent for some aspects of CST function but deficient in others. The effects of STN1-OBM do not align with the telomeric versus non-telomeric roles of CST, but instead separate out the different aspects of CST function both during telomere replication and in genome-wide repli- cation rescue. At telomeres, STN1-OBM cells are competent for C-strand fill-in following telo- merase action but they exhibit increased MTS which are indicative of deficienciesin the earlier process of telomere duplex replication. STN1-OBM cells are also competent to restart replica- tion via new origin firing following exogenous genome-wide replication stress. However, STN1-OBM is not able to prevent the accumulation of anaphase bridges during mitosis. The latter finding indicates a deficiencyin genome-wide resolution of endogenous replication stress because the anaphase bridges caused by CST depletion occur at both telomeric and non- PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 16 / 24 PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 Effect of STN1 Mutation on CST Function able to resolve unwanted DNA structure such as G quadruplexes (G4) [64]. The dynamic bind- ing model for CST action is appealing because it can explain why CST is involved in multiple steps during telomere replication and in the resolution of diverse forms of replication stress. It can also explain many of the phenotypes of CST depletion. For example, during telomere repli- cation, CST might aid in removal of G4 structure from the lagging strand during replication of the duplex DNA and it may engage pol α on the G-overhang to initiate C-strand fill-in follow- ing telomerase action. The role in G4 structure removal could explain why STN1 depletion leads to a slowing of telomere duplex replication with formation of MTS. Likewise,the role in pol α engagement could explain why C-strand fill-in is disrupted despite pol α remaining asso- ciated with the telomere [21,25]. The ability of CST to engage pol α to initiate DNA synthesis at dormant or late firing origins could also explain why STN1 depletion inhibits replication restart after genome-wide replication fork stalling. Moreover, resolution of DNA structure at G-rich or regions of repetitive sequence could underlie the role of CST in resolving endogenous replication stress at non telomeric loci [27,30,31]. While current models for CST function have focused on the regulation of DNA pol α, the large size of CTC1 suggests that CST will have many interaction partners. Thus, mammalian CST may well direct the actions of additional proteins involved in the resolution of replication stress. A broader understanding CST function will require the identification of these proteins and analysis of how CST modifies their ability to engage with stalled forks, replication origins or other replication intermediates. If having multiple DNA binding domains and a dynamic DNA binding mechanism is so important for CST function in mammals, one has to ask why S. cerevisiae Cdc13 uses only one OB fold to bind DNA [49] and S. pombe appears to lack a Cdc13/CTC1 subunit [65]. One pos- sibility is that the multiple DNA binding domains necessary for dynamic binding are provided through dimerization or alternative subunit stoichiometries such as those found in S. cerevisiae and C. glabrata [50,64]. An alternative answer could lie in the division of labor between CST and RPA and how this has evolved between organisms. In S. pombe, RPA cooperates with the helicase Pif1 to help resolve G4 structures at lagging strand telomeres [66,67] and a simple Stn1/Ten1 complex appears sufficient to regulate telomerase to pol α switching for C-strand fill-in [65,68]. Thus, a full CST complex with dynamic DNA binding properties may be unnec- essary for telomere maintenance. Perhaps a CST complex is also superfluous for genome-wide replication rescue because S. pombe RPA has adapted to function in these processes. While our work supports the multiple OB fold binding mechanism for mammalian CST, it also reveals significant differences between CST and RPA in the contribu- tions made by individual subunits during binding to ssDNA. For RPA, the only binding sites for short substrates correspond to the OB folds of RPA1 that bind proximal to the DNA 5’ end [41,42]. These OB folds also comprise the highest affinity binding sites. However, for CST, both CTC1 and STN1 contact short DNA substrates and STN1, which binds near the DNA 3’ end, is necessary to stabilize binding. These findings imply that the high affinity binding sites in CST are contributed by STN1 and CTC1 and they interact with DNA towards the 3’ end. While this architecture differs from that of RPA, it is well suited for CST to bind a telomeric 3’ overhang. Despite the above differences between the two protein complexes, RPA can still be used as a model to help us understand the relationship between CST function and its mechanism of DNA binding. The ability of RPA to act as a hub that directs the sequential loading and unload- ing of partners such as Rad51 and Rad52 or SV40 T-antigen and pol α stems from the dynamic nature of RPA binding to ssDNA [41,44,63]. Because RPA utilizes 4 OB-folds to bind DNA, individual OB folds can undergo rapid microscopic dissociation and re-association from the DNA without causing the whole protein to dissociate [43,44]. Instead the rapid dissociation and re-association of individual OB folds is what enables RPA to diffuse along DNA to melt DNA structure or load and unload partner proteins [45]. Given that mammalian CST is likely to bind DNA via a similar number of OB-folds, it is possible that CST binding is also dynamic. If so, microscopic dissociation of individual OB folds from ssDNA could enable CST to engage or disengage interaction partners from the DNA (Fig 7E). Like the CST complex from Candida glabrata, mammalian CST might also be PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 17 / 24 PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 Effect of STN1 Mutation on CST Function essentially as described[24]. At least 145 fibers and 5 images were scored for each independent experiment. See supplemental materials for more details. Co-immunoprecipitation. To detect TPP1 interaction with STN1, HEK293T cells were transfected with HA-mCherry-TPP1 [57] and FLAG-STN1 (WT or OBM) expression con- structs. To detect interaction between CST and DNA pol α, HEK293T cells were transfected with FLAG-CTC1, FLAG-STN1 (WT or OBM) and TEN1 expression constructs. Cells were extracted 72 hrs later with 20 mM Tris pH 8.0, 100 mM NaCl, 1 mM MgCl2, 0.1% NP-40 (Ige- pal). TPP1 was precipitated with HA antibody, STN1 and CTC1 were precipitated with FLAG beads (Sigma A2220). CST expression and purification. SF9 cells were co-infected with the baculovirusencod- ing FLAG-CTC1, His-STN1/STN1-OBM or TEN1 (all tags were N-terminal). Infected cells were lysed in 25 mM Tris-HCl pH 7.5, 500 mM NaCl, 0.5% NP-40, 1 mM PMSF and protease inhibitor cocktail. The supernatant was supplemented with 1 mM DTT and 25 mM imidazole and applied to nickel-sepharose beads (GE healthcare 17-5268-01). The beads were washed twice with 25 mM Tris pH 7.5, 500 mM NaCl, 0.5% NP40, 25 mM imidazole, 1 mM DTT and once with 25 mM Tris pH 7.5, 500 mM NaCl, 25 mM imidazole, 1mM DTT. The protein was eluted with 25 mM Tris 7.5, 500 mM NaCl, 100 mM imidazole, 10% glycerol, 1 mM DTT and then diluted 1:4 with 25 mM Tris pH 7.5, 175 mM NaCl, 10% glycerol, 1 mM DTT to bring the imidazole to 25 mM and NaCl to 300 mM. FLAG beads were added to the diluted protein and incubated for 1 hr at 4°C. Beads were washed with 25 mM Tris pH 7.5, 200 mM NaCl, 10% glycerol, 1mM DTT. The protein was eluted with 3X FLAG peptide (Sigma F4799) stored at 4°C. The concentration was determined by PAGE and silver staining using a BSA standard. Electrophoreticmobility shift assays and UV crosslinking. CST(WT) or CST (STN1-OBM) (0.5–20 nM) was incubated with 32P-labeled oligonucleotide (0.1 nM) in 25 mM Tris pH 8.0, 1 mM DTT, 150 mM NaCl for 30 min at RT. For UV cross-linking 20 nM CST was incubated with 2 nM 32P-labeled oligonucleotide for 30 min and then subject to cross-link- ing in a Stratagene Hybridization Oven for 30 min on ice at full power (7200mJ/cm2) using 6 UVA bulbs. Cell lines HeLa 1.2.11 STN1 knockdown (clone shSTN1-7), shSTN1 rescue (STN1-Res), control non- target (clone shNT-3) and TEN1 overexpressing cell lines were as describedpreviously [24,25,30]. To create the STN1-OBM cells, the three amino acid mutations (W89A, R139L and Y141A) were introduced by PCR mediated mutagenesis into the shRNA-resistant FLAG-STN1 allele previously used to make the shSTN1-Res cells [24]. shSTN1 cells were transfected with retrovirus encoding shRNA-resistant FLAG-tagged STN1-OBM and the Thy1.1 marker. Cells expressing STN1-OBM were isolated by FACS based on Thy1.1 expression and were re-sorted periodicallyto maintain expression. Analysis of telomere phenotypes and DNA fiber analysis. Telomere FISH was per- formed using FITC-(TTAGGG)3 probe as previously described[24]. Genomic DNA was iso- lated using the Promega Wizard Kit and G-overhang abundance was determined by non- denaturing in-gel hybridization with 32P-labeled (AATCCC)4 probe. For the DNA fiber analy- sis, cells were labeled with IdU and CldU, DNA spread and fibers stained and visualized 18 / 24 PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 Effect of STN1 Mutation on CST Function folds found in POT1, the three C-terminal OB folds resemble those of RPA70 while the central OB fold appears to resemble that of TPP1. folds found in POT1, the three C-terminal OB folds resemble those of RPA70 while the central OB fold appears to resemble that of TPP1. Filter binding assay to determine Kd,app and t½. Double-filter binding assays were per- formed as described[69,70] using a 72-well minifold vacuum manifold slot blot apparatus and nitrocellulose and HyBond XL filters (GE Healthcare). Phosphorimager screens were scanned on a TyphoonTrio phosphorimager (GE Healthcare) and the amount of bound versus free DNA was quantified using ImageQuantTL software. Graphpad prism software was used to for plotting and curve fitting (one site specific saturation binding equation for Kd and one phase exponential decay equation for t½). To determine Kd,app, CST (0.01–20 nM) was incubated with 32P-labeled oligonucleotide (0.01 nM) in binding buffer (25 mM Tris pH 8.0, 1 mM DTT, 150 mM NaCl) for 18 hrs at 4°C to reach reaction equilibrium. To determine the dissociation rate (t½), 20 nM CST was incubated with 0.1 nM 32P-labeled oligonucleotide for 1 hr at 4°C. 0.5 μM of the corresponding unlabeled oligonucleotide was then added. Samples were analyzed by filter binding assay after 0, 0.5, 1, 3, 6, 9 or 18 hrs incubation with cold competitor. Protein threading and structure prediction. CTC1 protein sequence was analyzed for structural homology to know proteins using Phyre2 and HHpred. The top hits identified with Phyre2 included homology between: (i) CTC1 a.a. 791–1109 and U. maydis RPA 70 a.a. 226– 560 (prob. 98%). This region of RPA 70 encompasses OB folds B and C and part of OB A. (ii) CTC1 a.a. 219–408 and the OB folds domains from human POT1 a.a. 18–246 (prob. 85%). (iii) CTC1 a.a. 608–703 and the OB fold of human TPP1 a.a. 117–213 (prob. 79%). Homology was also found between the above regions of CTC and OB-fold containing regions of other proteins (e.g. SSB). Similar results were seen with HHpred. Based on these findings, we suggest that CTC1 harbors a total of 6 OB folds. The two in the N-terminus are most similar to the OB PLOS Genetics | DOI:10.1371/journal.pgen.1006342 September 30, 2016 19 / 24 Supporting Information S1 Fig. (A) PCR and sequencing strategy to monitor cells for presence of the wild type sh-resis- tant STN1 allele versus STN1-OBM. The cartoon indicates relative location of exons in endoge- nous STN1 mRNA. Arrowhead indicates exon with mutations. Arrows indicate locations of primers used for PCR (black) or sequencing (dotted). (B) Telomere FISH of metaphase spreads from shSTN1, shNT, STN1-Res and STN1-OBM cells. Representative images show that STN1-OBM does not cause increased telomere fusion or telomere loss. White arrows, MTS; green, FITC-(C3TA2)3 probe; blue, DAPI. (C) Non-denaturing in-gel hybridization showing telo- meric restriction fragments from the indicated cell lines. Mean telomere lengths are shown at the bottom. Values represent the weight averaged mean from 3 or 4 independent experiments ± SD. (TIF) S2 Fig. (A) FACS analysis showing cell synchronization of shSTN1, STN1-OBM and STN1-Res cells used to analyze G-overhang length. (B) Co-immunoprecipitation of DNA pol α with CST. Extracts were from cells transfected with the indicated constructs. CST was precip- itated with FLAG beads, these were then heated to 50°C and loaded on the gel. Western blots were performed with antibody to Pol α, STN1, TEN1 or FLAG. The Western blots with STN1 and TEN1 antibody show only the overexpressed protein because the levels of endogenous pro- tein are too low to detect with the exposures that are shown. (TIF) S4 Fig. (A) Representative slot blots used to determine DNA binding affinity (Kd) for CST (WT) and CST(STN1-OBM) binding to NonTel-36 or TelG-18. DNA concentrations are shown in brackets. (B) Representative slot blot used to determine t½ for CST(WT) and CST (STN1-OBM) binding to NonTel-36 or TelG-18. Time of incubation with cold competitor DNA is shown in brackets. (TIF) S5 Fig. Photocrosslinking of CST subunits to unmodifiedor 3’ thiothymidinesubstituted TelG-18. CST(WT) or CST(STN1-OBM) was were incubated with unmodifiedor modified TelG-18, samples were irradiated with UVA, separatedin SDS gels and analyzedby phos- phorimaging.  indicates cross-linking products observedonly in some experiments. Markers on the phosphorimager scans were obtained by laying the gels on nitrocellulose membrane and marking the positions of the marker bands with radioactive ink. (TIF) References 1. Branzei D, Foiani M (2010) Maintaining genome stability at the replication fork. Nat Rev Mol Cell Biol 11: 208–219. doi: 10.1038/nrm2852 PMID: 20177396 2. Zeman MK, Cimprich KA (2014) Causes and consequences of replication stress. Nat Cell Biol 16: 2– 9. doi: 10.1038/ncb2897 PMID: 24366029 3. Stewart JA, Chaiken MF, Wang F, Price CM (2012) Maintaining the end: roles of telomere proteins in end-protection, telomere replication and length regulation. 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Characterization of a Heme-Regulated Non-Coding RNA Encoded by the prrF Locus of Pseudomonas aeruginosa
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Abstract Pseudomonas aeruginosa, an opportunistic pathogen, requires iron for virulence and can obtain this nutrient via the acquisition of heme, an abundant source of iron in the human body. A surplus of either iron or heme can lead to oxidative stress; thus, the Fur (ferric uptake regulator) protein blocks expression of genes required for iron and heme uptake in iron- replete environments. Fur also represses expression of two nearly identical genes encoding the 116- and 114-nucleotide (nt) long PrrF1 and PrrF2 RNAs, respectively. While other Pseudomonads encode for the two PrrF RNAs at separate genomic loci, PrrF1 and PrrF2 are encoded in tandem in all sequenced strains of P. aeruginosa. In this report we characterize a third longer transcript encoded by the prrF locus, PrrH, which is repressed by heme as well as iron. We mapped the PrrH RNA in PA01 using 59 rapid amplification of cDNA ends (RACE) and northern analysis, demonstrating the PrrH RNA is 325 nt in length. Accordingly, transcription of PrrH initiates at the 59 end of prrF1, proceeds through the prrF1 terminator and prrF1-prrF2 intergenic sequence (95 nt), and terminates at the 39 end of the prrF2 gene. We also present evidence that repression of PrrH by heme causes increased expression of previously identified PrrF-regulated genes, as well as newly identified iron- and heme-activated genes. Thus, the PrrH RNA appears to impart a novel heme regulatory mechanism to P. aeruginosa. Citation: Oglesby-Sherrouse AG, Vasil ML (2010) Characterization of a Heme-Regulated Non-Coding RNA Encoded by the prrF Locus of Pseudomonas aeruginosa. PLoS ONE 5(4): e9930. doi:10.1371/journal.pone.0009930 Editor: Laurent Re´nia, BMSI-A*STAR, Singapore Received November 21, 2009; Accepted March 9, 2010; Published April 8, 2010 Received November 21, 2009; Accepted March 9, 2010; Published April 8, 2010 Copyright:  2010 Oglesby-Sherrouse, Vasil. 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 Cystic Fibrosis Foundation (http://cff.org/) Grant OGLESB08F0 (to A.G.O.) and National Institutes of Health (http://nih.gov/) Grant R37-NIH AI15940 (to M.L.V.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Amanda.Oglesby@ucdenver.edu bacteria, including P. Abstract aeruginosa, this regulation is achieved by Fur (ferric uptake repressor), a 17-kDa iron-binding protein [6,7]. Under iron-replete conditions, the Fur protein becomes ferrated and binds to a 19-bp Fur Box sequence in the promoters of genes required for iron and heme uptake, thereby preventing their transcription. In P. aeruginosa, Fur also affects the expression of several genes encoding virulence traits, including toxins and extracellular proteases [8,9,10]. Most Fur regulation in P. aeruginosa occurs through the repression of sigma factors, which in turn activate the expression of genes for siderophore biosynthesis and uptake. For example, Fur binds to the promoter and represses expression of pvdS, encoding a sigma factor that directly activates expression of genes for pyoverdin biosynthesis (pvd) and uptake (fpv), exotoxin A (toxA), and a secreted protease which degrades iron-binding proteins (prpL) [8,11,12,13]. Binding of ferri-pyover- din to its outer membrane receptor, FpvA, leads to activation of PvdS, which is normally sequestered at the inner membrane by its anti-sigma factor, FpvR [14,15]. This paradigm of Fur-mediated regulation via sigma factors likely extends to the uptake systems for other iron sources. Amanda G. Oglesby-Sherrouse*, Michael L. Vasil epartment of Microbiology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States of America Characterization of a Heme-Regulated Non-Coding RNA Encoded by the prrF Locus of Pseudomonas aeruginosa Amanda G. Oglesby-Sherrouse*, Michael L. Vasil PLoS ONE | www.plosone.org Introduction Originally, the prrF locus of P. aeruginosa was thought to encode two differently-sized tran- scripts, with prrF2 encoding an iron-repressed 111-nt RNA and prrF1 encoding a 184-nucleotide (nt) RNA whose expression was affected by heme as well as iron [27]. More recently, the prrF1 and prrF2 genes were shown to encode transcripts of similar size, approximately 110-nt in length [16]. Elucidation of the sequence, expression, and regulatory role of each of these transcripts should shed light on the P. aeruginosa-specific arrangement of the prrF genes. sensor of intracellular heme levels, contributing to the mainte- nance of heme and iron homeostasis [38]. Furthermore, while hemO is repressed by iron [35], expression of bphO is unaffected by iron [36], suggesting these two heme oxygenases are expressed under different environmental conditions. The biliverdin com- pounds generated by each heme oxygenase differ [35,36], and in vitro studies have shown the direct delivery of heme by PhuS to HemO, but not BphO [39]. Thus, the functions of these two heme oxygenases in cellular physiology are likely distinct. Heme itself is also able to mediate damage to cells via its hydrophobic quality and oxidative reactivity; yet, while the mechanism of Fur-mediated iron regulation is well understood, heme regulation in P. aeruginosa has not been studied in depth. The study discussed herein characterizes the prrF-encoded small RNAs, aiming to better describe their role in iron and heme regulation in P. aeruginosa. In this report, we confirm that the prrF1 and prrF2 genes encode PrrF RNAs of similar sequence and size, and we describe our identification of the sequence encoding a longer heme-regulated RNA, here named PrrH. Additionally we demonstrate heme regulates expression of PrrH, possibly via an anti-termination mechanism at the prrF1 Rho-independent terminator. Furthermore, we begin to address the biological significance of the PrrH RNA and present evidence for its ability to mediate heme regulation of target mRNAs. Our findings yield new insights into the genetic organization of the prrF1, prrF2, and prrH genes in P. aeruginosa and introduce a new paradigm for heme regulation in gram-negative bacteria. g Many pathogenic bacteria mediate the acquisition of iron via the uptake and degradation of iron-porphyrin, i.e. heme, an abundant source of iron in the human body. Although the contribution of heme acquisition to P. aeruginosa infection has not been studied in depth, heme uptake in other pathogens is known to play an integral role in infection [28,29,30]. Introduction Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that causes serious infections in immuno-compromised individuals, such as burn victims and cancer patients, as well as persons with cystic fibrosis (CF). In order to cause disease, P. aeruginosa requires an abundance of iron, as evidenced by a multitude of studies [1,2,3,4]. In anaerobic environments, iron in its ferrous form is freely diffusible through the outer membrane and transported into the cytoplasm by inner membrane transport systems. The insolubility of ferric iron in aerobic environments, however, limits accessibility to this nutrient, and the sequestration of iron by host proteins from potential pathogens creates a substantial barrier to infection. To scavenge insoluble or host-bound iron, many bacteria use siderophores, low molecular weight iron-chelating compounds. Ferri-siderophore complexes are bound at the cell surface by specific outer membrane receptors and transported into the periplasm, where a periplasmic binding protein delivers the complex to an inner membrane transporter [5]. Once in the cytoplasm, the siderophore is degraded, releasing the iron for use in a multitude of cellular processes including respiration, gene regulation, and environmental sensing [5]. In addition to genes for iron uptake and virulence, Fur represses the expression of two nearly identical genes encoding the PrrF1 and PrrF2 small regulatory RNAs, respectively [16]. The PrrF RNAs are functionally homologous to the RyhB RNAs encoded by Escherichia coli, Shigella flexneri, Shigella dysenteriae, and Vibrio cholerae [17,18,19,20]. Additionally, iron-repressed homologs of PrrF have been identified in Neisseria meningitidis (NrrF) and Azotobacter vinelandii (ArrF) [21,22]. In E. coli, RyhB binds to complementary P. aeruginosa synthesizes and secretes at least two siderophores, pyoverdin and pyochelin, both of which have been shown to be important for pathogenesis of P. aeruginosa [1,4]. Although required for growth and virulence, the potential for iron-accelerated oxidative damage requires the uptake of iron and heme to be regulated in response to iron availability. In many gram-negative PLoS ONE | www.plosone.org April 2010 | Volume 5 | Issue 4 | e9930 1 Heme Regulation of PrrH sequences of target mRNAs, causing their degradation in an RNaseE- and Hfq-dependent manner [23,24,25]. RyhB can also stabilize at least one of its target mRNAs in E. coli, leading to its increased expression [26]. The most curious aspect of the PrrF RNAs is that they are encoded in tandem in P. aeruginosa strains, whereas all other sequenced Pseudomonads encode the two PrrF RNAs at distal genomic loci (Figure 1). Introduction P. aeruginosa mediates the uptake of heme via at least two systems: Phu (Pseudomonas heme uptake) and Has (heme assimilation system) [27]. The Phu system encodes for an outer membrane receptor that binds heme (phuR), a periplasmic heme binding protein (phuT), an inner membrane ATPase and permease (phuUV), and a cytoplasmic heme binding protein (phuS). The Has system, originally identified in Serratia marcescens, encodes for a secreted hemophore protein (hasA), which scavenges heme from hemoglobin [31,32], and an ATPase and permease that are required for hemophore secretion (hasDE) [33]. An outer membrane receptor encoded by hasR binds the hemophore and extracts the heme, which is then transported across the outer membrane [34]. The imported heme is then bound by PhuT and transported into the cytoplasm by the Phu inner membrane permease system. A potential third outer membrane heme receptor is encoded by gene PA1302 in PA01; work is currently underway to determine the role of this gene in heme acquisition. PhuS binds imported heme in the cytoplasm, and is thought to traffic heme to at least one of two heme oxygenases expressed by P. aeruginosa, encoded by hemO [35] and bphO [36], which degrade the heme moiety, releasing biliverdin, carbon monoxide, and iron [37]. PhuS is also believed to act as a The prrF locus encodes for a heme-regulated dimer of PrrF RNAs, here named PrrH Our lab previously identified an apparent 184-nucleotide (nt) transcript originating from the prrF locus of P. aeruginosa strain PA01 by RNase protection assay (RPA) [27]. However, these assays did not allow for the prrF-encoded RNAs encoded by this region to be precisely mapped, which is necessary for understand- ing the regulation and function of each transcript. To determine the transcriptional start sites of the RNAs encoded by the prrF region, we performed 59 rapid amplification of cDNA ends (RACE) using the primers PrrF.RACE1 and 2, designed to hybridize within the PrrF1 and PrrF2 RNAs as shown in Figure 2A. By this method, transcription of the PrrF1 RNA was Figure 1. Genetic organization of prrF regions from different Pseudomonads. The tandem gene organization of prrF1 and prrF2 is restricted to P. aeruginosa strains. Block arrows indicate directionality of the open reading frame, and orthologous genes are represented by the color and pattern of the arrow. Map not drawn to scale. doi:10.1371/journal.pone.0009930.g001 Figure 1. Genetic organization of prrF regions from different Pseudomonads. The tandem gene organization of prrF1 and prrF2 is restricted to P. aeruginosa strains. Block arrows indicate directionality of the open reading frame, and orthologous genes are represented by the color and pattern of the arrow. Map not drawn to scale. doi:10.1371/journal.pone.0009930.g001 PLoS ONE | www.plosone.org April 2010 | Volume 5 | Issue 4 | e9930 2 Heme Regulation of PrrH Figure 2. Identification of the PrrF and PrrH transcripts. A. Sequence of the prrF locus, showing the location of the transcriptional start sites for PrrF1, PrrF2, and PrrH determined by 59 RACE as described in the Materials and Methods. The coding sequence of PrrF1 and PrrF2 is indicated by bold, the Fur binding sites preceding each prrF gene are indicated by italics, and the PrrF1 and PrrF2 Rho-independent terminators are indicated by bolded italics. B. Northern analysis of the PrrF and PrrH RNAs as described in the Materials and Methods, performed with RNA from PA01 wild type, DprrF1, DprrF2, and DprrF1-2 strains grown for 18 hours in CM9 +1% glycerol. M – RNA size marker. C. Map of the prrF locus, showing the primer and probe binding sites for 59 RACE, northern analysis, and previous RNase protection assays [25]. doi:10.1371/journal.pone.0009930.g002 Figure 2. Identification of the PrrF and PrrH transcripts. A. The prrF locus encodes for a heme-regulated dimer of PrrF RNAs, here named PrrH Sequence of the prrF locus, showing the location of the transcriptional start sites for PrrF1, PrrF2, and PrrH determined by 59 RACE as described in the Materials and Methods. The coding sequence of PrrF1 and PrrF2 is indicated by bold, the Fur binding sites preceding each prrF gene are indicated by italics, and the PrrF1 and PrrF2 Rho-independent terminators are indicated by bolded italics. B. Northern analysis of the PrrF and PrrH RNAs as described in the Materials and Methods, performed with RNA from PA01 wild type, DprrF1, DprrF2, and DprrF1-2 strains grown for 18 hours in CM9 +1% glycerol. M – RNA size marker. C. Map of the prrF locus, showing the primer and probe binding sites for 59 RACE, northern analysis, and previous RNase protection assays [25]. doi:10.1371/journal.pone.0009930.g002 that should hybridize with both the PrrF1 and PrrF2 RNAs (shown in Figure 2C), three RNA species were apparent, two of which corresponded to the 116-nt and 114-nt PrrF1 and PrrF2 RNAs, respectively (Figure 2B). The third transcript was much longer than the anticipated size of 184 nt, migrating slower than the 300-nt marker band (Figure 2B). As was previously reported for the longer prrF-encoded RNA [27], this transcript was less abundant that the PrrF1 and PrrF2 transcripts (Figure 2B), and addition of either iron or heme to the growth medium eliminated detection of its expression by northern analysis (data not shown). The location of the riboprobe used for previous RPA analysis explains the apparent discrepancy in size estimation of this longer transcript: the distance from the PrrF1 transcriptional start site to the 59 end of the RPA riboprobe is 183 nt (Figure 2C), just one nucleotide difference in length from what was previously reported to be the size of the longer transcript. Thus, it appears that this longer RNA is generated when transcription of PrrF1 continues through its Rho-independent terminator, extends through the prrF1-prrF2 intergenic region (95 found to begin where previously estimated [16], 116 nt upstream from the predicted Rho-independent terminator of PrrF1, while transcription of the PrrF2 RNA begins 114 nt upstream of its predicted Rho-independent terminator (Figure 2A). No other transcriptional start sites were detected upstream of PrrF1, suggesting that the longer prrF-encoded transcript is transcribed beyond the prrF1 Rho-independent terminator. The prrF locus encodes for a heme-regulated dimer of PrrF RNAs, here named PrrH This idea was tested by 59 RACE using primers that bind downstream of the prrF1 terminator in the prrF1-prrF2 intergenic region (Figure 2A - PrrH.RACE1 and 2), yielding a product that, when sequenced, extended from the confirmed transcriptional start site of the PrrF1 RNA. These data demonstrate that transcription of prrF1 can continue through its putative Rho-independent terminator to generate a longer transcript. Although 59 RACE demonstrated where transcription of the longer prrF-encoded RNA begins, this technique did not clarify its 39 sequence. Therefore, northern analysis was used to estimate the sizes of the prrF-encoded RNAs. Upon hybridization with a probe PLoS ONE | www.plosone.org April 2010 | Volume 5 | Issue 4 | e9930 3 Heme Regulation of PrrH transcript specifically (prrF.for and prrH.rev), and a single probe was used to detect the PCR products amplified with either primer pair (Figure 3A). As expected, loss of either of the individual prrF genes eliminated PrrH expression (Figure 3B), while loss of the entire prrF locus was needed to eliminate PrrF expression (Figure 3C). Furthermore, addition of either iron or heme to our chelexed growth medium repressed PrrH expression (Figure 3B – 8.1-fold repression by iron and 7.2-fold repression by heme). In contrast, addition of protoporphyrin IX, an iron-free biosynthetic precursor of heme, to the growth medium caused no significant decrease in PrrH expression (data not shown), indicating repression by heme is not merely responsive to its porphyrin ring. Interestingly, heme also repressed PrrF expression (Figure 3C), possibly due to iron obtained from cytosolic heme degradation. nt), and ends at the prrF2 Rho-independent terminator (Figure 2C). Combined, these results indicate that the long PrrF transcript previously identified by RPA is actually 325 nucleotides in length and spans the entire prrF locus of P. aeruginosa (Figure 2C). Since this RNA is repressed by heme, we have named it PrrH for Pseudomonas RNA responsive to heme. Heme repression of PrrH does not require heme degradation In order to more thoroughly analyze PrrH expression, real time PCR (qRT-PCR) was employed to quantify the regulatory effects of iron and heme on the expression of the PrrF and PrrH RNAs. Primer pairs were designed to amplify the cDNA from all three prrF-encoded transcripts (prrF.for and prrF.rev) or the PrrH Figure 3. Quantification of PrrF and PrrH expression. A. Map of the PrrF-PrrH coding region showing the location of the primers and probes used for qRT-PCR. B-E. RNA was isolated from the indicated strains grown for 18 hours in CM9 +1% glycerol with no added iron (white bars), 40 mM hemin (hatched bars), or 100 mg/ml FeCl3 (black bars) and used for qRT-PCR analysis of the PrrH (B, D) and PrrF (C, E) RNAs as described in the Materials and Method. Error bars represent the standard deviation of three independent experiments. Asterisks (*) indicate expression was below detection levels. doi:10.1371/journal.pone.0009930.g003 PLoS ONE | www plosone org 4 April 2010 | Volume 5 | Issue 4 | e9930 Figure 3. Quantification of PrrF and PrrH expression. A. Map of the PrrF-PrrH coding region showing the location of the primers and probes used for qRT-PCR. B-E. RNA was isolated from the indicated strains grown for 18 hours in CM9 +1% glycerol with no added iron (white bars), 40 mM hemin (hatched bars), or 100 mg/ml FeCl3 (black bars) and used for qRT-PCR analysis of the PrrH (B, D) and PrrF (C, E) RNAs as described in the Materials and Method. Error bars represent the standard deviation of three independent experiments. Asterisks (*) indicate expression was below detection levels. doi:10.1371/journal.pone.0009930.g003 April 2010 | Volume 5 | Issue 4 | e9930 PLoS ONE | www.plosone.org 4 Heme Regulation of PrrH point normally used for PrrF expression analysis (Figure 4A). After entering logarithmic growth, the culture was sampled every two hours for RNA isolation, qRT-PCR and northern analysis. PA01 began secreting detectable amounts of pyoverdin (as measured by OD420) at a culture density of 0.15 (data not shown; indicated by an arrow in Figure 4A), at which point the growth rate decreased and the culture entered into stationary phase. This is likely when the intracellular iron stores of the cells were depleted, and pyoverdin production commenced in order to mediate iron acquisition. PrrH is expressed maximally in stationary phase Previous studies have shown that expression of most Fur-repressed genes in P. aeruginosa, including prrF1 and prrF2, is maximal in stationary phase cultures grown in low iron medium, when intracellular iron stores have become depleted. Since expression of PrrH is repressed by iron, the current studies have concentrated on stationary phase cultures to look at PrrH expression. However, other regulatory mechanisms may cause this RNA to be most optimally expressed at different growth phases. To address this issue, the expression of PrrH at various phases of growth was determined. PA01 was grown under iron-depleted conditions for 18 hours, the time Heme repression of PrrH does not require heme degradation Expression analysis of PrrF and PrrH expression by both qRT-PCR and northern blot (Figure 4A-B) demonstrated that de-repression of these RNAs coincided with onset of pyoverdin production. This data is in agreement with our model that PrrF1 and PrrH are transcribed from the same Fur-repressed promoter, and that both are expressed optimally under iron-depleted conditions. These data also demon- strate that maximal expression of the PrrH RNA occurs, as previously indicated for PrrF, in stationary phase at approximately 18 hours of growth. Hence, we continued to use this time point for further analysis of PrrH expression. Indeed, loss of the HemO heme oxygenase in a DhemO mutant reduced repression of PrrF by heme (Figure 3E), while, in contrast, causing a slight increase in heme repression of PrrH (Figure 3D). Residual heme repression of PrrF expression in the DhemO mutant is likely due to overlapping detection of the PrrH RNA by this primer-probe set, as well as effects from contaminating iron in the heme preparation. These data indicate that heme repression of PrrH is dependent on the entire heme moiety and distinguish heme regulation of the PrrH RNA from Fur-mediated iron regulation of the PrrF RNAs. Overall, our data indicate that expression of the PrrH RNA is repressed by iron, likely via the Fur protein, as well as heme via an unknown mechanism. PrrF target genes are regulated by heme in a prrF-dependent manner Several of the genes identified by this analysis encoded metabolic enzymes, including succinate dehydrogenase (PA1581-4; sdhCDAB), aconitase A (PA1562; acnA), aconitase B (PA1787; acnB), and methyl-aconitase A (PA0794; m-acnA). To determine if the PrrH RNA was able to mediate repression of these genes in response to heme, the ability of iron and heme to affect their expression was examined by qRT- PCR. Similar to what was previously reported for iron [16,40], expression of m-acnA, acnB, and sdhD was induced by heme in wild type PA01, although iron induction was stronger than heme induction for each of these genes (Figure 5A-C). These data may reflect the ability of iron to block expression of all three prrF- encoded RNAs, leading to complete de-repression of these target mRNAs, while addition of heme preferentially represses expres- sion of the PrrH RNA, still allowing for some PrrF-mediated repression of these genes. In order to test whether or not the PrrF and/or PrrH RNAs were responsible for the observed heme regulation of these target mRNAs, the regulatory effect of heme was examined in the prrF deletion mutants. As expected, deletion of either one of the prrF genes led to increased expression of acnB, m-acnA, and sdhD in low iron (Figure 5A-C), consistent with previous experiments per- formed in DTSB [16,40]. Moreover, deletion of the entire prrF locus caused complete de-repression of these genes in low iron, eliminating the effects of iron and heme on their expression (Figure 5A-C). Interestingly, expression of m-acnA was de- repressed by deletion of prrF2 in the presence of heme, while remaining unchanged in the presence of iron (Figure 5B), suggesting the PrrF2 RNA is expressed and represses expression of m-acnA in the presence of heme. Overall, these data demonstrate that heme mediates a regulatory effect on several previously-identified PrrF targets, and they suggest a role for either the PrrF and/or PrrH RNAs in this effect. Figure 5. Effect of heme on the expression of PrrF-regulated genes. RNA isolated from the indicated strains, grown for 18 hours in CM9 +1% glycerol with no added iron (white bars), 40 mM hemin (hatched bars), or 100 mg/ml FeCl3 (black bars) was used for qRT-PCR as described in the Materials and Methods. Error bars represent the standard deviation of expression of (A) acnB-PA1787, (B) m-acnA- PA0794, and (C) sdhD-PA1582 from at least three independent experiments. d i 10 1371/j l 0009930 005 The PhuR and HasR outer membrane heme receptors are important for heme-regulation of PrrH p g Heme uptake is likely an important mechanism by which P. aeruginosa acquires iron, as it possesses at least two heme acquisition systems. Yet we know little of how this bacterium responds to heme as a signaling molecule. As one of the few examples of a heme-regulated gene in P. aeruginosa, the mechanism by which PrrH is regulated by heme is of immense interest. To better understand how this regulation occurs, we tested mutants lacking one or both of the known outer membrane heme receptors for their ability to mediate heme repression of PrrH. While loss of either PhuR or HasR alone had no effect on heme repression of PrrH, deletion of both heme receptors caused a small increase in PrrH expression in the presence of heme (Figure 6A). Residual heme repression of PrrH in the DphuRDhasR mutant, albeit statistically insignificant (P.0.2 by student’s t test), could be mediated by a third putative heme outer membrane receptor encoded by PA1302; work is currently underway to determine the role of this gene in heme uptake and regulation. Since heme degradation is important for heme to have a regulatory effect on PrrF (Figure 3E), we hypothesized that PhuR and HasR would also be required for this regulation. In fact, loss of PhuR alone nearly eliminated heme repression of PrrF, and loss of both the PhuR and HasR heme receptors ablated the ability of heme to affect PrrF expression (Figure 6B). These data strengthen the idea Figure 5. Effect of heme on the expression of PrrF-regulated genes. RNA isolated from the indicated strains, grown for 18 hours in CM9 +1% glycerol with no added iron (white bars), 40 mM hemin (hatched bars), or 100 mg/ml FeCl3 (black bars) was used for qRT-PCR as described in the Materials and Methods. Error bars represent the standard deviation of expression of (A) acnB-PA1787, (B) m-acnA- PA0794, and (C) sdhD-PA1582 from at least three independent experiments. d i j l doi:10.1371/journal.pone.0009930.g005 that heme must be transported into the cytoplasm and degraded, releasing iron, in order to exert a regulatory effect on PrrF. Together, these data indicate that the PhuR and HasR heme receptors play a role in heme-regulated expression of PrrH. Furthermore, they demonstrate that heme repression of PrrH is mediated by a regulatory mechanism that is distinct from heme- dependent regulation of the PrrF RNAs. PrrF target genes are regulated by heme in a prrF-dependent manner Previously, we identified greater than 50 genes as candidate PrrF-regulated genes by microarray and qRT-PCR analysis Figure 4. PrrF and PrrH are maximally expressed during stationary phase in iron-depleted medium. A. PA01 was grown in CM9 +1% glycerol for 18 hours, taking culture density (A590) measurements every hour. After the cells began growing logarithmically (,5 hours) samples were taken for RNA isolation every two hours. Arrow indicates the onset of pyoverdin production. qRT-PCR analysis as described in the Materials and Methods was used to quantify PrrF (black bars) and PrrH (white bars) expression. B. RNA samples were also analyzed by northern blot as described in the Materials and Methods to detect the presence of the PrrF and PrrH RNAs. doi:10.1371/journal.pone.0009930.g004 Figure 4. PrrF and PrrH are maximally expressed during stationary phase in iron-depleted medium. A. PA01 was grown in CM9 +1% glycerol for 18 hours, taking culture density (A590) measurements every hour. After the cells began growing logarithmically (,5 hours) samples were taken for RNA isolation every two hours. Arrow indicates the onset of pyoverdin production. qRT-PCR analysis as described in the Materials and Methods was used to quantify PrrF (black bars) and PrrH (white bars) expression. B. RNA samples were also analyzed by northern blot as described in the Materials and Methods to detect the presence of the PrrF and PrrH RNAs. doi:10.1371/journal.pone.0009930.g004 April 2010 | Volume 5 | Issue 4 | e9930 PLoS ONE | www.plosone.org 5 Figure 5. Effect of heme on the expression of PrrF-regulated genes. RNA isolated from the indicated strains, grown for 18 hours in CM9 +1% glycerol with no added iron (white bars), 40 mM hemin (hatched bars), or 100 mg/ml FeCl3 (black bars) was used for qRT-PCR as described in the Materials and Methods. Error bars represent the standard deviation of expression of (A) acnB-PA1787, (B) m-acnA- PA0794, and (C) sdhD-PA1582 from at least three independent experiments. doi:10.1371/journal.pone.0009930.g005 Heme Regulation of PrrH Heme Regulation of PrrH [16,40]. These genes were identified as such when meeting the following three criteria: 1) induced by growth in high iron as compared to low iron media, 2) de-repressed in the DprrF1-2 mutant as compared to wild type when grown in low iron media, and 3) unchanged in the complemented DprrF1-2 mutant as compared to wild type in low iron media. The PhuR and HasR outer membrane heme receptors are important for heme-regulation of PrrH Next, we sought to correlate the heme acquisition requirements of heme-regulated PrrH expression with that observed for previously-identified PrrF targets. For this, we tested the ability PLoS ONE | www.plosone.org April 2010 | Volume 5 | Issue 4 | e9930 April 2010 | Volume 5 | Issue 4 | e9930 6 Heme Regulation of PrrH of heme to induce expression of acnB, m-acnA, and sdhD in our panel of heme utilization mutants. Simultaneous deletion of both the PhuR and HasR heme receptors caused noted defects in heme induction of both of acnB and sdhD (Figure 7A and C). Heme induction of acnB was impaired in the DphuR and DhemO mutants as well (Figure 7A); since heme is still capable of repressing expression of PrrH in the DhemO and DphuR mutants (Figures 3D and 6A, respectively), this data rules out a role for PrrH in repression of acnB expression. In contrast, heme induced expression of m-acnA in the single and double heme receptor mutants, although this induction was not statistically significant for the DphuRDhasR mutant due to variable m-acnA expression in the presence of heme (P.0.05 by student’s t test; Figure 7B). Furthermore, deletion of hemO had no effect on heme induction of m-acnA (Figure 7B), providing the strongest evidence yet for regulation by the PrrH RNA. Results of this analysis for sdhD were from the PrrF regulon. We have identified several mRNAs that Figure 6. PhuR and HasR are important for heme regulation of PrrH. Heme utilization mutants lacking one or both outer membrane heme receptors were grown for 18 hours in CM9 +1% glycerol with no added iron (white bars), 40 mM hemin (hatched bars), or 100 mg/ml FeCl3 (black bars). RNA was then isolated and analyzed by qRT-PCR, as described in the Materials and Methods, for the mutants’ ability to mediate heme and iron regulation of the PrrH (A) and PrrF (B) RNAs. Error bars show the standard deviation of at least three independent experiments. doi:10.1371/journal.pone.0009930.g006 Figure 7. PhuR and HasR are important for heme regulation of PrrF target mRNAs. Heme utilization mutants lacking one or both outer membrane heme receptors or the HemO heme oxygenase were grown for 18 hours in CM9 +1% glycerol with no added iron (white bars) or 40 mM hemin (hatched bars). The PhuR and HasR outer membrane heme receptors are important for heme-regulation of PrrH RNA was then isolated and analyzed by qRT-PCR for expression of (A) acnB-PA1787, (B) m-acnA- PA0794, and (C) sdhD-PA1582, as described in the Materials and Methods. Error bars show the standard deviation of at least three independent experiments. doi:10.1371/journal.pone.0009930.g007 cnB, m-acnA, and sdhD in our Simultaneous deletion of both caused noted defects in heme D (Figure 7A and C). Heme he DphuR and DhemO mutants is still capable of repressing nd DphuR mutants (Figures 3D ules out a role for PrrH in In contrast, heme induced e and double heme receptor s not statistically significant for able m-acnA expression in the tudent’s t test; Figure 7B). no effect on heme induction rtant for heme regulation of g one or both outer membrane urs in CM9 +1% glycerol with no n (hatched bars), or 100 mg/ml ed and analyzed by qRT-PCR, as ods, for the mutants’ ability to the PrrH (A) and PrrF (B) RNAs. n of at least three independent 6 Figure 7. PhuR and HasR are important for heme regulation of PrrF target mRNAs. Heme utilization mutants lacking one or both outer membrane heme receptors or the HemO heme oxygenase were grown for 18 hours in CM9 +1% glycerol with no added iron (white bars) or 40 mM hemin (hatched bars). RNA was then isolated and analyzed by qRT-PCR for expression of (A) acnB-PA1787, (B) m-acnA- PA0794, and (C) sdhD-PA1582, as described in the Materials and Methods. Error bars show the standard deviation of at least three independent experiments. doi:10.1371/journal.pone.0009930.g007 Figure 7. PhuR and HasR are important for heme regulation of PrrF target mRNAs. Heme utilization mutants lacking one or both outer membrane heme receptors or the HemO heme oxygenase were grown for 18 hours in CM9 +1% glycerol with no added iron (white bars) or 40 mM hemin (hatched bars). RNA was then isolated and analyzed by qRT-PCR for expression of (A) acnB-PA1787, (B) m-acnA- PA0794, and (C) sdhD-PA1582, as described in the Materials and Methods. Error bars show the standard deviation of at least three independent experiments. doi:10.1371/journal.pone.0009930.g007 Figure 6. PhuR and HasR are important for heme regulation of PrrH. Heme utilization mutants lacking one or both outer membrane heme receptors were grown for 18 hours in CM9 +1% glycerol with no added iron (white bars), 40 mM hemin (hatched bars), or 100 mg/ml FeCl3 (black bars). The PhuR and HasR outer membrane heme receptors are important for heme-regulation of PrrH RNA was then isolated and analyzed by qRT-PCR, as described in the Materials and Methods, for the mutants’ ability to mediate heme and iron regulation of the PrrH (A) and PrrF (B) RNAs. Error bars show the standard deviation of at least three independent experiments. doi:10.1371/journal.pone.0009930.g006 Figure 6. PhuR and HasR are important for heme regulation of of heme to induce expression of acnB, m-acnA, and sdhD in our panel of heme utilization mutants. Simultaneous deletion of both the PhuR and HasR heme receptors caused noted defects in heme induction of both of acnB and sdhD (Figure 7A and C). Heme induction of acnB was impaired in the DphuR and DhemO mutants as well (Figure 7A); since heme is still capable of repressing expression of PrrH in the DhemO and DphuR mutants (Figures 3D and 6A, respectively), this data rules out a role for PrrH in repression of acnB expression. In contrast, heme induced expression of m-acnA in the single and double heme receptor mutants, although this induction was not statistically significant for the DphuRDhasR mutant due to variable m-acnA expression in the presence of heme (P.0.05 by student’s t test; Figure 7B). Furthermore, deletion of hemO had no effect on heme induction of m-acnA (Figure 7B), providing the strongest evidence yet for regulation by the PrrH RNA. Results of this analysis for sdhD were less clear: while heme induction of sdhD was observed in the DphuR mutant, this induction was greatly affected by deletion of hemO (Figure 7C), possibly suggesting roles for both PrrH and PrrF in regulation of this target mRNA. Overall, these data suggest the PrrH RNA affects expression of m-acnA. Figure 7. PhuR and HasR are important for heme regulation of PrrF target mRNAs. Heme utilization mutants lacking one or both outer membrane heme receptors or the HemO heme oxygenase were grown for 18 hours in CM9 +1% glycerol with no added iron (white bars) or 40 mM hemin (hatched bars). RNA was then isolated and analyzed by qRT-PCR for expression of (A) acnB-PA1787, (B) m-acnA- PA0794, and (C) sdhD-PA1582, as described in the Materials and Methods. Error bars show the standard deviation of at least three independent experiments. doi:10.1371/journal.pone.0009930.g007 from the PrrF regulon. We have identified several mRNAs that share complementarity with the unique sequence of PrrH by in silico analysis using Target RNA (http://snowwhite.wellesley.edu/ targetRNA/). Discussion Heme acquisition plays an important role in pathogenesis for several bacteria and is hypothesized to confer a similar advantage to P. aeruginosa during infection. While heme can serve as a valuable iron source in the host, free heme is toxic due to its hydrophobic quality and re-dox potential. Therefore, it is expected that a heme monitoring system coordinates the expression of genes for heme transport, degradation, and biosynthesis. Here we describe a non-coding RNA encoded by the prrF locus, named PrrH, and demonstrate that its expression is repressed by heme as well as iron. PrrH is the first example of a non-coding RNA that is encoded by two identical adjacent genes, each of which encode smaller regulatory RNAs. We have separately shown that the PrrH RNA is synthesized by other P. aeruginosa strains that share this genetic structure, including several clinical isolates (unpublished data). Additionally, PrrH is the first described non-coding RNA target of heme regulation in P. aeruginosa and potentially serves as the first example of a heme-responsive regulatory RNA in any bacteria. While the precise mechanism by which heme modulates PrrH expression remains unknown, our study has uncovered several requirements for this regulation. Notably, PrrF2 transcrip- tion still occurs from its own Fur-regulated promoter in the absence of the prrF1 gene and promoter (Figure 2B), arguing against the idea that PrrF1 and PrrF2 are generated via processing of the longer PrrH RNA. Alternatively, our studies support a model in which PrrH expression occurs via anti-termination at the PrrF1 Rho-independent terminator. Rho-independent terminators consist of a GC-rich palindromic element followed by a string of U residues, both of which are required for efficient termination. Transcription of the U oligonucleotide causes the RNA polymerase (RNAP) to pause, allowing time for the formation of a GC-rich hairpin structure in the nascent RNA [41]. The hairpin structure, along with the weak A-U interactions of the nascent RNA with template DNA, destabilize the transcription elongation complex (TEC), and both the RNA and DNA are released from the RNAP [41]. RNA- binding proteins can affect termination at sequences both upstream and downstream of the TEC, either by stabilizing the hairpin structure to increase termination efficiency, or by disrupting this structure and acting as anti-terminators [41,42]. While the stem loops for Rho-independent termination of PrrF1 and PrrF2 transcription are identical, the sequences upstream and downstream of the PrrF1 and PrrF2 terminators vary (Figure 2A). The PhuR and HasR outer membrane heme receptors are important for heme-regulation of PrrH Heme induction of nirL was reduced, but not eliminated, in the DphuRDhasR mutant (Figure 8C), while deletion of hemO caused no significant loss in heme induction of nirL (Figure 8D), indicating that heme regulation of this target mRNA is not due to iron from heme breakdown. Furthermore, no obvious complementarity was identified between the PrrF sequence and the operon containing nirL, suggesting heme regulation of nirL is not due to interaction of this mRNA with the PrrF RNAs. Overall these studies suggest a role for PrrH in heme activation of nirL and support a model in which PrrH regulates gene expression via its unique sequence derived from the prrF1-prrF2 intergenic region. The PhuR and HasR outer membrane heme receptors are important for heme-regulation of PrrH Among these were two genes related to heme biosynthesis: nirL (Figure 8A) and thiE (not shown). To begin defining the role of PrrH in heme regulation, these genes were selected for qRT-PCR analysis to characterize their ability to be regulated by iron and heme. While expression of thiE was unchanged by the addition of heme or iron (data not shown), expression of nirL was induced by both iron and heme (Figure 8B- from the PrrF regulon. We have identified several mRNAs that share complementarity with the unique sequence of PrrH by in silico analysis using Target RNA (http://snowwhite.wellesley.edu/ targetRNA/). Among these were two genes related to heme biosynthesis: nirL (Figure 8A) and thiE (not shown). To begin defining the role of PrrH in heme regulation, these genes were selected for qRT-PCR analysis to characterize their ability to be regulated by iron and heme. While expression of thiE was unchanged by the addition of heme or iron (data not shown), expression of nirL was induced by both iron and heme (Figure 8B- Regulation of a novel heme- and iron-regulated gene via PrrH. The unique sequence of PrrH derived from the prrF1- prrF2 intergenic region offers the capability of this RNA to interact with and regulate the expression of a subset of mRNAs distinct PLoS ONE | www.plosone.org April 2010 | Volume 5 | Issue 4 | e9930 7 Heme Regulation of PrrH Figure 8. Expression of nirL is activated by heme in a PrrH- dependent manner. A. Complementarity between the PrrH unique sequence, derived from the prrF1-prrF2 intergenic region, and the nirL mRNA. The underlined sequence indicates the start codon for nirL translation. B-C. RNA isolated from wild type PA01 and the (B) prrF, (C) heme receptor, and (D) heme oxygenase mutants, grown for 18 hours in CM9 +1% glycerol with no added iron (white bars), 40 mM hemin (hatched bars), or 100 mg/ml FeCl3 (black bars), was used for qRT-PCR as described in the Materials and Method. Error bars represent the standard deviation of expression from three independent experiments. doi:10.1371/journal.pone.0009930.g008 D). Additionally, iron and heme induction of nirL were dependent upon the prrF locus (Figure 8B). PLoS ONE | www.plosone.org Discussion These sequence variations are conserved in all sequenced P. aeruginosa strains and may provide a means for preferentially targeting a hypothetical PrrH regulatory protein to the PrrF1 terminator or PrrH unique sequence. Expression analysis of PrrH and PrrF in a series of heme utilization mutants indicates that heme regulation of PrrH is independent of promoter-centric Figure 8. Expression of nirL is activated by heme in a PrrH- dependent manner. A. Complementarity between the PrrH unique sequence, derived from the prrF1-prrF2 intergenic region, and the nirL mRNA. The underlined sequence indicates the start codon for nirL translation. B-C. RNA isolated from wild type PA01 and the (B) prrF, (C) heme receptor, and (D) heme oxygenase mutants, grown for 18 hours in CM9 +1% glycerol with no added iron (white bars), 40 mM hemin (hatched bars), or 100 mg/ml FeCl3 (black bars), was used for qRT-PCR as described in the Materials and Method. Error bars represent the standard deviation of expression from three independent experiments. doi:10.1371/journal.pone.0009930.g008 PLoS ONE | www.plosone.org April 2010 | Volume 5 | Issue 4 | e9930 8 Heme Regulation of PrrH regulation by Fur (Figures 3 and 6). Thus, our findings have led to a putative model in which heme regulates expression of PrrH via anti-termination of PrrF1 transcription. Future studies into the protein and sequence requirements for PrrH expression will be needed to fully understand the mechanism by which heme regulates expression of PrrH. and prrF2 genes seems to allow for heme-regulated expression of PrrF target mRNAs, which otherwise would only be regulated by iron, as well as genes unique to the PrrH regulon. Additionally, our study demonstrates that heme regulation of PrrH occurs by a mechanism that is distinct from heme regulation of PrrF. In the original study demonstrating heme-regulated expression of PrrH, a knockout of the entire prrF locus led to a significant decrease in growth on heme or hemoglobin as a sole iron source, suggesting a role for PrrH-repressed mRNAs in heme utilization [27]. Future work into the role of the PrrH RNA in overall cell physiology should elucidate the selective pressures for these regulatory phenomena. g p In several bacteria, proteins involved in heme acquisition provide feedback regulation of the genes for heme uptake, much like the FpvA pyoverdin receptor and PvdS sigma factor control expression of pyoverdin biosynthesis genes. Bacterial strains, growth conditions, and genetic manipulations Bacterial strains used in this work are listed in Table 1. E. coli strains were routinely grown in Luria-Bertani (LB) broth or on LB agar plates, and P. aeruginosa strains were maintained in brain-heart infusion (BHI) broth or on BHI agar plates. For a defined high and low iron medium, a chelexed M9 (CM9) medium was developed by treating 10X M9 salts [50] with chelex and supplementing with 1% glycerol. FeCl3 was added to a final concentration of 100 mg ml21 as indicated; hemin and protoporphyrin IX were added to a final concentration of 40 mM. Antibiotics were used at the following concentrations (per milliliter): 100 mg of ampicillin, 15 mg of gentamicin, and 15 mg of tetracycline for E. coli and 750 mg of carbenicillin, 75 mg of gentamicin, and 150 mg of tetracycline for P. aeruginosa. The DhemO::gm mutant was generated by amplifying the altered hemO fragment from IA614 [35]. This fragment was cloned into PCR2.1 (Invitrogen), then sub-cloned into pEX18Tc [51] (pEX-hemO::gm). The resulting plasmid was conjugated from SM10 lpir [52] into PA01, and mutants were selected on gentamicin. Resolved mutants were isolated on sucrose and confirmed for loss of the plasmid backbone by lack of growth on tetracycline. The final mutant was confirmed by PCR of the hemO region. What is the biological function of PrrH? Our current model presumes that the PrrH regulon overlaps that of PrrF and includes oxidative stress protection, iron storage, and metabolic genes. Thus, the PrrF RNAs can repress these genes in response to iron, while PrrH can repress the same genes in response to either iron or heme. Expression data from analysis of the heme utilization mutants suggest the PrrH RNA is capable of repressing expression of at least one of these PrrF-regulated genes, m-acnA, under low iron conditions. Due to its unique sequence derived from the prrF1-prrF2 intergenic region, PrrH is likely to regulate a specific subset of genes, possibly involved in heme biogenesis or other cellular processes. In silico analysis of this intergenic region using the TargetRNA application (http://snowwhite.wellesley.edu/ targetRNA/) [47] has allowed for the identification of several putative PrrH-specific targets. We assessed the ability of heme and PrrH to regulate two of these targets and identified nirL as a novel iron- and heme-regulated gene. 59 rapid amplification of cDNA ends (RACE) 59 RACE (Invitrogen) was used to identify the transcriptional start sites of PrrF and PrrH as described in the kit’s instructions. Briefly, RNA was isolated from PA01 grown in iron-depleted conditions on RNeasy Mini Columns (Qiagen), and cDNA was generated using PrrF.RACE1 (CCCGGCAAAGTGCCGGGTC) or PrrH.RACE1 (CAGGTCAAGCCGGTTCTCATTCAT). The cDNA was tailed with dCTP using terminal deoxy-transferase, and a poly-G primer (Invitrogen) was combined with either PrrF.RACE1 or PrrH.RACE1 for PCR. A second PCR was carried out with a dilution of the first PCR, using nested primers – PrrF.RACE2 (AAAACCGTGATTAGCCTGATGAGGAG) or PrrH.RACE2 (ATTCCATCGCCAGCCGATG) – with an adap- tamer primer provided by Invitrogen. The PCR products from this reaction were analyzed by gel electrophoresis, and predominant products were purified, cloned into PCR2.1 and analyzed by sequencing. Discussion Gram-negative organisms use ECF sigma factors responsive to heme uptake through the Has system in Serratia marcescens and the Bhu system in Bordetella bronchispetica to activate expression of heme uptake systems [43,44]. Similarly, gram-positive bacteria utilize two- component regulatory systems (Chr and Hrr systems in Corynebac- terium diphtheriae and Hss system in Staphylococcus aureus) to mediate heme-responsive regulation of their associated heme uptake systems [45,46]. P. aeruginosa encodes for a putative ECF sigma factor and transmembrane sensor adjacent to the hasR gene, encoding one of the heme outer membrane receptors important for heme repression of PrrH. PA1302, encoding a putative outer membrane heme receptor, is also adjacent to a putative ECF sigma factor and transmembrane sensor. Thus, it is possible that interaction of heme with P. aeruginosa’s outer membrane heme receptors initiates a signaling cascade via one or both of these putative sigma factors, which in turn affect transcription of PrrH. Alternatively, periplasmic heme may be controlling the activity of an ECF sigma factor, possibly through interaction with an inner membrane anti-sigma factor, which ultimately leads to repression of PrrH transcription. Either scenario would allow heme to exert a regulatory affect on PrrH expression without transport into the cytoplasm, a necessity if cytoplasmic heme levels are relatively high already. Bacterial strains, growth conditions, and genetic manipulations The nirSMCFDLGHJEN gene cluster encodes for dissimilatory nitrite reductase (NIR; cyto- chrome cd1) and includes genes for the biosynthesis of heme d1, a prosthetic group of NIR [48]. Biosynthesis of heme d1 branches from the central heme biosynthetic pathway, with NirF, NirJ, and NirE catalyzing its production from uroporphyrinogen III [49]. Thus, repression of NIR production by PrrH under limiting heme concentrations may prioritize the function of the heme biosyn- thetic pathway. While further work is needed to understand the contribution of the PrrH RNA to NIR expression, the data presented here suggest this RNA plays a role in heme regulation in P. aeruginosa. PLoS ONE | www.plosone.org Acknowledgments For northern analysis, 10–20 mg of total RNA isolated on RNeasy Mini Columns was run on a 6% polyacrylamide denaturing (7M urea) gel then transferred to a BrightStarH membrane (Ambion) using a semi-dry transfer apparatus. A biotinylated probe to prrF1 was generated by PCR amplification using the following primers: PrrF1.NB.for (CGCGAGAT- CAGCCGGTAAGC) and PrrF1.NB.rev (GTGCCGGGTC- AAAAACCGTG). The probe was heat-denatured, labeled using the BrightStarH Psoralen Biotin nonisotopic labeling kit (Ambion), We would like to thank Rhea May and Angela Wilks for helpful discussion and critical reading of the manuscript. References 13. Cunliffe HE, Merriman TR, Lamont IL (1995) Cloning and characterization of pvdS, a gene required for pyoverdine synthesis in Pseudomonas aeruginosa: PvdS is probably an alternative sigma factor. J Bacteriol 177: 2744–2750. 1. Cox CD (1982) Effect of pyochelin on the virulence of Pseudomonas aeruginosa. Infect Immun 36: 17–23. 1. Cox CD (1982) Effect of pyochelin on the virulence of Pseudomonas aeruginosa. Infect Immun 36: 17–23. 2. Meyer J, Neely A, Stintzi A, Georges C, Holder I (1996) Pyoverdin is essential for virulence of Pseudomonas aeruginosa. Infect Immun 64: 518–523. 14. Beare PA, For RJ, Martin LW, Lamont IL (2003) Siderophore-mediated cell signaling in Pseudomonas aeruginosa: divergent pathways regulate virulence factor production and siderophore receptor synthesis. Mol Microbiol 47: 195–207. 3. Takase H, Nitanai H, Hoshino K, Otani T (2000) Requirement of the Pseudomonas aeruginosa tonB gene for high-affinity iron acquisition and infection. Infect Immun 68: 4498–4504. p p p y 15. Lamont IL, Beare PA, Ochsner U, Vasil AI, Vasil ML (2002) Siderophore- mediated signaling regulates virulence factor production in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 99: 7072–7077. 4. Takase H, Nitanai H, Hoshino K, Otani T (2000) Impact of siderophore production on Pseudomonas aeruginosa infections in immunosuppressed mice. Infect Immun 68: 1834–1839. 16. Wilderman PJ, Sowa NA, FitzGerald DJ, FitzGerald PC, Gottesman S, et al. (2004) Identification of tandem duplicate regulatory small RNAs in Pseudomonas aeruginosa involved in iron homeostasis. Proc Natl Acad Sci U S A 101: 9792–9797. 5. Neilands JB (1995) Siderophores: structure and function of microbial iron transport compounds. J Biol Chem 270: 26723–26726. 6. Prince RW, Cox CD, Vasil ML (1993) Coordinate regulation of siderophore and exotoxin A production: molecular cloning and sequencing of the Pseudomonas aeruginosa fur gene. J Bacteriol 175: 2589–2598. 17. Masse E, Gottesman S (2002) A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli. Proc Natl Acad Sci U S A 99: 4620–4625. g 7. Prince RW, Storey DG, Vasil AI, Vasil ML (1991) Regulation of toxA and regA by the Escherichia coli fur gene and identification of a Fur homologue in Pseudomonas aeruginosa PA103 and PA01. Mol Microbiol 5: 2823–2831. 18. Murphy ER, Payne SM (2007) RyhB, an iron-responsive small RNA molecule, regulates Shigella dysenteriae virulence. Infect Immun 75: 3470–3477. 19. Real time PCR Strains were grown at 37uC for 18 hours in CM9 or DTSB and supplemented with the indicated amounts of FeCl3 or hemin. Total RNA was isolated on RNeasy Mini Columns and DNase- treated with RNase-free DNaseI (New England Biolabs). cDNA was prepared from 50 ng of RNA using the ImProm II Reverse Transcription System (Promega). Real time PCR reactions were This study has yielded insights into why P. aeruginosa has maintained the unique genetic structure encoding the PrrF1 and PrrF2 RNAs. That is, the tandem gene arrangement of the prrF1 PLoS ONE | www.plosone.org April 2010 | Volume 5 | Issue 4 | e9930 9 Heme Regulation of PrrH Table 1. Bacterial strains used in this study. Table 1. Bacterial strains used in this study. Strain Description Source/Reference PA01 Wild type P. aeruginosa strain; originally isolated from a human wound in 1955 in Australia [53] DprrF1 Deletion mutant of prrF1 in PA01 [16] DprrF2 Deletion mutant of prrF2 in PA01 [16] DprrF1-2 Deletion mutant of entire prrF locus in PA01 [16] DphuR::gm Deletion mutant with phuR gene replaced by gentamicin cassette in PA01 [27] DhasR::tc Deletion mutant with hasR gene replaced by tetracycline cassette in PA01 [27] DphuRDhasR DphuR::gm mutant with hasR gene replaced by tetracycline cassette in PA01 [27] DhemO::gm DhemO::gm mutation from strain IA614 [35] moved into the wild type PA01 background This study DH5a E. coli cloning strain; endA1 hsdR17 supE44 thi-1 recA1 gyrA relA1 D (lacZYA-argF)U169 deoR [Q80 dLacD (lacZ)M15] [50] SM10 lpir E. coli strain used for conjugation; pirR6K [52] doi:10.1371/journal.pone.0009930.t001 doi:10.1371/journal.pone.0009930.t001 carried out in a LightCyclerH 480 using the LightCyclerH 480 RNA Master Hydrolysis Probes master mix (Roche), and data was analyzed using the LightCyclerH 480 software. Relative amounts of cDNA were normalized by dividing the expression values by the relative amounts of omlA cDNA in each sample. and hybridized to the blot overnight at 42uC. The membrane was washed using the Ambion Northern Max Low Stringency and High Stringency wash solutions according to the manufacturer’s instructions. Detection of the biotinylated probes was carried out using the BrightStarH BioDetectTM nonisotopic detection kit (Ambion). Author Contributions Conceived and designed the experiments: AGOS MV. Performed the experiments: AGOS. Analyzed the data: AGOS. Wrote the paper: AGOS. Funding, advising: MV. Conceived and designed the experiments: AGOS MV. Performed the experiments: AGOS. Analyzed the data: AGOS. Wrote the paper: AGOS. Funding, advising: MV. References Prevost K, Salvail H, Desnoyers G, Jacques JF, Phaneuf E, et al. (2007) The small RNA RyhB activates the translation of shiA mRNA encoding a permease of shikimate, a compound involved in siderophore synthesis. Mol Microbiol 64: 1260–1273. 40. Oglesby AG, Farrow JM, Lee JH, Tomaras AP, Greenberg EP, et al. (2008) The influence of iron on Pseudomonas aeruginosa physiology: a regulatory link between iron and quorum sensing. J Biol Chem 283: . pp 15558–15567. 27. Ochsner UA, Johnson Z, Vasil ML (2000) Genetics and regulation of two distinct haem-uptake systems, phu and has, in Pseudomonas aeruginosa. Microbiology 146 (Pt1): 185–198. 41. Nudler E, Gottesman ME (2002) Transcription termination and anti- termination in E. coli. Genes Cells 7: 755–768. 42. Stulke J (2002) Control of transcription termination in bacteria by RNA-binding proteins that modulate RNA structures. Arch Microbiol 177: 433–440. ( ) 28. Brickman TJ, Vanderpool CK, Armstrong SK (2006) Heme transport contributes to in vivo fitness of Bordetella pertussis during primary infection in mice. Infect Immun 74: 1741–1744. 43. Vanderpool CK, Armstrong SK (2003) Heme-responsive transcriptional activation of Bordetella bhu genes. J Bacteriol 185: 909–917. 29. Henderson DP, Payne SM (1994) Vibrio cholerae iron transport systems: roles of heme and siderophore iron transport in virulence and identification of a gene associated with multiple iron transport systems. Infect Immun 62: 5120–5125. 44. Rossi MS, Paquelin A, Ghigo JM, Wandersman C (2003) Haemophore- mediated signal transduction across the bacterial cell envelope in Serratia marcescens: the inducer and the transported substrate are different molecules. Mol Microbiol 48: 1467–1480. 30. Hagan EC, Mobley HL (2009) Haem acquisition is facilitated by a novel receptor Hma and required by uropathogenic Escherichia coli for kidney infection. Mol Microbiol 71: 79–91. 45. Bibb LA, Kunkle CA, Schmitt MP (2007) The ChrA-ChrS and HrrA-HrrS signal transduction systems are required for activation of the hmuO promoter and repression of the hemA promoter in Corynebacterium diphtheriae. Infect Immun 75: 2421–2431. 31. Letoffe S, Redeker V, Wandersman C (1998) Isolation and characterization of an extracellular haem-binding protein from Pseudomonas aeruginosa that shares function and sequence similarities with the Serratia marcescens HasA haemophore. Mol Microbiol 28: 1223–1234. 46. Stauff DL, Torres VJ, Skaar EP (2007) Signaling and DNA-binding activities of the Staphylococcus aureus HssR-HssS two-component system required for heme sensing. J Biol Chem 282: 26111–26121. 32. Alontaga AY, Rodriguez JC, Schonbrunn E, Becker A, Funke T, et al. References Oglesby AG, Murphy ER, Iyer VR, Payne SM (2005) Fur regulates acid resistance in Shigella flexneri via RyhB and ydeP. Mol Microbiol 58: 1354–1367. 8. Wilderman PJ, Vasil AI, Johnson Z, Wilson MJ, Cunliffe HE, et al. (2001) Characterization of an endoprotease (PrpL) encoded by a PvdS-regulated gene in Pseudomonas aeruginosa. Infect Immun 69: 5385–5394. 20. Mey AR, Craig SA, Payne SM (2005) Characterization of Vibrio cholerae RyhB: the RyhB regulon and role of ryhB in biofilm formation. Infect Immun 73: 5706–5719. 9. Ochsner UA, Vasil AI, Vasil ML (1995) Role of the ferric uptake regulator of Pseudomonas aeruginosa in the regulation of siderophores and exotoxin A expression: purification and activity on iron-regulated promoters. J Bacteriol 177: 7194–7201. 21. Jung Y-S, Kwon Y-M (2008) Small RNA ArrF regulates the expression of sodB and FeSII genes in Azotobacter vinelandii. Current Microbiology 57: 593–597. 10. Ochsner UA, Vasil ML (1996) Gene repression by the ferric uptake regulator in Pseudomonas aeruginosa: cycle selection of iron-regulated genes. Proc Natl Acad Sci U S A 93: 4409–4414. 22. Mellin JR, Goswami S, Grogan S, Tjaden B, Genco CA (2007) A novel Fur- and iron-regulated small RNA, NrrF, is required for indirect Fur-mediated regulation of the sdhA and sdhC genes in Neisseria meningitidis. J Bacteriol 189: 3686–3694. 11. 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THE CURRENT STANDING OF INDIAN HEALTHCARE AND THE PHARMACIST
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ABSTRACT India is a large country and is ranked as the second most populous nation in the world. It is difficult to ensure the upkeep and effective operation of healthcare systems in a nation with such a large population. Recent surveys and reports from around the world indicate that India does not have a healthy health index. According to the Ministry of Health and Family Welfare, the health index of some southern Indian states such as Kerala, Tamilnadu, and Telangana is good, while the health index of some northern Indian states such as Bihar and Uttar Pradesh is poor. The healthcare systems in India face a number of challenges, such as a lack of awareness, a lack of access to healthcare, a shortage of healthcare professionals, high treatment costs, and a lack of accountability. Pharmacists have sufficient knowledge and skills to educate people and the general public about treatment, diagnosis, prevention, and mitigation of health problems. They are able to provide patients with sound advice in a variety of areas, including the appropriate use of medications, dietary recommendations, communicable and non- communicable diseases, pathogens and diseases related to them, and more. By involving pharmacists in health awareness programmes, patient counselling, and eradication programmes, one of the options available to raise the health index is to reduce the number of preventable illnesses. In this manner, those in need and those living in poverty, whether they are located in an urban or rural setting, will have access to medical care. The current health index of India, the challenges faced by Indian healthcare systems, and the ways in which pharmacists can contribute to the improvement of healthcare systems were all examined and discussed in this study. KEYWORDS: Health index of India, Ranking of Indian states in health index, Challenges of Indian healthcare systems, Role of pharmacists in improving health index. 2021, placing it 101st out of 157 countries in the world according to the rating of health and health systems.[2] According to the health index published by Ministry of health and family welfare in association with Niti Ayog and The World bank, the ranking of different states of India in the year 2019-20 is given in table 1 below. wjpmr, 2022,8(12), 84-87 SJIF Impact Factor: 5.922 wjpmr, 2022,8(12), 84-87 Ma Research Research Article ISSN 2455-3301 Wjpmr Mahato. World Journal of Pharmaceutical and Medical Research wjpmr, 2022,8(12), 84-87 SJIF Impact Factor: 5.922 Research Article ISSN 2455-3301 Wjpmr WORLD JOURNAL OF PHARMACEUTICAL AND MEDICAL RESEARCH www.wjpmr.com Mahato. World Journal of Pharmaceutical and Medical Research wjpmr, 2022,8(12), 84-87 SJIF Impact Factor: 5.922 Research Article ISSN 2455-3301 Wjpmr WORLD JOURNAL OF PHARMACEUTICAL AND MEDICAL RESEARCH www.wjpmr.com Medical Research SJIF Impact Factor: 5.922 Research Article ISSN 2455-3301 Wjpmr SJIF Impact Factor: 5.922 wjpmr, 2022,8(12), 84-87 THE CURRENT STANDING OF INDIAN HEALTHCARE AND THE PHARMACIST B. Pharmacy College Rampura, At Rampura, PO Kakanpur, Taluka Godhra, District Panchmahal, Gujarat-388713. *Corresponding Author: Tapan Kumar Mahato B. Pharmacy College Rampura, At Rampura, PO Kakanpur, Taluka Godhra, District Panchmahal, Gujarat-388713. Corresponding Author: Tapan Kumar Mahato B. Pharmacy College Rampura, At Rampura, PO Kakanpur, Taluka Godhra, District Panchmahal, Gujarat-388713. B. Pharmacy College Rampura, At Rampura, PO Kakanpur, Taluka Godhra, District Panchmahal, Gujarat-388713. INTRODUCTION What's encouraging is that most efforts to raise people's awareness have led to positive results. A review of the effectiveness of interventions on the reproductive health of adolescents showed that girls became much more aware of health problems, environmental health, nutrition and their children's health after interventions. The message is clear: we need to try to make those we work with more aware, and we need to get younger people to believe in the power of education to change behaviour. 3. Absence (Crisis of manpower): When talking about how to provide health care, it's important to talk about the people who do the work. Do we have enough people, have they been properly trained, are they spread out evenly and do they have a good attitude about doing their jobs? The workforce is not spread out in the best way, since most people prefer to work in places with better infrastructure and more opportunities for family life and growth. Even though most of the money spent on health care in the country comes from the private sector, the state-run health sector is still the only choice for many people in rural and near-urban areas. People are less likely to look for health care when there isn't a qualified person at the point of delivery after they have travelled a long way to get there. Since most health care services are provided by the private sector, there have been many programmes that try to use private expertise to give public health care services. The newest idea is the new nationwide plan to accredit private providers to provide services that the government will pay for. In an ideal world, this should lead to better coverage, but does it also mean that the public health system isn't good enough? It's time to make a policy on health human power that every Indian is cared for by a sensitive, trained and skilled healthcare worker. 2. Access (Lack): The Oxford dictionary says that "access" to healthcare means "the right or chance to use or benefit from" healthcare. How much access does our population have to good healthcare? is a very important one. A paper from 2002 says that "access" depends on things like the availability, supply and use of health care services. INTRODUCTION The global healthcare security (GHS) index evaluates the ability of nations to gather information in advance of diseases and pandemics. With a score of 42.8 and a change of -0.8 from 2019, India received a ranking of 66 out of 195 nations in the GHS Index 2021 report and data.[1] India received a health index score of 67.1 in Table 1: Ranks of larger states, smaller states and union territories in the year 2019-20.[3] Larger states (19) excluding West Bengal) Smaller states (08) Union territories (07) excluding Ladakh Rank State Rank State Rank UT 1 Kerala 1 Mizoram 1 Dadra & Nagar haveli and Daman & Diu 2 Tamilnadu 2 Tripura 2 Chandigarh 3 Telangana 3 Sikkim 3 Lakshadweep 4 Andhra Pradesh 4 Goa 4 Puducherry 5 Maharashtra 5 Meghalaya 5 Delhi 6 Gujarat 6 Manipur 6 Jammu & Kashmir 7 Himachal Pradesh 7 Arunachal Pradesh 7 Andaman and Nicobar 8 Punjab 8 Nagaland 9 Karnataka 10 Chhattisgarh 11 Haryana Table 1: Ranks of larger states, smaller states and union territories in the year 2019-2 www.wjpmr.com │ Vol 8, Issue 12, 2022. │ ISO 9001:2015 Certified Journal │ 84 84 84 World Journal of Pharmaceutical and Medical Research Mahato. 12 Assam 13 Jharkhand 14 Odisha 15 Uttarakhand 16 Rajasthan 17 Madhya Pradesh 18 Bihar 19 Uttar Pradesh Five “A” explains the challenges in Indian healthcare system health care possible. We should find and analyse possible access barriers in the financial, geographical, social and system-related areas of getting good healthcare. Five “A” explains the challenges in Indian healthcare system 1. Awareness (Lack): How aware is the Indian population of health issues that are important to them? Two studies found that only one-third of pregnant women knew enough about how to breastfeed to do it right. A study of adolescent girls in urban Haryana found that only 11.3% knew the right information about key reproductive health issues. A review article on geriatric morbidity found that 20.3% of the people who took part knew what caused common illnesses and how to avoid them. Why does the Indian population not know much about health? There may be many reasons for this, such as a low level of education, low functional literacy, a lack of emphasis on education in the healthcare system and a low priority on health among the population. Pharmacist and its role According to pharmacy Act 1948, "A registered pharmacist is a person whose name is currently on the list of pharmacists in the state where he or she lives or where he or she is currently practising or running a pharmacy business." What does the term "pharmacist" refer to? Table 2 below tells about the meaning of each alphabet used in the word Pharmacist. Table 2: Expansion of each alphabet used in the word Pharmacist. Table 2: Expansion of each alphabet used in the word Pharmacist. 1 P Patience 6 A Administrator 2 H Honesty 7 C Courageous 3 A Alertness 8 I Intelligent 4 R Research 9 S Studious 5 M Motivator 10 T Thinker INTRODUCTION Health professional - having adequate knowledge of health.[5] 5. Accountability (Lack): Accountability has been defined as the steps and processes that one party uses to explain and take responsibility for its actions. People in the health care field are responsible to give the patients best service they deserve. There may also be a spiritual or religious dimension. Nearly 70% of the population in India lacks access to vital pharmaceuticals for a variety of reasons including the lack of health experts and inappropriate professional advice regarding the use of drugs. The current number of pharmacists in India can contribute significantly to the improvement of drug accessibility and safe use. The health of the people in our great country is at risk because of the five problems listed above. Let's be aware of these and other challenges and get ready to face them, keeping in mind that the fight against illness is the fight against everything that is bad for humanity.[4] Worldwide, pharmacists are the third largest group of healthcare workers. The majority of pharmacists in India work in community settings which are the first point of interaction between community members and healthcare. The role of pharmacists should undergo a significant shift that will help fill the gaps in healthcare and significantly contribute to national health efforts. Even while pharmacists play a crucial role in providing improved healthcare, it is sad that the majority of patients do not distinguish between a grocery store clerk and a pharmacist. To provide improved health care, it is imperative that pharmacists become an integral component of the healthcare system. INTRODUCTION Even in places where services are "available" they may not be used as much as they could be because of financial, organisational, social and cultural barriers. Physical reach is one of the most important factors that determine access, which is defined as "the ability to get to a health care facility within 5 km of where you live or work." Using this definition, a 2012 study in India found that only 37% of people in rural areas could get to in- patient facilities within 5 km, while 68% could get to out-patient facilities. In 2012, Krishna and Ananthapur said that in general, the farther away from towns someone lives and the more rural their life is, the more likely they are to get sick, go hungry, get weak, and die young. Even if a health care centre is physically easy to get to, how good is the care it gives? Is this care available all the time? As people who think about community medicine and public health, we need to encourage people to talk about what makes access to 4. Affordability (Cost): How much does health care cost in India and more importantly, how many people can pay for it? In India, everyone knows that the private sector is the most important player in health care. Almost 75% of healthcare costs come out of people's own pockets and high healthcare costs are a major reason why people are poor. The problem is made worse by the lack of rules in the private sector, which means that the quality and cost of services vary. The public sector offers low-cost or free health care but it is seen as unreliable and of average quality, so it is usually not the first choice unless someone can't pay for private care. Local and national efforts can solve the problem of how to make health care more affordable. Nationally, the government needs to spend more on health, this will give the rural and poor areas a much-needed boost to their infrastructure and hopefully, make it easier for people to get the services, facilities of health care. Everyone in the health care system needs to be aware of costs. Wasteful spending, options that cost a lot and tests/procedures that aren't needed should be avoided. 85 www.wjpmr.com │ Vol 8, Issue 12, 2022. 85 ISO 9001:2015 Certified Journal World Journal of Pharmaceutical and Medical Research Mahato. i. Medications should be used correctly In situations where it is necessary, a pharmacist is able to provide the patient with counselling to provide advice on the proper use of medications as well as information on how they should be stored. By educating patients on how to make appropriate use of medications and maintaining sound pharmacy practises, a pharmacist has the potential to play an indispensable role in the provision of healthcare. Pharmacist take the time to educate patients about the medications that have been prescribed to them can considerably boost a patient's level of awareness regarding the appropriate application of medications from 56% to 90%. Mission: “The patient receives the appropriate medication at the appropriate time, in the appropriate quantity and in the appropriate manner”. It is evident that medicine has been manufactured by the specialised individual whom we refer to as a pharmacist. Humans have basic needs for food, clothing and shelter but today's greatest basic requirement is medicine. According to a 1963 discussion at a British pharmaceutical conference, a pharmacist is regarded as an expert on drugs and that "without medicine there is no life, it acts as Sanjivani for human beings." Such medications are created by pharmacists, who are also the first person in the health care system. The current workforce of pharmacists in India has the potential to make a significant contribution toward enhancing both access to medications and the safe administration of those medications. The field of pharmacy as a whole: In the form of patient interactions, pharmacists are able to reflect on all facets of society. The contribution of pharmacists to the improvement of healthcare in India In the following sectors of healthcare provision across the country, the participation of pharmacists has the potential to play an important role: i. Medications should be used correctly CONCLUSION In the context of healthcare in India, there is a lack of utilisation of both community pharmacy practise and pharmacies in general. At most cases, pharmacists who are employed in community pharmacies do not offer patient counselling services to their customers. In order to acknowledge the important role that pharmacists play in delivering higher-quality medical care, the government and various health organisations need to collaborate closely with the associations of pharmacists, during which they should discuss their shared experiences, as well as draught appropriate guidelines. Since pharmacists are involved in every aspect of the society's health and the construction of an excellent health care system, they must continually strive to expand their expertise while adhering to ethical standards. Therefore, pharmacists are the one and only highly skilled medical practitioners who serve as the backbone of the nation's health care system. This helps to ensure that the nation's health care system remains strong. DISCLOSURE OF CONFLICT OF INTEREST DISCLOSURE OF CONFLICT OF INTEREST The author declare that there is no conflict of interest. The author declare that there is no conflict of interest. REFERENCES 1. Advancing collective action and accountability amid global crisis 2021. Global health security index. Johns Hopkins Bloomberg School of Public health. http://www/ghsindex.org iv. Drug therapy customization One of the most important developments in contemporary medical practise is something called "personalization of medication therapy" which can also be explained as the process of tailoring a patient's drug selection and dosage to their specific needs. A patient can receive counselling from a pharmacist in situations where a physician is too busy on topics such as drug information, Pharmaco-economics and alternative therapy, moral support and so on. Pharmacist is able to retain the specifics of the patient's medical history including any allergies and other information that may be relevant to the treatment which makes it possible to execute the notion of individualization in drug therapy. ii. Dietary advice Community pharmacists have the potential to become an excellent resource for ensuring that patients receive adequate nutrition by advising patients on their fundamental dietary requirements, helping children break unhealthy eating habits, recommending a special diet for those who suffer from food allergies or diabetes and taking part in campaigns held in rural areas to educate people about the importance of maintaining healthy and well-balanced diets. To ensure that patients have a better overall state of health, pharmacists can disseminate information such as the reduced risk of stroke among fish Artist – Designer of drug dosage form Lawyer – having a basic understanding of drug laws and regulations Lawyer – having a basic understanding of drug laws and regulations Engineer - being technically knowledgeable Entrepreneur - with strong managerial, accounting, marketing and counselling skills Entrepreneur - with strong managerial, accounting, marketing and counselling skills www.wjpmr.com │ Vol 8, Issue 12, 2022. │ ISO 9001:2015 Certified Journal │ Vol 8, Issue 12, 2022. │ ISO 9001:2015 Certified Journal Vol 8, Issue 12, 2022. ISO 9001:2015 Certified Journal 86 86 World Journal of Pharmaceutical and Medical Research Mahato. eaters, the potential for symptoms of hypervitaminosis to disrupt menstrual cycles, the potential for Nutraceuticals and dietary supplements to provide a wide range of health benefits and much more. iii. Educating individuals regarding sexually transmitted diseases According to the most recent information that the government has released in 2019, it is projected that there were approximately 23.49 lakh persons living with HIV/AIDS (PLHIV) in India in 2019. Antiretroviral therapy is a treatment for HIV that comes at a high price and is out of reach for a sizeable section of the world's population. Pharmacists can contribute to the fight against HIV/AIDS by increasing public awareness and disseminating information on the virus, its mode of transmission, risk factors and various preventative strategies. ACKNOWLEDGMENTS Because of the papers that are cited in the references, I was able to write this article and I would like to offer my sincere gratitude to the authors of those publications. How might pharmacists participate more in healthcare? 2. Health and health systems ranking of countries worldwide in 2021. By health index score. https://www.statista.com/statistics/1290168/health- index-of-countries-worldwide-by-health-index- score/, 2021 Pharmacists can help fill healthcare shortages and provide a platform for professional development in India due to a shortage of medical professionals and poorly qualified providers. Pharmacists should learn programme development, implementation, monitoring, and 3. Healthy states progressive India. Report on the ranks of states and union territories. Health index sound by 2019-20. https://www.niti.gov.in/sites/default/files/2021- 12/NITI-WB_Health_Index_Report_24-12-21.pdf evaluation to effectively manage community health. Implementation science is being used to understand how to apply evidence to public health challenges including drug safety and mobile health as technology and innovation improve. Implementation research can offer the government with data to integrate pharmacists into public health care. Pharmacists will need to understand how and why pharmacy services may improve the health and well-being of individuals in resource-limited situations as they enter public health. Dual-trained pharmacists and public health experts are needed. A few Indian pharmacy universities offer Pharm.D./MPH dual degree programmes, although most pharmacy students just briefly learn about public health. Only a few pharmacy courses focus on public health and few textbooks do so. Thus, Pharmacists need public health and Pharmacoepidemiology studies in pharmacy school.[6] 4. Kasthuri A. Challenges to healthcare in India – The five A’s. Indian J Community Med. 2018; 43: 141-3. 5. Kokane JV, Avhad PS. Role of Pharmacist in Health Care System. The Journal of Community Health Management, 2016; 3(1): 37-40. 6. Nikkhil KM. Pharmacists can play a Critical Role in Enhancing Healthcare Delivery in India. ETHealthWorld. September 25, 2021. https://health.economictimes.indiatimes.com ISO 9001:2015 Certified Journal 87 w.wjpmr.com │ Vol 8, Issue 12, 2022. Vol 8, Issue 12, 2022. 87
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ЖИНОЯТ ПРОЦЕССУАЛ МУНОСАБАТЛАРДА ГУМОН ҚИЛИНУВЧИ, АЙБЛАНУВЧИ ЁКИ СУДЛАНУВЧИГА АДВОКАТЛАР ТОМОНИДАН ЮРИДИК ЁРДАМ КЎРСАТИШ БЎЙИЧА ХАРАЖАТЛАРНИ ДАВЛАТ ҲИСОБИГА ЎТКАЗИШДА ИНСОН ОМИЛИНИНГ УСТИВОРЛИГИ
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Ибрагимов Акмал Ишмуратович “CONSILIUM” Адвокатлик фирмаси адвокати https://doi.org/10.5281/zenodo.7568244 Аннотация: Мақолада жиноят процессуал муносабатларда гумон қилинувчи айбланувчи ёки судланувчига адвокатлар томонидан юридик ёрдам кўрсатиш бўйича харажатларни давлат ҳисобига ўтказишда инсон омилининг устиворлигини такомиллаштириш масалаларига эътибор берилган. Таянч сўзлар: жиноят процессуал муносабатлар, гумон қилинувчи айбланувчи ёки судланувчи,ички ишлар органлари, адвокатлар томонидан юридик ёрдам кўрсатиш, харажатларни давлат ҳисобига ўтказиш, процессуал ҳаракат. Ўзбекистон Республикаси Вазирлар Маҳкамасининг «Адвокатлар томонидан кўрсатилган юридик ёрдам учун давлат ҳисобидан ҳақ тўлаш механизмини такомиллаштириш чора-тадбирлари тўғрисида» 2008 йил 20 июнь кунидаги 137- сонли қарорига[1] асосан, гумон қилинувчи, айбланувчи ёки судланувчига адвокатлар томонидан юридик ёрдам кўрсатиш бўйича харажатларни давлат ҳисобига ўтказиш тартиби белгиланган. Ўзбекистон Республикаси Жиноят-процессуал кодексининг 50-моддасига, шунингдек Ўзбекистон Республикасининг «Адвокатура тўғрисида»ги[2] Қонунининг 11- моддасига ва Ўзбекистон Республикаси Вазирлар Маҳкамасининг «Адвокатлар томонидан кўрсатилган юридик ёрдам учун давлат ҳисобидан ҳақ тўлаш механизмини такомиллаштириш чора-тадбирлари тўғрисида» 2008 йил 20 июнь кунидаги 137-сонли қарорига[3] асосан, гумон қилинувчи, айбланувчи ёки судланувчи адвокатлар томонидан кўрсатилган юридик ёрдам учун харажатлардан озод қилинган ҳолларда юридик ёрдам учун харажатлар давлат ҳисобига ўтказилган. Гумон қилинувчи, айбланувчи ёки судланувчига адвокат томонидан юридик ёрдам кўрсатиш бўйича харажатларни давлат ҳисобига ўтказиш учун асос бўлиб, юридик ёрдам учун ҳақ тўлашдан озод қилиш тўғрисидаги суриштирувчи, терговчи, прокурорнинг қарори, суднинг ажрими ҳисобланади. Оиласида ҳар бир оила аъзосига тўғри келадиган ялпи ойлик даромад юридик ёрдам кўрсатилганлиги учун ҳақ тўлашдан озод қилиш тўғрисида қарор (ажрим) қабул қилинган кундаги энг кам ойлик иш ҳақининг 1,5 бараваридан ортиқ бўлмаган шахслар адвокатлар томонидан юридик ёрдам кўрсатилганлиги учун гумон қилинувчи, айбланувчи ёки судланувчининг иш жойидан берилган охирги ўн икки ойлик даромади тўғрисидаги маълумотнома мавжуд бўлган тақдирда, уни илова қилган ҳолда яшаш жойидаги маҳалла, овул, қишлоқ фуқаролар йиғини ёки фуқаролар йиғини томонидан ваколатланган комиссия томонидан берилган хулосага асосан ҳақ тўлашдан озод қилинади ва харажатлар давлат ҳисобига ўтказилади. Ҳужжатларни тўплаш мажбурияти суриштирувчи, терговчи, прокурор ва судга юклатилади. Ушбу ҳужжатлар суриштирувчи, терговчи, прокурор ва судга гумон Page 51 EURASIAN JOURNAL OF LAW, FINANCE AND APPLIED SCIENCES INTERNATIONAL SCIENTIFIC JOURNAL SPECIAL SERIES « OUTCOMES IN CRIMINAL-PROCEDURAL RELATIONS» UIF = 8.3 | SJIF = 5.961 қилинувчи, айбланувчи ва судланувчи ёки уларнинг қариндошлари томонидан ҳам тақдим этилиши мумкин. Кечиктириб бўлмайдиган тергов ҳаракатларини амалга ошириши шарт бўлган муддатларда кўрсатилган ҳужжатларни тўплаш имконияти бўлмаса суриштирувчи, терговчи, прокурор бу ҳужжатларни келгусида тўплаш билан гумон қилинувчи ва айбланувчини юридик ёрдам учун тўловдан озод этишга ҳақлидир. Гумон қилинувчи, айбланувчи ва судланувчини юридик ёрдам учун тўловдан озод этишга асослар мавжуд эмаслиги фактлари аниқланса, адвокатлар томонидан кўрсатилган юридик ёрдам бўйича харажатларини қоплаш ҳукм қилинганга юклатилади. Гумон қилинувчи, айбланувчи ва судланувчи ҳимоячидан воз кечиши билан боғлиқ процессуал ҳаракатда адвокат иштирок этиши ҳолларида, шунингдек, Ўзбекистон Республикаси Жиноят-процессуал кодекси 52-моддасининг[4] иккинчи қисмига мувофиқ жиноят ишида ҳимоячининг қатнашиши мажбурий деб ҳисобланган ҳолларда ҳам юридик ёрдам учун ҳақ тўлаш бўйича харажатлар давлат ҳисобига ўтказилади. Адвокатлар томонидан юридик ёрдам кўрсатилганлиги учун адвокатлик тузилмаси тегишли молия органига мурожаат қилгандан кейин ўттиз кундан кечикмай Молия вазирлигининг қарор (ажрим) чиқарган орган жойлашган жойдаги Ғазначилиги ҳудудий бўлинмаси орқали Давлат бюджетининг харажатлари параметрларида назарда тутилган маблағлар ҳисобидан ҳақ тўланиши ушбу Низомга иловага мувофиқ шакл бўйича кўрсатилган юридик ёрдам тўғрисидаги маълумотномага асосан амалга оширилади. Суриштирув жараёнида, дастлабки тергов босқичида, биринчи инстанция судларида ва апелляция, кассация ёки назорат тартибида кўрилаётган ишларда адвокатлар томонидан юридик ёрдам кўрсатилганлиги учун тўлов ҳар бир кун учун энг кам ойлик иш ҳақининг 50 фоизи миқдорида амалга оширилади. Икки ва ундан ортиқ шахсларга битта жиноий иш бўйича адвокатлар томонидан юридик ёрдам кўрсатилганлиги учун тўлов ҳар бир ишонч билдирувчига, белгиланган тўлов миқдорининг 80 фоизи ҳисобида амалга оширилади. Суд мажлисининг ҳар бир танаффус куни учун тўлов адвокатлар томонидан юридик ёрдам кўрсатилганлиги учун, белгиланган тўлов миқдорининг 50 фоизи ҳисобида амалга оширилади. Дам олиш ва байрам (ишланмайдиган) кунлари адвокатлар томонидан юридик ёрдам кўрсатилганлиги учун тўлов энг кам ойлик иш ҳақининг 100 фоизи миқдорида, тунги вақтда эса энг кам ойлик иш ҳақининг 75 фоизи миқдорида амалга оширилади. Адвокатлар томонидан юридик ёрдам кўрсатиш билан боғлиқ бўлган хизмат сафари ва транспорт харажатлари миқдори адвокатлик тузилмаси томонидан белгиланган тартибда аниқланади ҳамда маълумотномада кўрсатилади. Адвокатнинг ишда иштирок этиши суриштирувчи, терговчи, прокурор ёки суд томонидан Ўзбекистон Республикаси Адвокатлар палатасининг ҳудудий бошқармаси томонидан кўрсатилган адвокатлик тузилмасига мурожаатнома юбориш йўли билан таъминланади. Мурожаатномада адвокатнинг шахси кўрсатилмаслиги керак. EURASIAN JOURNAL OF LAW, FINANCE AND APPLIED SCIENCES INTERNATIONAL SCIENTIFIC JOURNAL SPECIAL SERIES « OUTCOMES IN CRIMINAL-PROCEDURAL RELATIONS» UIF = 8.3 | SJIF = 5.961 двокатнинг аниқ бир ишни юритишга ваколати ордер билан тасдиқланади. Юридик ёрдам кўрсатилганлиги учун ҳақ тўлашдан озод қилиш тўғрисида суриштирувчи, терговчи, прокурорнинг қарори, суднинг ажрими ҳимоячининг жиноий ишда иштирок этиши бошланиши билан бир вақтда чиқарилади. Гумон қилинувчи, айбланувчи ва судланувчининг ҳимоячидан воз кечиши билан боғлиқ процессуал ҳаракатда адвокатнинг иштирок этиши ҳолларида юридик ёрдам учун тўловдан озод этиш тўғрисидаги қарор (ажрим) адвокат иштирок этган процессуал ҳаракат тугатилгандан сўнг чиқарилади. Ҳар битта процессуал ҳаракатда, маълумотнома, яъний иш рақами, жиноий ишда иштирок этган адвокатнинг фамилияси, исми ва шарифи, ишонч билдирувчи (ҳимоя остидаги шахс)нинг фамилияси, исми ва шарифи, иштирок кунлари, тўлов миқдори ва адвокатлик тузилмасининг банк реквизитлари албатта кўрсатилган ҳолда уч нусхада тузилади ва адвокатлик тузилмаси раҳбари томонидан имзоланади ҳамда унинг муҳри билан тасдиқланади. Маълумотномада адвокат томонидан юридик ёрдам кўрсатиш билан боғлиқ бошқа маълумотлар ҳам кўрсатилиши мумкин. у р ўр у Адвокат жиноий ишда узоқ вақт иштирок этган ҳолда (бир ойдан ортиқ) маълумотнома ҳар ойда тузилади. Юридик ёрдам кўрсатилганлиги учун ҳақ тўлашдан озод қилиш тўғрисида қарор (ажрим) чиқарган шахс ҳар ойда ёки суриштирув жараёни, дастлабки тергов босқичи тугаган куни ёки суднинг тегишли қарори чиқарилган куни маълумотномани ўзининг имзоси ва суриштирув, тергов ёки суд органи муҳри билан тасдиқлаши шарт. Маълумотноманинг бир нусхаси адвокатлик тузилмасида қолади, иккинчи нусхаси маълумотномани тасдиқлаган шахсга ишга қўшиш учун берилади, учинчи нусхаси эса юридик ёрдам кўрсатилганлиги учун ҳақ тўлашдан озод қилиш тўғрисидаги қарор (ажрим) билан бирга адвокатлик тузилмаси томонидан тегишли ҳудудий молия органига юборилади. Молия вазирлигининг Ғазначилиги ҳудудий бўлинмаси адвокатлар томонидан кўрсатилган юридик ёрдам учун тўловларни адвокатлик тузилмасининг мурожаатномаси тегишли молиявий органга келиб тушган кундан бошлаб, ўттиз кун муддатдан кечиктирмасдан маълумотномага ва юридик ёрдам кўрсатилганлиги учун ҳақ тўлашдан озод қилиш тўғрисидаги қарор (ажрим)га асосан адвокатлик тузилмасининг банк ҳисоб рақамига пул маблағларини ўтказиб бериш йўли билан тўлаб беради. Гумон қилинувчи, айбланувчи ёки судланувчига адвокатлар томонидан юридик ёрдам кўрсатиш бўйича харажатларни давлат ҳисобига ўтказишда адвокатлик тузилмасидан қўшимча ҳужжатлар талаб қилинишига йўл қўйилмайди. Молия вазирлигига тегишли ҳудудий молия органларининг мурожаатлари асосида адвокатлар томонидан кўрсатилган юридик ёрдам учун ҳақ тўлаш мақсадли бюджет маблағлари республика бюджетидан тегишли қуйи бюджетига белгиланган тартибда пайдо бўладиган бюджетлараро ўзаро муносабатлар тартибга солинган тақдирда ўтказилади. Мухтасар қилиб айтганда, айбланувчи ёки судланувчига адвокатлар томонидан юридик ёрдам кўрсатиш бўйича харажатларни давлат ҳисобига ўтказиш тартиби Volume 2 Issue 12, December 6, Special Issue 2022 ISSN 2181-2853 EURASIAN JOURNAL OF LAW, FINANCE AND APPLIED SCIENCES INTERNATIONAL SCIENTIFIC JOURNAL SPECIAL SERIES « OUTCOMES IN CRIMINAL-PROCEDURAL RELATIONS» UIF = 8.3 | SJIF = 5.961 бўйича самарали механизм яратилган бўлиб,ушбу механизм доирасида бугунги кунда инсон омилининг хар нарсадан устун эканлигини кўришимиз мумкин. References: 1. Ўзбекистон Республикаси Вазирлар Маҳкамасининг 2008 йил 20 июнь кунидаги 137-сонли қарори // https://lex.uz/docs/1363850 2. Ўзбекистон Республикасининг «Адвокатура тўғрисида»ги Қонун// https://lex.uz/docs/54503#55841 3. Ўзбекистон Республикаси Вазирлар Маҳкамасининг 2008 йил 20 июнь кунидаги 137-сонли қарори // https://lex.uz/docs/1363850 4. Ўзбекистон Республикаси Жиноят-процессуал кодекси Т-2014й /111460#253615 References: 1. Ўзбекистон Республикаси Вазирлар Маҳкамасининг 2008 йил 20 июнь кунидаги 137-сонли қарори // https://lex.uz/docs/1363850 2. Ўзбекистон Республикасининг «Адвокатура тўғрисида»ги Қонун// https://lex.uz/docs/54503#55841 3. Ўзбекистон Республикаси Вазирлар Маҳкамасининг 2008 йил 20 июнь кунидаги 137-сонли қарори // https://lex.uz/docs/1363850 4. Ўзбекистон Республикаси Жиноят-процессуал кодекси Т-2014й /111460#253615 Volume 2 Issue 12, December 6, Special Issue 2022 ISSN 2181-2853 Volume 2 Issue 12, December 6, Special Issue 2022 Page 54
https://openalex.org/W2127955885
https://publications.goettingen-research-online.de/bitstream/2/25842/1/13414_2012_Article_279.pdf
English
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The Tölz Temporal Topography Study: Mapping the visual field across the life span. Part II: Cognitive factors shaping visual field maps
Attention, perception & psychophysics
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Atten Percept Psychophys (2012) 74:1133–1144 DOI 10.3758/s13414-012-0279-y Atten Percept Psychophys (2012) 74:1133–1144 DOI 10.3758/s13414-012-0279-y The Tölz Temporal Topography Study: Mapping the visual field across the life span. Part II: Cognitive factors shaping visual field maps Dorothe A. Poggel & Bernhard Treutwein & Claudia Calmanti & Hans Strasburger Published online: 24 April 2012 # The Author(s) 2012. This article is published with open access at Springerlink.com Published online: 24 April 2012 Keywords Temporal processing . Visual field maps . Alertness . Divided attention . Visual search . Aging Abstract Part I described the topography of visual perfor- mance over the life span. Performance decline was explained only partly by deterioration of the optical apparatus. Part II therefore examines the influence of higher visual and cogni- tive functions. Visual field maps for 95 healthy observers of static perimetry, double-pulse resolution (DPR), reaction times, and contrast thresholds, were correlated with measures of visual attention (alertness, divided attention, spatial cueing), visual search, and the size of the attention focus. Correlations with the attentional variables were substantial, particularly for variables of temporal processing. DPR thresholds depended on the size of the attention focus. The extraction of cognitive variables from the correlations between topographical variables and participant age substantially reduced those correlations. There is a systematic top-down influence on the aging of visual functions, particularly of temporal variables, that largely explains performance decline and the change of the topography over the life span. Keywords Temporal processing . Visual field maps . Alertness . Divided attention . Visual search . Aging Cognitive influences on visual performance Cognitive processes change over the life span. A typical pattern is an increase of cognitive performance until early adulthood, caused by maturation of the nervous system, fol- lowed by a decline toward senescence (Craik & Bialystok, 2006). While this prototypical development is not found for all functions (e.g., not for crystallized intelligence), particularly any measures of speeded or fluid performance tend to follow that pattern (e.g., fluid intelligence or speeded attention (Aizpurua & Koutstaal, 2010). Attentional capacity is also reduced with increasing age (Groth & Allen, 2000; Madden, 2007). This has an indirect influence on the size of the attention focus that can be maintained for efficient visual processing (Groth & Allen, 2000; Pesce, Guidetti, Baldari, Tessitore, & Capranica, 2005). Visual information traveling from the retina to the primary visual cortex and beyond is processed and shaped by the structure and functional specialization of the visual system. In addition to bottom-up processes, however, visual processing at lower stages in the pathway is profoundly influenced by top- down modulation originating from higher visual areas and from networks representing cognitive functions such as such as attention and memory (Bressler, Tang, Sylvester, Shulman, & Corbetta, 2008; Noesselt et al., 2002; Soto, Hodsoll, Rotsh- tein, & Humphreys, 2008). Attention, in particular, has been shown to modify behavioral performance and neuronal activa- tion as far down as V1 and even subcortical brain regions (Giesbrecht, Woldorff, Song, & Mangun, 2003; Kastner, Pinsk, De Weerd, Desimone, & Ungerleider, 1999; Mangun, Buonocore, Girelli, & Jha, 1998; Mangun & Fannon, 2007; Natale, Marzi, Girelli, Pavone, & Pollmann, 2006; Schneider & Kastner, 2009). Cognitive influences on visual processing vary with stimulus characteristics, but also with task demands (Eimer, Kiss, Press, & Sauter, 2009; Toth & Assad, 2002; Travers & West, 2008). While there is an extensive literature on the effects of visual performance decline over age on cognitive functions (Groth & Allen, 2000; Pesce et al., 2005), there is, to our knowledge, no systematic study showing the influence of cognitive aging on visual function and visual field maps (cf. Owsley, 2011; Werner, Peterzell, & Scheetz, 1990). As was shown in Part I of this study, visual performance is distributed across the visual field in a characteristic way for a specific visual function. Cognitive influences on visual performance The center and periphery of the visual field show large differences of visual performance, usually a lower performance in the periphery than in the center of the visual field (see the Introduction and Discussion in Part I). Focusing attention at a specific location within the visual field can change these performance maps in healthy observers (Carrasco, 2006), as well as in patients with partial vision loss (Poggel, Kasten, Müller-Oehring, Bunzenthal, & Sabel, 2006). The effectiveness of focusing attention depends, among many other factors, on the size of the attention focus; the larger the size of the attended area, the lower is the processing capacity and, hence, the lower is the gain from focusing attention at that location (Castiello & Umiltà, 1990). Hypotheses Given these interconnections of visual processing, cognitive factors, and aging, it is likely that the changes of visual field maps over the life span observed in Part I of this study are caused not only by the deterioration of the optical apparatus and neural structures in the visual pathway, but also by higher visual and cognitive functions. As was mentioned above, the size of the attention focus is relevant for visual processing. In addition, the individual’s general alertness and ability to divide attention between different locations or sour- ces of information are prerequisites for efficient handling of visual tasks. The ability to shift attention covertly (as assessed, for instance, in the Posner (1980) paradigm) and overtly (visual search) is also likely related to visual performance—in partic- ular, for temporal variables that are the main focus in this study. Background and motivation of the study Cognitive processes change over the life span. A typical pattern is an increase of cognitive performance until early adulthood, caused by maturation of the nervous system, fol- lowed by a decline toward senescence (Craik & Bialystok, 2006). While this prototypical development is not found for all functions (e.g., not for crystallized intelligence), particularly any measures of speeded or fluid performance tend to follow that pattern (e.g., fluid intelligence or speeded attention (Aizpurua & Koutstaal, 2010). Attentional capacity is also reduced with increasing age (Groth & Allen, 2000; Madden, 2007). This has an indirect influence on the size of the attention focus that can be maintained for efficient visual processing (Groth & Allen, 2000; Pesce, Guidetti, Baldari, Tessitore, & Capranica, 2005). related deterioration of speeded performance has been related to functional and structural changes in the brain (Birren & Fisher, 1995; Eckert, Keren, Roberts, Calhoun, & Harris, 2010; Falkenstein et al., 2006; Haier et al., 2005; Spear, 1993). Background and motivation of the study Background and motivation of the study This second part of the Tölz Temporal Topography Study describes how cognitive variables, particularly measures of visual attention, are related to the psychophysical data pre- sented in Part I. The study is based on an extensive data set of visual field maps and cognitive variables acquired within the Vision Lab of the Generation Research Program at Bad Tölz, with the goal of describing and analyzing the topography of visual performance and its changes over the life span. For reasons of length, the study is presented in two parts. Part I describes and interprets topographical variables of temporal processing and light detection, their relationship to each other, and their variation over the life span. In Part II, cognitive variables, particularly visual attention measures, are related to the psychophysical data presented in Part I. D. A. Poggel: C. Calmanti: H. Strasburger Generation Research Program (GRP), Ludwig Maximilian University Munich, Human Science Center, Bad Tölz, Germany e-mail: strasburger@uni-muenchen.de D. A. Poggel: C. Calmanti: H. Strasburger Generation Research Program (GRP), Ludwig Maximilian University Munich, Human Science Center, Bad Tölz, Germany e-mail: strasburger@uni-muenchen.de A main result of Part I was the dissimilarity of topographies and their different change with age. Time-related parameters fell into two groups, one reflecting processing speed (reaction time [RT]), and the other reflecting sensitivity or signal-to- noise ratio (double-pulse resolution [DPR]). The increase of RT over age happened rather uniformly over all visual field positions. DPR was unrelated to RT and, instead, resembled perimetric luminance thresholds in both topography and aging. Particularly for DPR thresholds, the periphery of the visual field showed a stronger reduction of performance than did foveal field positions, but interindividual variation was high, and age was not a good predictor of performance. Hence, other factors that co-vary with age—such as cognitive performance D. A. Poggel: H. Strasburger Dept. of Medical Psychology and Medical Sociology, Georg August University Göttingen, Göttingen, Germany B. Treutwein IuK, Ludwig-Maximilian University Munich, Munich, Germany D. A. Poggel (*) Hanse-Wissenschaftskolleg Institute of Advanced Study, Lehmkuhlenbusch 4, 27753 Delmenhorst, Germany e-mail: dapoggel@gmail.com Atten Percept Psychophys (2012) 74:1133–1144 1134 parameters—may influence visual maps and their change over the life span. related deterioration of speeded performance has been related to functional and structural changes in the brain (Birren & Fisher, 1995; Eckert, Keren, Roberts, Calhoun, & Harris, 2010; Falkenstein et al., 2006; Haier et al., 2005; Spear, 1993). Aging of visual and cognitive performance We therefore hypothesized the following: A large number of studies have shown a decline of visual performance in the elderly (Brabyn, Schneck, Haegerstrom- Portnoy, & Lott, 2001; Fiorentini, Porciatti, Morrone, & Burr, 1996; Haegerstrom-Portnoy, Schneck, & Brabyn, 1999; Owsley, 2011; Rubin et al., 2001; Schneck, Haegerstrom- Portnoy, Lott, Brabyn, & Gildengorin, 2004; West et al., 2002). This particularly applies to any type of speeded pro- cessing, like the measurement of RTs or other indicators of temporal-information processing (Falkenstein, Yordanova, & Kolev, 2006; Haier, Jung, Yeo, Head, & Alkire, 2005). Age- 1. Cognitive functions—in particular, attentional functions— influence the topography of visual functions, especially with respect to variables of temporal processing. 2. Cognitive factors, particularly attentional functions, can differentially influence the shape of visual field maps, depending on the cognitive task demands involved in mea- suring a particular visual variable. 3. Due to their aging, cognitive functions exert a differential influence on topographical tests of visual performance at different stages in life. 1135 Atten Percept Psychophys (2012) 74:1133–1144 4. The shape of a visual field map and its variation across the life span is the result of an interaction of optical and lower-level visual functions and cognitive processes. was performed in a darkened room with 30-cm viewing distance (see Fig. 1 of Part I). Each observer performed ten blocks of trials with a specific eccentricity of the peripheral stimuli (i.e., ring radius) set for each block. Each eccentricity (2.5°, 5°, 10°, 15°, and 20°) was tested twice. Test speed and duration were controlled by the observer responding in a self- paced manner. Sample The sample consisted of 95 paid volunteers (26 of them male) between 10 and 90 years of age (mean age: 47.8 years; see Table 1, Part I). All observers had normal or corrected- to-normal vision. Severe dementia, impairments of attention or other cognitive functions, and depression or other psy- chiatric disorders, as well as brain lesions and the presence of visual impairment at any level of the visual pathway, were exclusion criteria. In particular, the older participants were asked about potential ophthalmic diseases, and we inquired about the most recent eye exam to ascertain that participants were clinically inconspicuous. All observers (or their parents for minors) gave their informed consent for participation. The study design had been approved by the ethics committee of the Ludwig-Maximilian University, Munich, Germany, and testing procedures were in accor- dance with the tenets of the Declaration of Helsinki. For perimetric testing, observers were examined with the G2 program implemented on the Octopus 101 Perimeter (Interzeag/ Haag-Streit, Wedel, Germany) to determine luminance detec- tion thresholds within the central 30° of the visual field. Tests were performed separately for each eye, with a test duration of 10–12 min per eye. Fixation was controlled by an infrared- sensitive camera integrated into the sphere. The observer pressed a response button upon detecting a stimulus presented in the periphery (background luminance: 10 cd/m²). The G2 test uses a simple adaptive procedure for the independent variation of stimulus luminance at the 59 test positions and includes catch trials. Test conditions and general setting The experimenter was present throughout the test session(s), observed the participant’s performance and gaze position in a mirror, and interrupted the test when he or she appeared tired. Topographical variables Thresholds of double-pulse resolution (DPR) were measured using an apparatus and psychophysical technique developed by Treutwein (1989, 1995, 1997); Treutwein & Rentschler (1992). In an adaptive, nine-alternative forced choice task, the observer identified the noncontinuous stimulus in an array of nine stimuli so that the minimum detectable tempo- ral gap between two light pulses was determined. Testing Method RT maps were assessed with a high-resolution (474 positions) computer-based campimetric test (Nova Vision, Magdeburg, Germany) under the same testing conditions (darkened room, 30-cm viewing distance) as those for DPR threshold measure- ment (display size: ± 27° × ± 22.5°; cf. Fig. 2 of Part I). Participants reacted to the presentation of small, suprathreshold white-light stimuli on a dark background by pressing a key on the computer keyboard. Total duration of this test was 20 min. RTs corresponding to the visual field positions for DPR thresh- olds were used for analysis. Test conditions and general setting Testing took place under standardized conditions for all observers. Total testing time was approximately 7 h per participant, with some interindividual variation due to the different duration of the thresholding tests. With very few exceptions, the tests were performed in several sessions of 1.5- to 3.5-h duration, usually over a period of 1–2 weeks. All the variables mentioned in Parts I and II were examined in all participants; that is, for participants, the separation into a basic vision and a cognitive part of the experiment was not apparent. Participants were allowed to take breaks at any time to avoid excessive fatigue. The experimenter was present throughout the test session(s), observed the participant’s performance and gaze position in a mirror, and interrupted the test when he or she appeared tired. Testing took place under standardized conditions for all observers. Total testing time was approximately 7 h per participant, with some interindividual variation due to the different duration of the thresholding tests. With very few exceptions, the tests were performed in several sessions of 1.5- to 3.5-h duration, usually over a period of 1–2 weeks. All the variables mentioned in Parts I and II were examined in all participants; that is, for participants, the separation into a basic vision and a cognitive part of the experiment was not apparent. Contrast thresholds for the recognition of characters were tested with the software R_Contrast (Strasburger, 1997), which uses ML-PEST as the thresholding algorithm (Harvey, 1997). The general setting was the same as that for the computer-based RT measurement. Contrast thresholds were determined for the central (foveal) stimulus position and on the horizontal and vertical meridians at ± 10° eccentricity. On a gray background (36 cd/m2), the digits from 0 to 9 were presented at 2.4° visual angle height (viewing distance: 43 cm) for 100 ms each in randomized order. Initially, the digits were white, and the contrast to the background was reduced with every correct answer and, for an incorrect response, was increased again. The answers were given verbally, and the experimenter entered the responses into the software using the computer keyboard. A run was ended when the (estimated) 95% confidence interval of the Michelson contrast threshold value undercut 0.2 log units. Duration of a test run was 5–10 min. Participants were allowed to take breaks at any time to avoid excessive fatigue. Data analysis In a first step, raw data were analyzed separately for each test. Parametric tests (t-test and GLM/ANOVA) were used for the comparison of means. Correlations between test parameters were computed using Pearson’s r. Additionally, the influence of cognitive variables was extracted from the correlation of age with the topographical/temporal variables by using the partial correlation technique to explore the influence of cog- nitive factors on visual field maps. We calculated the common variance (R2) of two variables from the original correlation value, as well as the common variance (Rpart 2) remaining after the cognitive factor had been extracted as the square of the partial correlation. The absolute amount of extracted variance for a specific cognitive variable was then determined as the difference between R2 and Rpart 2. The relative amount of extracted variance was further calculated as (R2 −Rpart 2)/R2 (e.g., 100% relative extracted variance means that all the common variance was extracted). Attentional variables Three functions of attention were assessed by a computer- based test battery TAP (“Test Battery for Attentional Perfor- mance,” a standard tool for assessment of attentional functions in German-speaking countries [Zimmermann & Fimm, 1995]). In the alertness subtest, a central visual stimulus was presented with versus without an acoustic cue preceding its presentation by 100 ms. In cued as well as in uncued runs, the observer’s task was to press a key as quickly as possible in response to the appearance of the stimulus. Cued and uncued RTs and their difference were registered. Four runs were performed in an A–B–B–A design. The test took approxi- mately 5 min for all four runs. Data were extracted from the RT maps only at the DPR test positions. In the perimetric maps, luminance threshold values at the DPR-grid positions were determined by inter- polation, because the stimulus positions in perimetry did not correspond to those of the DPR test. All statistical testing was performed using SPSS software (Version 12.0, Chicago, IL). The alpha level was set to .05 (two tailed). For a detailed description of the analysis of the topographical variables, see Part I. Divided attention was examined with a dual visual– auditory subtest. The visual task consisted of monitoring a display of 8 × 8 positions that were filled by either dots or crosses. The stimulus pattern changed every 1.5 s and, at random intervals, formed a square configuration of crosses on the screen. This was the critical visual stimulus to be detected by the observer and indicated by a keypress. Simultaneously with the visual task, a series of alternating high and low tones was presented, and the observer was instructed to monitor the tones for a break in the sequence—that is, two high tones or two low tones—which was the critical acoustic stimulus. RTs to the critical visual and acoustic stimuli, as well as hits and misses, were recorded. Total test duration was approximately 10 min. Saccadic exploration We examined the search field—that is, the distribution of speed and accuracy of saccadic eye movements toward peripheral targets—with a subroutine of the Nova Vision test battery. The Atten Percept Psychophys (2012) 74:1133–1144 1136 one indicated by the cue). The RT difference between the two conditions was registered as cue benefit. Total test duration was approximately 10 min. basic setting was the same as that for the RT mapping de- scribed above. Participants were instructed to keep their eyes on the fixa- tion mark in the center of the screen and then to make eye movements toward a stimulus in the periphery of the visual field that was presented simultaneously with an acoustic sig- nal. The stimulus was the digit “2,” “3,” or “8,” presented in random order at 284 positions in the visual field. Observers responded by pressing one of three keys on the computer keyboard (arrow left, arrow down, arrow right) without taking their gaze off the screen. After the response, they returned their gaze to the fixation point. Saccadic RTs for each position were registered by the program and plotted in the same way as the RTs from the computer-based visual field test. Topographical attention effect in DPR threshold maps DPR thresholds were always measured simultaneously at all nine positions in each block. This included the central (foveal) position, which remained constant over all blocks of testing. Since the physical position of the central test location did not change, one would expect constant foveal DPR thresholds, independent of the variation of the periph- eral test locations. However, as we have shown earlier (Fig. 4 of Poggel, Treutwein, Calmanti, & Strasburger, 2006), the central DPR thresholds increase with the radius of the display. The pattern and the average rate of increase were almost exactly the same as those for the increase of DPR thresholds at the peripheral positions. Since the stim- ulus characteristics and all other test conditions were iden- tical, we interpreted the increase of central thresholds with increasing display size as the effect of attentional processing capacity being distributed over a larger area (see Poggel, Treutwein, et al., 2006, for a detailed discussion). Our results on perimetric thresholds of light detection replicated earlier findings of increasing thresholds toward the periphery. Mean luminance thresholds were significantly affected by age: The youngest participants showed the best performance, and the three oldest age groups had increased thresholds, as compared with the remainder of the sample. For perimetric performance the effect of age was more systematic and much stronger than for the other variables examined in this study. In contrast to DPR thresholds, how- ever, there was no significant interaction between the aging and eccentricity effect. Only singular elderly participants showed a disproportionately stronger increase of perimetric thresholds in the periphery than did the other age groups. Mean contrast thresholds for character recognition (R_Contrast) were higher at 10° eccentricity than in the fovea. Mean thresholds over all tested positions increased slightly but significantly with age, and there was a significant interac- tion of age and eccentricity. Interestingly, while the attention effect at the central stim- ulus location was pronounced and highly significant for the total group of observers, it was absent for the youngest par- ticipants (10–20 years) and was very small in participants between 20 and 40 years of age (see Fig. 7 in Poggel, Treut- wein, et al., 2006). For all other age groups—that is, from 40 to 90 years—the increase of central DPR thresholds with increasing radius of the test display was large and highly significant. Topographical attention effect in DPR threshold maps The attention effect was particularly pronounced for the oldest age group of 80- to 90-year-old participants, who also showed the steepest increase of DPR thresholds toward the periphery. Thus, the topographical variables of temporal and light/ contrast processing showed different forms of visual field maps and also differing aging processes. Topographical variables Topographical variables Results on the topographical variables from Part I (DPR, RT, perimetry, R_Contrast) are briefly summarized here for their comparison with the cognitive variables. The figures in Part I illustrate the effects described below. Results on the topographical variables from Part I (DPR, RT, perimetry, R_Contrast) are briefly summarized here for their comparison with the cognitive variables. The figures in Part I illustrate the effects described below. Thresholds of DPR increased toward the periphery of the visual field, with a steep increase from the center to 2.5° eccen- tricityandashallow,steadyriseofthresholdsbeyond5°.Agehad a significant effect on mean DPR thresholds: The best perfor- mance was observed in participants between 20 and 30 years of age; up to the age of 60, there was hardly any increase of mean thresholds, but we observed a sharp increase in the 70s and 80s. The form of the DPR threshold maps changed over the life span; thatis,therewasaninteractionbetweenageandeccentricity,with olderparticipantsshowingasteeperinclineofthresholdsbeyond 5° of eccentricity (see Figs. 5 and 6, Part I); that is, there was a To test the ability to covertly shift attention to a visual field location indicated by a cue and the costs and benefits of attending to a cued visual field location (Posner, 1980), a third subtest of the TAP was employed (Posner paradigm). The observer fixated a central fixation mark and made a speeded response to a cross appearing at a fixed position in either the left or the right visual field. Before the presentation of a target, an arrow above the central fixation mark indicated the target position with 80% validity. RTs were measured for valid trials (target presented at the position indicated by the arrow) and invalid trials (target presented at the position opposite to the 1137 Atten Percept Psychophys (2012) 74:1133–1144 medium to high, and particularly, the ability to shift attention —overtly (saccadic RTs) or covertly (Posner paradigm)—had a close connection with the topographical variables. stronger reduction of performance in the periphery than in the center of the visual field for older observers. Simple visual RTs showed a shallow, evenly distributed increase from the center to the periphery of the visual field. The aging effect on mean RTs was significant, with best performance in the 30s and a noticeable increase after 60 years. The (overall) interaction of age and eccentricity missed significance; the increase of RTs toward the periphery was steeper in the two oldest age groups, however. TAP = test of attentional performance; p values in parentheses Saccadic exploration (overt attention) The mean RT in the saccadic exploration test (mean over all observers and test positions ± SEM) was 851.4 ms ± 17.5 ms. There was a significant increase of search times over the life span, with the best performance in the 20- to 29-year-old participants (697.5 ± 28.7 ms) and the lowest in the oldest group [1,216.8 ± 66.9 ms; analysis of variance over age groups: F(7, 85) 0 13.0, p < .001, η2 0 .52]. The effect of age was also reflected in the substantial correlation of mean search time and observer age, r(93) 0 .64, p < .001. In the following, attentional variables were extracted from the correlations of the main topographical variables with age, to explore their influence on visual field maps (cf. the corresponding section on each variable below). When saccadic RTs were extracted, the age correlations were substantially reduced for all topographical variables except the campimetric RT map (Table 2), the effect being most pronounced for DPR. (Table 3) and were considerably lower than the correlations of search times with age. When RTs from the TAP Alertness Test were extracted from the correlations of the main topographical variables with age, the common variance of age with DPR thresholds, with campimetric RT, and with perimetric thresholds, respectively, was somewhat reduced (8%–10% extracted variance), while R_Contrast remained largely unchanged (Table 4). Correlation of topographical and cognitive variables Table 1 shows a summary of all correlations between cognitive and topographical variables. Almost all correlations were Table 1 Pearson correlations of topographical variables with cognitive parameters Subtest Variable DPR Thresholds Simple RT in Campimetric Test Perimetric Thresholds Character Recognition Contrast Thresholds (R_Contrast) TAP Alertness overall mean RT .35 (.001) .35 ( < .001) −.25 (.015) .32 (.002) mean RT difference cued−uncued −.19 (.061) −.07 (.534) .13 (.212) −.09 (.387) TAP Divided Attention mean overall RT .34 (.001) .30 (.004) −.20 (.055) .24 (.019) mean number of correct detections −.39 (<.001) −.44 ( < .001) .07 (.488) −.18 (.089) mean visual RT .34 (.001) .14 (.182) −.26 (.013) .22 (.033) mean auditory RT .34 (.001) .27 (.009) −.07 (.513) .21 (.048) TAP Posner Paradigm mean RT valid trials .54 (<.001) .46 ( < .001) −.39 ( < .001) .43 ( < .001) mean RT invalid trials .50 (<.001) .51 ( < .001) −.30 (.004) .42 ( < .001) cue benefit (mean difference valid−invalid) .43 (<.001) .49 ( < .001) −.21 (.046) .37 ( < .001) Saccadic exploration mean search RT .62 (<.001) .32 (.002) −.47 ( < .001) .49 ( < .001) TAP = test of attentional performance; p values in parentheses Table 1 Pearson correlations of topographical variables with cognitive parameters Atten Percept Psychophys (2012) 74:1133–1144 1138 Table 3 Correlations of attentional subtests in the TAP with age (N095) TAP Subtest TAP Variable Correlation With Age: r (93) p Alertness Median RT all trials .32 < .001 Median RT uncued trials .33 < .001 Median RT cued trials .28 .006 Divided attention Median RT all trials .22 .036 Number of correct resp. −.19 .063 Median visual RT .18 .075 Median auditory RT .20 .054 Covert attention shift (Posner paradigm) Median RT valid trials .55 < .001 Median RT invalid trials .47 < .001 Difference (cueing benefit) .37 < .001 Table 3 Correlations of attentional subtests in the TAP with age (N095) Nontopographical attentional variables from the TAP Alertness The average RT in the alertness subtest of the TAP over all observers and trials was 267.7 ms (± 6.1). Without an acoustic cue, observers took an average of 272.1 ms (± 6.0) to respond to the visual stimulus, while on trials with an acoustic cue, the average RTamounted to 266.8 ms (± 7.0). Thus, there was an overall advantage of 5.3 ms (± 3.6) of cued over uncued trials. Divided attention The median RT of all observers in the divided attention task amounted to 698.9 ms (± 10.6), with 878.4 ms (± 14.9) for the visual subtask and 565.7 ms (± 12.1) for the auditory subtask. The average number of correct detections of critical events (both modalities) was 27.8 (± 0.5). Overall, RTs in the TAP Alertness Test increased over the life span [ANOVA, F(7, 87) 0 2.21, p 0 .041, η2 0 .15], but not monotonously. The best performance was obtained in participants between 30 and 39 years of age, and the lowest in the oldest age group (80–90 years). The same pattern was found for uncued and for cued RTs developing over the life span. Although the correlations of RTs in the TAP Alertness Test with age were all significant, they were not substantial Over the life span, overall, RTs in the TAP Divided Atten- tion Test significantly increased [ANOVA: F(7, 87) 0 4.04, p 0 .001, η2 0 .25], reaching from 634.0 ms (± 24.2) for the group of 20- to 29-year-olds to 836.1 ms (± 41.9) for the group of 80- to 90-year-olds. Nontopographical attentional variables from the TAP Similarly, there was a reduction Table 2 Pearson correlations of topographical variables with age and corresponding partial correlations where saccadic exploration time is partialled out Variable Correlation With Age: Partial Correlation With Extracted Variance Pearson’s r (p) Age r (p), Saccadic Reaction Absolute: R2 −Rpart 2 R2 Times Partialled Out Relative: (R2 −Rpart 2) /R2 DPR thresholds .62 ( < .001) .35 ( 0 .001) 26.2% 38.4% 68.2% RT campimetric test .16 (.118) −.05 ( 0 .628) 2.3% 2.6% 88.5% Perimetric thresholds −.68 ( < .001) −.55 ( < .001) 16.0% 46.2% 34.6% R_Contrast .50 ( < .001) .31 ( 0 .002) 15.4% 25.0% 61.6% 1139 Atten Percept Psychophys (2012) 74:1133–1144 Table 4 Correlations of topographical variables with age and partial correlations where response times in the TAP Alertness Test are extracted Topographic Variable Correlation With Age: Partial Correlations— Extracted Variables Alertness Median RT Alertness Median RT Difference Cued−Uncued Trials r (p) All Trials R2 DPR thresholds .65 ( < .001) rpart (p) .57 ( < .001) .63 ( < .001) 42.3% % absolute variance extracted 9.8% 2.6% % relative variance extracted 23.2% 6.1% RT campimetric test .30 (.003) rpart (p) .05 (.610) .16 (.120) 9.0% % absolute variance extracted 8.8% 6.4% % relative variance extracted 97.8% 71.1% Perimetric thresholds −.71 ( < .001) rpart (p) -0.65 ( < 0.001) -0.68 ( < 0.001) 50.4% % absolute variance extracted 8.2% 4.2% % relative variance extracted 16.3% 8.3% R_contrast thresholds .47 ( < .001) rpart (p) .45 ( < .001) .50 ( < .001) 22.1% % absolute variance extracted 1.8% −2.9% % relative variance extracted 8.1% −4.5% aphical variables with age and partial correlations where response times in the TAP Alertness Test are extracted found, similar to the influence of alertness, some reduction of the common variance for the topographical variables (up to a quarter, relative to the original shared variance), again with the exception of R_Contrast (Table 5). of the number of correctly identified critical events with age [ANOVA: F(7, 87) 0 3.43, p 0 .003, η2 0 .22], ranging from 29.4 (± 1.4) for participants in their 30s to 20.2 (± 1.9) for participants in their 80s. Nontopographical attentional variables from the TAP Participants in their 20s, although showing the fastest responses on both valid and invalid trials, profited least from the cue (20–30 years: 326.2 ± 23.6 ms). The absolute increase of the cueing benefit over the life span was significant [ANOVA: F(7, 87) 0 4.14, p 0 .001, η2 0 .25]. However, that increase could be due to a multiplicative effect of overall slowed RT with increasing age: When the relative increase of the cueing benefit was calculated using the ratio (RTvalid −RTinvalid)/RTmean, there was no signifi- cant change over the life span [ANOVA: F(7, 85) 0 1.31, p 0 .256, η2 0 .10]. Of all the subtests of the attention test battery, the variables of the Posner paradigm showed the highest correlations with observer age (Table 2). RTs for invalidly cued trials [ANOVA: F(7, 87) 0 6.14, p < .001, η2 0 .34; best performance: 584.2 ms (± 34.6); lowest performance: 896.4 ms (± 60.0)]. The greatest ben- efit of valid over invalid cues with respect to RT differences was found in the oldest group (80–90 years: 492.8 ± 40.9 ms). Participants in their 20s, although showing the fastest responses on both valid and invalid trials, profited least from the cue (20–30 years: 326.2 ± 23.6 ms). The absolute increase of the cueing benefit over the life span was significant [ANOVA: F(7, 87) 0 4.14, p 0 .001, η2 0 .25]. However, that increase could be due to a multiplicative effect of overall slowed RT with increasing age: When the relative increase of the cueing benefit was calculated using the ratio (RTvalid −RTinvalid)/RTmean, there was no signifi- cant change over the life span [ANOVA: F(7, 85) 0 1.31, p 0 .256, η2 0 .10]. Of all the subtests of the attention test battery, the variables of the Posner paradigm showed the highest correlations with observer age (Table 2). the outer visual field. For DPR thresholds, we thus further extracted the influence of cognitive variables from correlations for the inner and outer ranges separately (Table 7) and found a strong reduction of shared variance for overt (saccadic explo- ration) and covert attention variables, but only minor effects for alertness and divided attention were extracted. For those cog- nitive variables that had an influence on the common variance between age and DPR thresholds, the effect was somewhat stronger for the outer parts of the DPR threshold map (Table 7). Nontopographical attentional variables from the TAP Overall, age dependency of the topographical variables is partly mediated by attentional factors. The nontopographical variables of alertness and divided attention partly mediate age dependency of DPR thresholds and campimetric RTs, and less so perimetry, but not R_Contrast; overt attention mediates age dependency for all topographical variables except RTs; covert attention more strongly mediates the age dependency of all four topographical variables. Again, the variables of covert attention were extracted from the correlation between the topographical variables and age. We found that the common variance of age with DPR thresholds was, in absolute values, the one most strongly affected by extracting covert attention variables, with more than half of the shared variance extracted in relative values (Table 6). Campimetric RT’s moderate cor- relation with age was almost completely mediated by covert attention variables; that is, close to 100% of the originally shared variance was extracted. The extraction of covert attention variables also had a large effect on perimetric thresholds and on R_Contrast (Table 6). Nontopographical attentional variables from the TAP Similarly, the visual RTs [ANOVA: F(7, 87) 0 2.79, p 0 .011, η2 0 .18] and auditory RTs [ANOVA: F(7, 87) 0 1.85, p 0 .088, η2 0 .13] increased over the life span, with optimal performance for participants in their 20s and lowest performance in the oldest age group. Covert attention The median RTs of all observers in the covert attention task (Posner paradigm) were 305.4 ms (± 6.8) for valid and 699.8 ms (± 16.4) for invalid trials. The mean difference between invalid and valid trials amounted to 394.1 ms (± 10.6). RTs on valid trials increased with age [ANOVA: F(7, 87) 0 8.16, p < .001, η2 0 .40; best performance: 258.0 ms (± 13.9) in the 20s group; lowest performance: 403.6 ms (± 24.0) in the 80s group], as did Correlations of parameters of divided attention with age were relatively low, and only some became significant (Table 3). We extracted the influence of divided attention from the correlation between age and the topographical variables and Table 5 Correlations of topographical variables with age and partial correlations where response times in the TAP Divided Attention Test are extracted Topographic Variable Correlation With Age: Partial Correlations—Extracted Variables Divided Attention Median RT, All Trials Divided Attention, mean Number of Correct Detections r (p) R2 DPR thresholds .65 ( < .001) rpart (p) .59 ( < .001) .60 ( < .001) 42.3% % absolute variance extracted 7.4% 6.3% % relative variance extracted 17.5% 14.9% RT campimetric test .30 (.003) rpart (p) .05 (.610) .09 (.403) 9.0% % absolute variance extracted 8.8% 8.2% % relative variance extracted 97.8% 91.1% Perimetric thresholds −.71 ( < .001) rpart (p) −.66 ( < .001) −.68 ( < .001) 50.4% % absolute variance extracted 6.9% 4.2% % relative variance extracted 13.7% 8.3% R_Contrast thresholds .47 ( < .001) rpart (p) .47 ( < .001) .48 ( < .001) 22.1% % absolute variance extracted 0% 0% % relative variance extracted 0% 0% Atten Percept Psychophys (2012) 74:1133–1144 1140 RTs for invalidly cued trials [ANOVA: F(7, 87) 0 6.14, p < .001, η2 0 .34; best performance: 584.2 ms (± 34.6); lowest performance: 896.4 ms (± 60.0)]. The greatest ben- efit of valid over invalid cues with respect to RT differences was found in the oldest group (80–90 years: 492.8 ± 40.9 ms). Atten Percept Psychophys (2012) 74:1133–1144 Atten Percept Psychophys (2012) 74:1133–1144 Atten Percept Psychophys (2012) 74:1133–1144 1141 Atten Percept Psychophys (2012) 74:1133–1144 Table 7 Correlations of DPR in the inner and outer visual field with age, and partial correlations where cognitive variables are extracted n s Variable Mean DPR Thresholds Inner Visual Field (0° – 10°) Mean DPR Thresholds Outer Visual Field (10° – 20°) Correlation with age r (p) .52 ( < .001) .59 ( < .001) 27.0% 34.8% Partial correlation variables extracted Saccadic exploration mean RT .23 (.031) .34 (.001) Absolute extracted variance 21.8% 23.3% Relative extracted variance 80.7% 67.0% TAP Alertness median RT all trials .47 ( < .001) .54 ( < .001) Absolute extracted variance 5.0% 5.7% Relative extracted variance 18.5% 16.4% TAP Alertness mean RT difference cued-uncued trials .53 ( < .001) .60 ( < .001) Absolute extracted variance −1.1% −1.2% Relative extracted variance −4.1% −3.4% Divided attention median RT all trials .48 ( < .001) .56 ( < .001) Absolute extracted variance 4.0% 3.5% Relative extracted variance 14.8% 10.1% TAP Divided Attention mean number of correct detections .49 ( < .001) .57 ( < .001) Absolute extracted variance 3.0% 2.3% Relative extracted variance 11.1% 6.6% TAP Covert Attention (Posner paradigm) mean RT valid trials .36 ( < .001) .43 ( < .001) Absolute extracted variance 14.1% 16.3% Relative extracted variance 52.2% 46.8% TAP Covert Attention (Posner paradigm) mean RT invalid trials .41 ( < .001) .46 ( < .001) Absolute extracted variance 10.2% 13.7% Relative extracted variance 37.8% 39.4% TAP Covert Attention (Posner paradigm) mean RT difference valid−invalid trials .45 ( < .001) .50 ( < .001) Absolute extracted variance 6.8% 9.8% Relative extracted variance 25.2% 28.2% literature before integrating the results from Part I and II of the study. of the size of the attention focus on temporal resolution and an increasingly stronger effect of visual attention in the elderly. We therefore hypothesized that the topographies of the visual variables assessed in Part I of this study and their respective change over the life span not only reflect the functionality of the visual system per se, but also are influ- enced by higher visual and cognitive functions. In this second part of the Tölz Temporal Topography Study, we thus presented correlation patterns of the topographical var- iables introduced in Part I with a selected set of cognitive variables. 1. Table 7 Correlations of DPR in the inner and outer visual field with age, and partial correlations where cognitive variables are extracted Discussion The first part of this study on visual field mapping over the life span had shown large differences between the visual field topographies of DPR thresholds, RT in campimetric testing, perimetric thresholds, and contrast thresholds of character recognition. In addition, the patterns of aging of those four main variables were markedly different, and we concluded that experimental or clinical testing that is limited to the fovea conceals important aspects of the development of visual functions over the life span. An earlier analysis of DPR thresholds in observers of the same sample (Poggel, Treutwein, et al., 2006) had further revealed a strong effect For DPR thresholds, the correlation with age was slightly lower in the inner visual field (up to 10°) than in the outer visual field (10° – 20°). This reflected the steeper increase with age for Table 6 Correlations of topo- graphical variables with age and partial correlations where response times in the TAP covert attention test (Posner paradigm) are extracted Variable Correlation With Age: Partial Correlations—Variables Extracted r (p) Posner Median RT, Valid Trials Posner Median RT, Invalid Trials Posner Cue Benefit (Mean RT Difference Valid−Invalid Trials) DPR thresholds .65 (< .001) .45 ( < .001) .49 ( < .001) .54 ( < .001) 42.3% 22.0% 18.2% 13.1% 52.0% 43.0% 31.0% RT campimetric test .30 (.003) −.13 (.229) −.13 (.232) −.05 (.621) 9.0% 7.3% 7.3% 8.8% 81.1% 81.1% 98.8% Perimetric thresholds −.71 (< .001) −.60 (< .001) −.64 (< .001) −.66 ( < .001) 50.4% 14.4% 9.5% 6.9% 28.6% 18.8% 13.7% R_Contrast thresholds .47 ( < .001) .35 ( < .001) .40 ( < .001) .44 ( < .001) 22.1% 9.8% 6.1% 2.7% 44.3% 27.6% 12.2% Atten Percept Psychophys (2012) 74:1133–1144 Influence of cognitive variables on visual field maps Almost all variables of cognitive functions—alertness, divided attention, covert attention shift (Posner paradigm; Posner, 1980; Zimmermann & Fimm, 1995), and overt attention shift (saccadic RTs)—were correlated with the topographical variables measured in Part I. The correlations where higher for variables of temporal processing (DPR thresholds, RTs in campimetric testing) than for perimetric luminance and contrast thresholds of character recognition. In addition, a strong influence of the size of the attention focus In the following, we will first relate our findings to the hypotheses stated in the introduction and to the 1142 Atten Percept Psychophys (2012) 74:1133–1144 had been shown for DPR thresholds (Poggel, Treutwein, et al., 2006). These results confirm the first part of our hypothesis— that is, that there is an influence of cognitive variables on visual field maps. Thus, the visual field map of a specific function is shaped not only by the properties of the visual system, but also by cognitive factors. Attentional influences on temporal processing (Yeshurun, 2004; Yeshurun & Levy, 2003) and perimetric thresholds (Wall et al., 2004) have been reported by other authors, but to our knowledge, there has not been a systematic study relating different aspects of attention to visual field maps of different visual functions. had been shown for DPR thresholds (Poggel, Treutwein, et al., 2006). These results confirm the first part of our hypothesis— that is, that there is an influence of cognitive variables on visual field maps. Thus, the visual field map of a specific function is shaped not only by the properties of the visual system, but also by cognitive factors. While there is extensive literature on the influence of cognitive variables on a wide range of perceptual performance measures, this study is, to our knowledge, the first to system- atically show the influence of cognitive measures on the topography of visual field maps—in particular, of temporal functions. That spatial attention has an effect on (local) tem- poral resolution has been shown, for example, by Yeshurun and Levy (2003), who found a decrease of temporal resolution performance under attended conditions. Their paradigm, however, uses short-term cueing (i.e., transient attention; Nakayama & Mackeben, 1989), while our results stem from long-term monitoring of the visual field (i.e., from sustained attention). Atten Percept Psychophys (2012) 74:1133–1144 Our findings are also in accordance with a study by Wall, Woodward, and Brito (2004), who demonstrated reduced sensitivity in perimetric measurements with an increasing workload (and less attentional resources available for light detection) in healthy individuals. 3. Differential influence of cognitive factors over the life span All of the cognitive variables measured in the Tölz Tem- poral Topography Study systematically depended on the observer’s age. For most measures, young adults around 20 to 30 years of age showed the best performance, and over the life span performance significantly declined, with the oldest participants usually showing the lowest perfor- mance. Similarly, when the influence of overt attention (saccadic exploration) or covert attention (TAP Posner par- adigm–spatial attention test), respectively, was extracted from the correlations of the topographical variables with age, the correlations were strongly reduced. Alertness and divided attention, in contrast, had only a small effect. Thus, a large part of the variation observed for the topographical variables over the life span seems not to stem from low-level visual factors, such as a deterioration of the visual system or of optical structures in the elderly, but rather, from top-down influences on visual processing. The unusually large difference between invalid and valid trials of the covert attention test (Posner paradigm) that we found in our study may stem, in part, from the age of our observers, many of whom were much older than those in most cognitive psychological experiments. In addition, for some observers, there might have been a contamination with eye movements, which may have led to good RTs on valid trials, but longer RTs and an overall increased variance of RTs on invalid trials. 4. Shape of visual field maps and their change over the life span depend on an interaction of visual functions and cognitive processes For DPR thresholds, we further compared the correlations with age separately for the inner (< 10°) and outer (10° – 20°) visual fields. There was a slightly higher correlation for the outer than for the inner positions. Both correlations with age were reduced by partialling out cognitive factors, especially covert attention variables, again with a slightly stronger effect for the outer visual field positions. This effect and the influ- ence of the size of the attention focus on temporal resolution mentioned above help to explain why the periphery “ages” somewhat more than the fovea and inner visual field with respect to DPR. Depending on the demands of a visual field measure on cognitive functions, cognitive aging may have differential effects on the visual field center and periphery. 2. Differential influence of cognitive factors on visual field topography due to cognitive test demands In contrast, overt attention mediates age dependency for all topographical variables except RTs, and covert attention more strongly mediates the age dependency of all four topographical variables. Apparently, saccadic exploration (overt attention) and the Posner paradigm of the TAP (covert attention) reflect the ability to efficiently distribute and spatially shift around attentional resources in the visual field. Over the life span, these variables exert a strong influence on visual map topography because, along with the visual system, the cognitive resources and their flexibility are affected by aging. Acknowledgments This research was supported by grants from the Deutsche Forschungsgemeinschaft to Hans Strasburger (“Funktionelle Charakterisierung des Gesichtsfeldes bei zerebralen Teilschädigungen mittels Methoden der Psychophysik,” STR 354/3-1, and STR 354/7-1). We thank Sebastian Rummel and Dr. Jan Churan for help with techni- cal equipment and data analysis and Luise Schulze, Andrea Geyer, and Inge and Patrick Das Gupta for help with data acquisition and data management. Prof. Ernst Pöppel and PD Dr. Marc Wittmann provided encouragement and a spirited and pleasant environment for research at the GRP. We particularly thank Dr. Manfred MacKeben for helpful comments on the manuscript. In summary, cognitive variables—in particular, the size of the spatial focus of attention, as well as the ability of shifting it across the visual field, overtly and covertly—have a profound influence on visual field maps, particularly on the topography of temporal processing, and their change over the life span. Open Access This article is distributed under the terms of the Crea- tive Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. In Part I of this study we saw that observer age is not the most important predictor of visual performance in the topo- graphical variables assessed here. Instead, the cognitive status of a person and the level of attentional capability may be more important for determining visual performance and the topography of visual field maps. First, there appears to be an influence of cognitive factors on the general level of performance—that is, the average across the visual field. Second, there are some aspects of attention selectively shap- ing the periphery and the center of the visual field. 2. Differential influence of cognitive factors on visual field topography due to cognitive test demands 2. Differential influence of cognitive factors on visual field topography due to cognitive test demands The measures of attentional functions selected here assess many aspects of the complex conglomerate of attention. Inter- estingly, the divided attention task in the TAP had only a weak connection with performance in the topographical tests. This is likely due to that the test assesses the ability to divide attention between modalities (visual and auditory), rather than within the visual modality (between different visual field locations or different visual subtasks). A purely visual test of divided atten- tion might have been more strongly related to the topographical variables. The change of visual field topography over the life span is at least partly mediated by attentional factors. Alertness and divided attention partly mediate age dependency of DPR thresholds and campimetric RTs, but less so of perimetry, and not at all of R_Contrast. Partial correlation analysis revealed that the TAP subtests of alertness and divided atten- tion were confounded with general RT measures. Hence, by the way these variables are measured in the TAP, they appear not to reflect purely cognitive function but are more influ- enced by visual RTs. This would explain their predominant The highest among the correlations of cognitive with topographical variables (particularly those reflecting tempo- ral processing) were those with the ability to overtly and covertly shift attention across the visual field. The variance of the topographical variables explained by RTs in the Posner paradigm or saccadic responses (Table 1) came close to the percentage explained by participant age (see Part I). 1143 Atten Percept Psychophys (2012) 74:1133–1144 mediation of the age dependency of RTs in the campimetric test. Due to the confoundedness with RT, the influence of the TAP measures alertness and divided attention is visible only in the topography of time-related variables like DPR thresh- olds and campimetric RTs. including the periphery of the visual field into functional characterization and the necessity of co-investigating the cog- nitive status of the observer, particularly in an elderly population. It seems that temporal processing encompasses various mech- anisms (see the differences of visual field maps between RTs and DPR thresholds) and that these mechanisms are differen- tially affected by aging—that is, a reduced visual, cognitive, and general neural functionality. 2. Differential influence of cognitive factors on visual field topography due to cognitive test demands Attentional functions may be used to compensate for a deterioration of perceptual difficulties (Li & Lindenberger, 2002) due to reduced functionality of the optical and neural parts of the visual system with increasing age (Bellis, 1933; Birren & Fisher, 1995; Falkenstein et al., 2006; Haier et al., 2005; Owsley, Sekuler, & Siemsen, 1983; Plainis, Murray, & Chauhan, 2001; Schmidt, Galuske, & Singer, 1999; Spry & Johnson, 2001; Tyler & Hamer, 1993; Weale, 1963; see Schiefer et al., 2001, for a review). References Aizpurua, A., & Koutstaal, W. (2010). 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P., . . . and for the SEE Project Team. (2001). The
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Issue 6 / Part 3 e/view/swj06-03-028 712 2020-06-03-028 Issue 6 / Part 3 e/view/swj06-03-028 712 2020-06-03-028 Issue 6 / Part 3 e/view/swj06-03-028 712.2020-06-03-028 SWorldJournal https://www.sworldjournal.com/index.php/swj/article/view/swj06-03-028 DOI: 10.30888/2663-5712.2020-06-03-028 Вступ. Ефективна та збалансована система внутрішніх і зовнішніх комунікацій є однією з необхідних передумов якісного виконання своїх функцій організаціями усіх типів та форм власності. Комунікативна діяльність органів державної влади, яка забезпечується відповідною системою публічного менеджменту, полягає у здійсненні інформаційної взаємодії, спрямованої на виконання функцій публічного управління та адміністрування. Пошук, збір, опрацювання інформації, прийняття на її основі управлінських рішень, як і здійснення будь-яких управлінських впливів та взаємодій взагалі [1, с.11], забезпечується шляхом використання формальних комунікативних каналів, визначених на державному рівні або регламентованих на рівні органу публічної влади, а також через неформальні, неофіційні канали, наприклад різноманітні месенджери та соціальні мережі. Державні органи, так само як і приватні організації, потребують ефективної системи комунікації, яка дозволить раціонально використовувати наявні людські, фінансові та матеріальні ресурси [1; 2]. Основний текст ISSN 2663-5712 УДК 35:316.77 MODERN TRENDS IN THE FORMATION OF THE CORPORATE COMMUNICATION SYSTEM IN PUBLIC AUTHORITIES СУЧАСНІ ТЕНДЕНЦІЇ ФОРМУВАННЯ СИСТЕМИ КОРПОРАТИВНИХ КОМУНІКАЦІЙ В ДЕРЖАВНИХ ОРГАНАХ КОМУНІКАЦІЙ В ДЕРЖАВНИХ ОРГАНАХ Arkhypova Ye.O. / Архипова Є.О. PhD in phil., as.prof. / к.філос.н., доц. ORCID: 0000-0002-1640-1488 Hula M.S. / Гула М. master's student / магістрантка. National Technical University of Ukraine “Igor Sikorsky Kyiv Polytechnic Institute”, Kyiv, Prosp. Peremohy, 37, 03056 Національний технічний університет «Київський політехнічний інститут ім. Ігоря Сікорського», Київ, пр-т Перемоги, 37, 03056 Arkhypova Ye.O. / Архипова Є.О. PhD in phil., as.prof. / к.філос.н., доц. ORCID: 0000-0002-1640-1488 Hula M.S. / Гула М. Arkhypova Ye.O. / Архипова Є.О. PhD in phil., as.prof. / к.філос.н., доц. ORCID: 0000-0002-1640-1488 Hula M.S. / Гула М. master's student / магістрантка. National Technical University of Ukraine “Igor Sikorsky Kyiv Polytechnic Institute”, Kyiv, Prosp. Peremohy, 37, 03056 Національний технічний університет «Київський політехнічний інститут ім. Ігоря Сікорського», Київ, пр-т Перемоги, 37, 03056 Hula M.S. / Гула М. master's student / магістрантка. National Technical University of Ukraine “Igor Sikorsky Kyiv Polytechnic Institute”, Kyiv, Prosp. Peremohy, 37, 03056 Національний технічний університет «Київський політехнічний інститут ім. Ігоря Сікорського», Київ, пр-т Перемоги, 37, 03056 у master's student / магістрантка. National Technical University of Ukraine “Igor Sikorsky Kyiv Polytechnic Institute”, Kyiv, Prosp. Peremohy, 37, 03056 Національний технічний університет «Київський політехнічний інститут ім. Ігоря Сікорського», Київ, пр-т Перемоги, 37, 03056 Анотація. В роботі розглянуто сучасні тенденції формування системи корпоративних комунікацій в органах публічної влади. Проаналізовано досвід використання в державно- управлінському секторі України та інших держав систем електронного документообігу та управління контентом (АСКОД, ДОК ПРОФ, FossDoc, ЕСМ Alfresco, Government Transaction Portal). Визначено переваги, які надає державним органам використання вільного програмного забезпечення для управління контентом. Окреслено практику використання публічними органами інших комунікативних інструментів: корпоративних сайтів, соціальних мереж, месенджерів. Ключові слова: комунікація, державні органи, органи публічної влади, система електронного документообігу, управління контентом, сайт, месенджер ISSN 2663-5712 Основний текст Основна перевага АСКОД www.sworldjournal.com www.sworldjournal.com 76 Issue 6 / Part 3 Issue 6 / Part 3 SWorldJournal Issue 6 / Part 3 зводиться до відсутності потреби у встановленні додаткового програмного забезпечення, а доступ до серверу здійснюється, як правило, через мережу Інтернет. Окрім цього, вона орієнтована на роботу через WEB-доступ, що є актуальним для організацій з територіально-розподіленою організаційною структурою. Окрім електронного цифрового підпису, система передбачає можливість штрих-кодування документів, що суттєво мінімізує вплив «людського фактора» при обробці документації та зменшує навантаження на співробітників канцелярії. зводиться до відсутності потреби у встановленні додаткового програмного забезпечення, а доступ до серверу здійснюється, як правило, через мережу Інтернет. Окрім цього, вона орієнтована на роботу через WEB-доступ, що є актуальним для організацій з територіально-розподіленою організаційною структурою. Окрім електронного цифрового підпису, система передбачає можливість штрих-кодування документів, що суттєво мінімізує вплив «людського фактора» при обробці документації та зменшує навантаження на співробітників канцелярії. З практичної точки зору впровадження АСКОД доречно в тих органах державної влади, де левова частка роботи припадає на надання адміністративних послуг (відділи державної реєстрації актів цивільного стану, Державна служба України з безпеки на транспорті, Державна служба морського та річкового транспорту України, митна та податкова служби тощо). Це зумовлюється функціональними можливостями ідентифікації та обробки документів, що потрібні для надання адміністративних послуг: система визначає шаблон документу, вид послуги, перелік документації, яка необхідна для її отримання. З практичної точки зору впровадження АСКОД доречно в тих органах державної влади, де левова частка роботи припадає на надання адміністративних послуг (відділи державної реєстрації актів цивільного стану, Державна служба України з безпеки на транспорті, Державна служба морського та річкового транспорту України, митна та податкова служби тощо). Це зумовлюється функціональними можливостями ідентифікації та обробки документів, що потрібні для надання адміністративних послуг: система визначає шаблон документу, вид послуги, перелік документації, яка необхідна для її отримання. За основу електронного документообігу в таких установах, як Державна регуляторна служба, Державна прикордонна служба, Міністерство охорони здоров’я, Національне агентство запобіганню корупції, Державна екологічна інспекція було обрану іншу платформу – ДОК ПРОФ 2.0. Реєстрація документації в системі, зазвичай, здійснюється шляхом проставляння штрих- коду у відповідності із номенклатурою справи. ДОК ПРОФ 2.0 дає змогу здійснювати контроль виконання, зокрема система здатна інформувати (нагадувати) про необхідність здійснення певної дії з документом за три дні до дедлайну. Програма передбачає підготовку документів у форматі Microsoft Word або OpenOffice.org із застосуванням визначеного шаблону. На нашу думку, це формує не лише єдиний підхід до підготовки документації, але й дисциплінує виконавців, підвищує рівень їх корпоративної культури. Основний текст Запровадження системи електронного документообігу та платформ, які дозволяють вирішувати задачі організації різноманітних інформаційних потоків у комплексі, є нагальною потребою організацій сучасного типу. Усунення застарілих методів обробки інформації та кореспонденції, оцифровування www.sworldjournal.com ISSN 2663-5712 75 Issue 6 / Part 3 SWorldJournal Issue 6 / Part 3 відомостей та даних, автоматизація процесів в державно-управлінській діяльності – це ключові завдання, які поставлені перед органами публічного управління в частині оптимізації комунікаційних процесів. Більше того, розвиток електронного документообігу та е-взаємодії є частиною Концепції розвитку електронного урядування в Україні, схваленої розпорядженням КМУ від 20.09.2017 № 649-р [3]. р [ ] Ключову роль на рівні кожного окремого органу публічного управління відіграє система внутрішніх комунікацій. У роботі публічних органів розрізняють потоки вхідної документації (надходять від суб’єктів господарювання, громадян, інших органів державної влади і в подальшому скеровують до структурних підрозділів після резолюції керівника); вихідної (розроблені установою та надсилаються адресатам, що не входять до складу цього органу); внутрішньої (документи установи чи організації, які циркулюють виключно в її межах). Кожна документальна одиниця рухається у власному потоці, розробляється і маршрутизується різними суб’єктами (користувачами). Спільна електронна платформа дає змогу (за наявності доступу та дозволу) відслідковувати рух документів та зв’язки, що встановлюються в рамках документообігу. Система електронного документообігу покликана здійснювати централізоване управління документацією (оперативна заміна чи модифікація форми документу, контроль стану та термінів виконання тощо); маршрутизацію (автоматична передача документу на розгляд та візування); забезпечувати колективну роботу та розмежування прав доступу. Крім того, система електронного документообігу спроможна забезпечити інтеграцію із іншими електронними корпоративним системами, в тому числі бухгалтерськими, аналітичними та фінансовими [4, с.33-34]. Найпростіша система електронного документообігу в державно-управлінському секторі має бути адаптованою до специфіки роботи органу публічного управління та реалізовувати звичний алгоритм документообігу; підтримувати функції розсилки та візування; передбачати можливість зовнішнього контролю та бути придатною до інтегрування з іншими системами. Розглянемо більш детально специфіку роботи із деякими системами електронного документообігу, які наразі застосовують органи публічного управління. З найбільш поширених виділяємо АСКОД (розробник – ІнфоПлюс), ДОК ПРОФ 2.0 (СВІТ Проф), OPTIMA-WorkFlow (Optima), Док Проф (Ситроникс), MasterDOC (Банкомзв’язок), Megapolis. Документообіг (Софтлайн). АСКОД – це система електронного документообігу, яка завдяки своїм функціонально-технічним можливостям дозволяє нагромаджувати та систематизувати інформацію, що початково являється неструктурованою, підтримувати ділові процеси, котрі орієнтовані на роботу із документацією, та аналізувати відомості в частині документообігу [5]. Наразі зазначену систему експлуатує багато міських та районних рад, ДСНС, Державна архівна служба України, Український центр оцінювання якості освіти, Федерація профспілок України тощо. ISSN 2663-5712 www.sworldjournal.com Основний текст Централізоване збереження документації відбувається у стислому форматі, а виконавці за потереби можуть залишити текстову копію документу на своїх ПК. Через систему можна надсилати пропозиції співвиконавцям, службові записки, залишати коментарі та зауваження. Тестуючи дану систему ми відзначили ґрунтовно продуманий моніторинг процесів та формування статистичних звітів. Так, дозволяється відслідковувати, порівнювати чи узагальнювати дані в режимі реального часу. Окрім цього, розробник здійснив кілька вдалих технологічних рішень, що доречні і в роботі державних та муніципальних органів, а саме заповнення полів форми оптичним олівцем, графічну форму представлення даних, сканування складних документів. Окремо зупинимося на системах електронного документообігу для тих державних установ, які працюють за принципом «єдиного вікна», тобто передбачають можливість централізованої подачі усього пакету необхідних документів для оформлення дозвільних документів одній посадовій особі. За таким принципом працюють, наприклад, центри надання адміністративних www.sworldjournal.com www.sworldjournal.com ISSN 2663-5712 77 Issue 6 / Part 3 SWorldJournal Issue 6 / Part 3 послуг (ЦНАПи). В комунікативних системах ЦНАПів циркулює значний обсяг документів та іншої інформації, що створюється та обробляється у процесі надання адміністративних послуг населенню чи юридичним особам, тому їх зручність та функціональність має надзвичайно важливе значення. послуг (ЦНАПи). В комунікативних системах ЦНАПів циркулює значний обсяг документів та іншої інформації, що створюється та обробляється у процесі надання адміністративних послуг населенню чи юридичним особам, тому їх зручність та функціональність має надзвичайно важливе значення. Багато ЦНАПів використовують систему FossDoc з модулем ЦНАП [6], який не лише спрямований на автоматизацію діловодства, але й функціонує як корпоративний портал. Переваги, які отримує ЦНАП у системі зовнішніх та внутрішніх комунікації із застосуванням даної системи, полягають, зокрема, у суттєвому скороченні часу при оформлення заявок від суб’єктів, котрі вже були попередньо зареєстровані в системі (користувалися послугами ЦНАП); можливості автоматично заповнення, здійснення пошуку та друку всіх необхідних форм; моментальній обробці заяв; здійсненні обліку повторних звернень громадян; наявності потужного багатокритеріального пошукового сервісу; обліку роботи реєстраторів, що допомагає об’єктивно оцінювати її результати їх роботи. Аналіз вітчизняних систем електронного документообігу дає змогу виділити їх особливість – відсутність вільного програмного забезпечення. Співставляючи їх із закордонними програмними продуктами, відзначаємо цілком протилежну ситуацію: широке використання інформаційно- комунікативних платформ на базі відкритого вихідного коду. Одним із таких рішень, впроваджених у органах публічного управління інших держав, є ЕСМ Alfresco – програмне забезпечення для управління контентом організації. Воно дає змогу користувачам ефективно і оперативно управляти корпоративним інформаційним контентом. ISSN 2663-5712 www.sworldjournal.com Основний текст Широкі функціональні можливості передбачають підтримку електронного документообігу, можливість управляти проєктами, оптимізувати окремі державно-управлінські процеси, вести електронний архів, публікувати відомості в мережах, взаємодіяти як із колегами, так і зі спеціалістами інших органів публічної влади. Відкритий код, гнучкість, модульність платформи, широкий спектр інтеграцій та додаткових модулів дозволяють оптимізувати роботу платформи під запити і потреби конкретної організації. Відзначимо, що додаткові функції та підтримка у роботі із програмою притаманні комерційній системі Alfresco Content Service (на відміну від безкоштовної версії Alfresco Community Edition). До числа користувачів цього програмного забезпечення належать Брістольська міська рада та поліція Хемпшира (Англія), департамент Жиронда, Спільнота Агломерації Монпельє та Бюджетна дирекція (Франція), Муніципалітет Керетаро (Мексика), уряд Нідерландів, Федеральна авіаційна адміністрація та Департамент охорони здоров’я Говарду (США), а також багато інших організацій публічної сфери по всьому світу [7]. Розглянемо детальніше специфіку застосування ЕСМ Alfresco у зарубіжних органах публічної влади та державних організаціях. Так, комунікативну систему Alfresco використовує науково-дослідний центр NASA в Ленглі. Цей Центр надає послуги із структурного, аероакустичного та аеронавігаційного тестування, дослідні послуги для своїх департаментів, комерційних організацій та органів публічної влади. www.sworldjournal.com ISSN 2663-5712 ISSN 2663-5712 78 Issue 6 / Part 3 Issue 6 / Part 3 SWorldJournal Issue 6 / Part 3 Прийнявши рішення про недоцільність подальшої експлуатації попередньої програми aeroCOMPASS через застарілу систему захисту інформації та складнощі, які виникали при роботі з новими форматами цифрової документації, NASA дослідило суттєву кількість цифрових платформ, що забезпечують як роботу із документацією, так і корпоративне спілкування, та зупинилися на програмі Alfresco . На їх думку, у порівнянні з аналогічними системами Alfresco мало ряд переваг, зокрема: − відкритість та цифрова безпечність електронної системи для колективної роботи спеціалістів державної науково-дослідної організації; − відкритість та цифрова безпечність електронної системи для колективної роботи спеціалістів державної науково-дослідної організації; − розширений функціонал, який зручно використовувати в колективній роботі; − розширений функціонал, який зручно використовувати в колективній роботі; − зрозуміла візуалізація, легка для сприйняття система налаштувань, відсутність суттєвих труднощів в усуненні технічних помилок; − можливість перенесення усіх попередньо накопичених документів та сайтів у систему; − можливість перенесення усіх попередньо накопичених документів та сайтів у систему; − наявність доступу до корпоративного контенту більше ніж 3 000 працівників [7]. − наявність доступу до корпоративного контенту більше ніж 3 000 працівників [7]. Американська асоціація страхових послуг, яка також належить до державних структур, активно користується у свої роботі контент-послугами, що надаються ЕСМ Alfresco, і пакетом OpenContent Management Suite, що дає змогу покращити корпоративну роботу із формами полісів й іншими видами страхових документів. ISSN 2663-5712 Основний текст Система дозволяє Американській асоціації страхових послуг швидко реагувати на нормативні і законодавчі зміни. Інтеграція з Workshare Compare дозволяє динамічне внесення змін до документів. Ще одним органом, який використовує Alfresco у своїй діяльності, є Департамент охорони здоров’я округу Говард, Колумбія [7]. Як державна установа, він керується стандартами та нормами, які вимагають зберігати офіційні документи протягом багатьох років, а іноді довічно. Це призвело до того, що багато документів зберігалося поза межами Департаменту. Для того, щоб знайти певну інформацію, співробітники повинні були прийти у сховище та вручну шукати потрібний файл. Співробітники Департаменту охорони здоров’я створювали документи локально або на спільних дисках, а положення безпеки на цих дисках не дозволяли іншим знаходити необхідні документи. Як результат, департаменти не могли самостійно обмінюватися даними або надавати доступ для спільної роботи із документами, не звертаючись за допомогою до ІТ-відділу. Кілька років тому департамент почав шукати способи оцифрування всіх своїх даних. Потрібна була основа для створення документів, а також спосіб управління вже наявним масивом документів (в тому числі сканованими копіями), централізованому в одному місці. Агентству потрібно було гнучке, нестандартне рішення із наявними полями метаданих, для налаштування яких не було потрібно багато часу. Оцінивши кілька рішень, агентство обрало ЕСМ Alfresco як рішення для управління документами через відкриту архітектуру. Залучивши партнерські мікро-стратегії Alfresco, вони також скористалося перевагами інтеграційного www.sworldjournal.com www.sworldjournal.com 79 Issue 6 / Part 3 Issue 6 / Part 3 SWorldJournal Issue 6 / Part 3 рішення Microsoft Office, яке поєднує простоту використання додатків Microsoft Office та потужність Alfresco, щоб забезпечити агентство необхідними інструментами для захисту конфіденційних документів. Агентства транспорту, громадської інформації, політики охорони здоров'я та фіскальний департамент мали трохи пізніше також перейти на використання Alfresco. Наразі всі документи та електронні листи, передаються або створюються працівниками в межах Alfresco. Щомісяця створюється від 200 до 300 сторінок вмісту, які архівуються у Alfresco. Це важливе рішення для Говарду, яке використовується щоденно у відповідних адміністративно- управлінських процесах. Ще один приклад – Брістольська міська рада у Великобританії, де впровадження в роботу системи Alfresco стало наслідком скорочення фінансування держустанови. Раціоналізація роботи в міській раді та функціонування Alfresco дало змогу автоматизувати опрацювання електронних документів; забезпечити обмін даними як на рівні міської ради, так із іншими органами публічної влади; модернізувати методику взаємодії з персоналом (в тому числі в умовах дистанційної роботи, що стало особливо корисним в умовах пандемії COVID-2019); поширити використання цифрової звітності. ISSN 2663-5712 ISSN 2663-5712 Основний текст В свою чергу, у Голландії більше двох десятків років функціонує єдина система для обміну електронною документацією – Government Transaction Portal, що забезпечує взаємодію працівників як в межах одного, так і з усіма органами публічного управління. Ця система має два інтерфейси: один – доступний виключно для громадян та здебільшого стосується надання адміністративних послуг / консультацій / роз’яснень фізичними особами та бізнесу; інший – забезпечує корпоративну взаємодію уповноважених на здійснення державно-управлінських завдань членів колективу. Що стосується використання комунікаційних мереж, які дозволяють взаємодіяти різним органам державної влади, то слід згадати National Computer Board, котра функціонує в Угорщині. Ще з 2008 року вона поєднує всі відділи, управління, відомства, які є структурними підрозділами Міністерства оборони та Міністерства внутрішніх справ. За допомогою цієї цифрової платформи в режимі on-line правоохоронні органи можуть швидко комунікувати між собою, з цивільними органами управління і системою охорони здоров’я. Наведені приклади засвідчують переваги використання подібних рішень на основі вільного програмного забезпечення та доцільність їх впровадження в органах публічного управління та адміністрування в Україні. Українськими державними організаціями також використовуються комунікаційні платформи змішаного типу, інтерфейс яких дозволяє обмінюватися інформацією як між різними територіальними відомствами чи підрозділами одного державного органу, так і здійснювати зовнішню комунікацію, представляючи певну інформацію у формі реєстру чи відкритих даних. До таких платформ відносять різні портали та сервіси, що знаходяться у розпорядженні окремих служб, зокрема, Інспекційний портал (державний нагляд та контроль в частині безпеки праці), електронна система Добровільної реєстрації українських громадян при подорожах за кордон («ДРУГ»), система www.sworldjournal.com www.sworldjournal.com 80 Issue 6 / Part 3 SWorldJournal Issue 6 / Part 3 «Лікар онлайн» тощо. «Лікар онлайн» тощо. «Лікар онлайн» тощо. «Лікар онлайн» тощо. До переліку потужних елементів у системі державно-управлінських корпоративних комунікацій відносять і корпоративні соціальні мережі, які розглядаються як комунікативні засоби, діяльність яких забезпечується за рахунок мережі Інтернет. Корпоративні сайти зазвичай використовуються центральними органами влади і досить рідко – органами місцевого самоврядування. Для взаємодії вони частіше застосовують Facebook, так як він дозволяє створювати та вести персональні профілі, тематичні сторінки та групи, анонсувати певні заходи, коментувати події, публікувати результати. Майже кожне міністерство і більшість органів виконавчої влади мають сторінки у Facebook, у більшості також є Twitter, чотири Міністерства публікують тематичні відео на YouTube. Більш простою альтернативою корпоративних комунікацій є електронна пошта. У цьому контексті Google має значну кількість переваг, зокрема безкоштовність, здатність до інтеграції із іншими платформами, систематичне оновлення та україномовний інтерфейс. Корисними для налагодження зручного робочого середовища є вбудована система орфографічної перевірки, підтримка різних мов, фільтрація спаму та пошук відповідної кореспонденції за її змістом. Наявність акаунту в Google дає змогу скористуватися іншими популярними додатками від Google (календар, карти, диск, чати, перекладач, форми та інші) та налагодити колективну роботу з документами, що є важливим для публічного управління та адміністрування. Зокрема, гугл-форми активно використовуються для налагодження як внутрішніх, так і зовнішніх комунікацій, адже дають змогу проводити анкетування чи анонімне опитування для будь-якого кола осіб (співробітників чи серед зовнішньої аудиторії) швидко систематизувати та аналізувати інформацію. Ще одним популярним інструментом, який використовується радше у неформальних комунікаціях, являються месенджери, зокрема, Viber, Telegram, WhatsApp, Skype, WeChat, Imo.im, Discord. Три перші платформи, які є найбільш популярними, дозволяють формувати групи та окремі чати, долучати до вже існуючих груп інших членів колективу. Обмін, як правило, здійснюється у текстовому форматі, але передбачена можливість надсилання аудіоповідомлень та відеозаписів. Відмінність ділового спілкування у месенджерах від комунікацій в усному форматі чи через електронну пошту, зазвичай полягає в тому, що повідомлення носять фрагментарний характер. Схема корпоративного спілкування у месенджерах носить характер такої комунікативної техніки, як «мозковий штурм», що практикується в усному спілкуванні. Прикладом платформ, що дозволяють реалізувати взаємодію фахівців публічного сектору, також можна назвати мобільні додатки, що, як правило, застосовують у соціальних сферах. Так, «FixMyStreet» – це проект англійського фонду «UK Citizens Online Democracy», що дає змогу громадянам повідомити місцеву владу про негаразди на дорогах (неякісне покриття, сміття, розбиті ліхтарі тощо), а останнім – передати фахівцям в роботу отримане повідомлення. Цей досвід згодом перейняла Канада, Німеччина та США. «Лікар онлайн» тощо. www.sworldjournal.com ISSN 2663-5712 81 Issue 6 / Part 3 Issue 6 / Part 3 SWorldJournal Issue 6 / Part 3 Issue 6 / Part 3 Розвантаження системи комунікацій в країнах ЄС та США здійснюється за рахунок роботи так званих «гонгів», які працюють при міністерствах та центральних органах виконавчої влади. За своєю організаційно-правовою формою «гонги» є громадськими організаціями, котрі приналежні до державних установ, але в той же час – є незалежними від політичного істеблішменту з точки зору підпорядкованості чи керівництва. Йдеться про Бюро консультації громадян у Великій Британії, Чехії, Польщі та Литві; Центр волонтерства у Данії (діє при міністерстві, яке опікується питаннями соціальної політики) тощо. Їх робота спрямована на надання оперативного консультування щодо заповнення різних форм, заявок, потрібних для отримання адміністративних послуг; вони взаємодіють із громадянами і тоді, коли останнім необхідна допомога у веденні переговорів; долучаються до врегулювання проблемних питань в сфері зайнятості та міграції. Іншими словами, діяльність цих бюро дає змогу скоротити час спілкування працівників публічного органу та громадянина за рахунок попередньої «підготовки» громадянина. 6. Автоматизація ЦНАП та ВДР. URL: https://fossdoc.com/tsnap (дата звернення: 02.12.2020). 6. Автоматизація ЦНАП та ВДР. URL: https://fossdoc.com/tsnap (дата звернення: 02.12.2020). 7. Alfresco: Customers Stories. URL: https://www.alfresco.com/customers (дата звернення: 02.12.2020). 7. Alfresco: Customers Stories. URL: https://www.alfresco.com/customers (дата звернення: 02.12.2020). References 1. Dreshpak V.M., 2015. Komunikatsiyi v publichnomu upravlinni [Communications in public administration]. Dnipropetrovsk: DRIDU NADU. 1. Dreshpak V.M., 2015. Komunikatsiyi v publichnomu upravlinni [Communications in public administration]. Dnipropetrovsk: DRIDU NADU. 1. Dreshpak V.M., 2015. Komunikatsiyi v publichnomu upravlinni [Communications in public administration]. Dnipropetrovsk: DRIDU NADU. 2. Arkhypova Ye. O. and Dmytrenko N.O., 2016, Effective communication between public authorities and the public as a condition of the development of a democratic society. Investytsiyi: praktyka ta dosvid, no.1, pp. 92-96. 2. Arkhypova Ye. O. and Dmytrenko N.O., 2016, Effective communication between public authorities and the public as a condition of the development of a democratic society. Investytsiyi: praktyka ta dosvid, no.1, pp. 92-96. 3. The concept of e-government development in Ukraine, 2017. Available at: https://zakon.rada.gov.ua/laws/show/649-2017-р#Text [Accessed 02.12.2020]. 3. The concept of e-government development in Ukraine, 2017. Available at: https://zakon.rada.gov.ua/laws/show/649-2017-р#Text [Accessed 02.12.2020]. 4. Belska T.V., 2019. Dokumentoobih v publichnomu upravlinni [Document circulation in public administration]. Kharkiv: KhNUMG. 4. Belska T.V., 2019. Dokumentoobih v publichnomu upravlinni [Document circulation in public administration]. Kharkiv: KhNUMG. 5. ASKOD products, 2020. Available at: https://infoplus.ua/uk/page/products [Accessed 02.12.2020]. 5. ASKOD products, 2020. Available at: https://infoplus.ua/uk/page/products [Accessed 02.12.2020]. 6. Automation of CNAP and VDR, 2020. Available at: https://fossdoc.com/tsnap [Accessed 02.12.2020]. 6. Automation of CNAP and VDR, 2020. Available at: https://fossdoc.com/tsnap [Accessed 02.12.2020]. 7. Alfresco: Customers Stories, 2020. Available at: https://www.alfresco.com/customers [Accessed 02.12.2020]. р Висновки В статті були розглянуті сучасні тенденції формування системи корпоративних комунікацій в органах публічної влади. Показано, що вітчизняні та зарубіжні органи публічного управління та адміністрування використовують різноманітні комунікативні інструменти. Окрім основних формальних комунікативних каналів, визначених нормативно-правовими актами або регламентованих на рівні конкретного органу публічної влади активно використовуються і неформальні інструменти комунікації. Одним з основних механізмів, який дозволяє налагодити та оптимізувати внутрішні інформаційні взаємодії є системи електронного документообігу. Відзначено, що комплексне програмне забезпечення на основі відкритого коду має значно ширші можливості для управління контентом організації завдяки модульності та можливості інтегрування додаткових програм, функцій і розширень. Література: р ур 1. Дрешпак В.М. Комунікації в публічному управлінні. Д.: ДРІДУ НАДУ, 2015. 168с. 1. Дрешпак В.М. Комунікації в публічному управлінні. Д.: ДРІДУ НАДУ, 2015. 168с. 2. Архипова Є.О., Дмитренко Н.О. Ефективна комунікація між органами державної влади та громадськістю як умова розвитку демократичного суспільства. Інвестиції: практика та досвід. 2016. №1. С. 92-96. 2. Архипова Є.О., Дмитренко Н.О. Ефективна комунікація між органами державної влади та громадськістю як умова розвитку демократичного суспільства. Інвестиції: практика та досвід. 2016. №1. С. 92-96. у р 3. Концепція розвитку електронного урядування в Україні, схвалена розпорядженням Кабінету Міністрів України від 20.09.2017. № 649-р. URL: https://zakon.rada.gov.ua/laws/show/649-2017-р#Text (дата звернення: 02.12.2020). 4. Бєльська Т. В. Документообіг в публічному управлінні. Харків. нац. ун-т міськ. госп-ва ім. О.М. Бекетова. Харків: ХНУМГ ім. О. М. Бекетова, 2019. 40 с. 5. Продукти АСКОД. URL: https://infoplus.ua/uk/page/products (дата звернення: 02.12.2020). www.sworldjournal.com ISSN 2663-5712 ISSN 2663-5712 82 SWorldJournal Issue 6 / Part 3 Abstract. Abstract. Introduction. An efficient and balanced system of internal and external communications is one of the necessary prerequisites for the quality realization of its functions by the organizations of all types and forms. The aim of this paper is to consider the modern trends in the formation of the system of corporate communications in public authorities. Main part. The experience of using of electronic document management system and content management systems in the public administration sector of Ukraine and other countries is analyzed. Emphasis is placed on the requirements for electronic document management systems for government agencies that operate on the principle of "single window". The advantages that the use of free enterprise content management software to state bodies are determined. It is noted that complex software based on open source has much more opportunities for content management of the organization due to the modularity and the ability to integrate additional programs, functions and extensions. The practice of using other communicative tools by public bodies is outlined: corporate sites, social networks, messengers. Conclusion. It is shown that domestic and foreign public administration bodies use various communication tools. In addition to the main formal communication channels defined by regulations or regulated at the level of a particular public authority, informal communication tools are also actively used. Keywords: communication, government bodies, public authorities, electronic document management system, content management, website, messenger. ISSN 2663-5712 www.sworldjournal.com www.sworldjournal.com 83
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Distinctive Profile of IsomiR Expression and Novel MicroRNAs in Rat Heart Left Ventricle
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Received November 13, 2012; Accepted May 3, 2013; Published June 14, 2013 Copyright:  2013 McGahon 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 research was partly supported by a British Heart Foundation (BHF; http://www.bhf.org.uk) grant to AC (PI) and DAS (Co-I) (PG110/039/28365); and partly supported by a Biotechnology and Biological Sciences Research Council (BBSRC; http://www.bbsrc.ac.uk) grant to DAS (BB/H005498/1). The Trinity Genome Sequencing Laboratory is a core facility funded by Science Foundation Ireland and supported by the Trinity Centre for High Performance Computing. 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: anthony.collins@qub.ac.uk Analysis of sequencing-based miRNA expression data has revealed that individual miRNAs exist not just as a single canonical sequence but exhibit a range of sequence variants or ‘isomiRs’ [52–55]. These isomiRs can have additions or trunca- tions at their 59 or 39 end, or internal single-base differences. The variation between isomiRs occurs during biogenesis due to cleavage by the RNaseIII enzymes Dicer and/or Drosha at alternative sites or post-transcriptional editing. The availability of the rat whole genome sequence makes it possible to differentiate isomiRs of a single microRNA from closely related microRNAs with very similar sequences that originate from separate miRNA genes, designated ‘a’, ‘b’, etc. (for some microRNAs several genes exist which result in the same mature sequence and in this case it is not possible to determine whether specific isomiRs are formed preferentially from one gene). The distribution of isomiRs indicates that they are not randomly generated, and may have (patho)physiological importance [56–58]. Indeed, specific isomiRs have been shown to be functional and to have target gene repertoires that differ from those of their corresponding canonical Mary K. McGahon, Janet M. Yarham, Aideen Daly, Jasenka Guduric-Fuchs, Lyndsey J. Ferguson, David A. Simpson, Anthony Collins* Mary K. McGahon, Janet M. Yarham, Aideen Daly, Jasenka Guduric-Fuchs, Lyndsey J. Ferguson, David A. Simpson, Anthony Collins* Centre for Vision and Vascular Science, Queen’s University Belfast, Belfast, County Antrim, United Kingdom Centre for Vision and Vascular Science, Queen’s University Belfast, Belfast, County Antrim, United Kingdom Abstract MicroRNAs (miRNAs) are single-stranded non-coding RNAs that negatively regulate target gene expression through mRNA cleavage or translational repression. There is mounting evidence that they play critical roles in heart disease. The expression of known miRNAs in the heart has been studied at length by microarray and quantitative PCR but it is becoming evident that microRNA isoforms (isomiRs) are potentially physiologically important. It is well known that left ventricular (patho)physiology is influenced by transmural heterogeneity of cardiomyocyte phenotype, and this likely reflects underlying heterogeneity of gene expression. Given the significant role of miRNAs in regulating gene expression, knowledge of how the miRNA profile varies across the ventricular wall will be crucial to better understand the mechanisms governing transmural physiological heterogeneity. To determinine miRNA/isomiR expression profiles in the rat heart we investigated tissue from different locations across the left ventricular wall using deep sequencing. We detected significant quantities of 145 known rat miRNAs and 68 potential novel orthologs of known miRNAs, in mature, mature* and isomiR formation. Many isomiRs were detected at a higher frequency than their canonical sequence in miRBase and have different predicted targets. The most common miR-133a isomiR was more effective at targeting a construct containing a sequence from the gelsolin gene than was canonical miR-133a, as determined by dual-fluorescence assay. We identified a novel rat miR-1 homolog from a second miR-1 gene; and a novel rat miRNA similar to miR-676. We also cloned and sequenced the rat miR-486 gene which is not in miRBase (v18). Signalling pathways predicted to be targeted by the most highly detected miRNAs include Ubiquitin-mediated Proteolysis, Mitogen-Activated Protein Kinase, Regulation of Actin Cytoskeleton, Wnt signalling, Calcium Signalling, Gap junctions and Arrhythmogenic Right Ventricular Cardiomyopathy. Most miRNAs are not expressed in a gradient across the ventricular wall, with exceptions including miR-10b, miR-21, miR-99b and miR-486. Citation: McGahon MK, Yarham JM, Daly A, Guduric-Fuchs J, Ferguson LJ, et al. (2013) Distinctive Profile of IsomiR Expression and Novel MicroRNAs in Rat Heart Left Ventricle. PLoS ONE 8(6): e65809. doi:10.1371/journal.pone.0065809 Editor: Fabio Martelli, IRCCS-Policlinico San Donato, Italy Received November 13, 2012; Accepted May 3, 2013; Published June 14, 2013 Introduction MicroRNAs (miRNAs) are single-stranded non-coding RNAs consisting of 18–25 nucleotides (nts) that negatively regulate target gene expression through mRNA cleavage or translational repres- sion [1]. In recent years miRNAs have been implicated in the control of expression of many proteins in development and pathology. In particular there is mounting evidence that they play critical roles in diseases of the heart [2–4], and they have great potential as circulating biomarkers of cardiac damage [5]. The rat heart has provided a useful model for the study of heart physiology and disease for many years, and this utility has been extended to investigations into the role of miRNAs. Numerous recent studies have used the rat heart and rat heart cells to demonstrate the involvement of miRNAs in ischaemia [6–20], hypertrophy [21–29], heart failure [30–34], fibrosis [35–38], apoptosis [6,10,17,39–43], metabolism [44,45], angiogenesis [11,46–48], drug action [11,15,16,49–51] and arrhythmogenesis [35,51]. 1 PLOS ONE | www.plosone.org June 2013 | Volume 8 | Issue 6 | e65809 MicroRNAs in Rat Heart Left Ventricle ligated with adapters, reverse transcribed into cDNA and Figure 1. Length distribution analysis of small RNA sequences in 3 mid-myocardial rat samples. Reads greater than 12 nt are included. Red bars represent reads annotated to known rat miRNAs (miRBase v18), green bars represent reads annotated to miRNAs in other species (novel orthologs) and black bars represent unannotated small RNA sequences. doi:10.1371/journal.pone.0065809.g001 MicroRNAs in Rat Heart Left Ventricle miRNAs [59,60]. Recent data suggests the possibility of isomiR- specific gene targeting in the heart [61,62]. The vast majority of myocardial miRNA expression profiling studies to date have used microarray technologies or quantitative PCR (qPCR). Although much useful data has emerged from these studies, these techniques, like all methodological approaches, have limitations. Microarrays and qPCR assays are designed to detect and quantify molecular species that are already known to exist; novel sequences are not detected. IsomiRs would either be indistinguishable from the canonical sequence or not detected at all. The former is likely in microarray-based methods while the latter could apply in qPCR, depending on the type of isomiR. In contrast to the aforementioned methods ‘next-generation’ or ‘deep’ sequencing is able to detect and quantify novel sequence variants as well as those already known. Introduction We therefore used next- generation sequencing to characterise the expression profile of miRNAs in rat heart left ventricular wall, identify novel miRNAs and novel orthologs not previously characterised in this species and determine the relative expression of isomiRs of individual miRNAs. To our knowledge this is the first report of miRNA deep sequencing data from rat heart using the Illumina platform. We detected several novel rat miRNAs and multiple novel isomiRs of known miRNAs, many of which were detected at a higher frequency than the corresponding mature sequence recorded in miRBase [63,64]. The well established heterogeneity of cardiomyocyte physiology across the left ventricular wall influences both normal heart function and pathophysiology [65,66] and likely reflects underly- ing heterogeneous gene expression. Given the significant role of miRNAs in regulating gene expression, knowledge of how the miRNA profile varies across the ventricular wall will be crucial to understand the mechanisms governing transmural physiological heterogeneity. Data describing transmural miRNA expression is very limited and we therefore undertook the first comprehensive determination of miRNA expression profiles at different locations across the left ventricular wall. We have found that most miRNAs are not expressed in a gradient across the ventricular wall, with the exceptions of miR-10b, miR-21, miR-99b and miR-486. Ethics Statement This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health; and the United Kingdom Animals (Scientific Procedures) Act, 1986. The protocol was approved by the Animal Research Ethics Committee of Queen’s University Belfast (Project Licence: PPL 2683). Figure 1. Length distribution analysis of small RNA sequences in 3 mid-myocardial rat samples. Reads greater than 12 nt are included. Red bars represent reads annotated to known rat miRNAs (miRBase v18), green bars represent reads annotated to miRNAs in other species (novel orthologs) and black bars represent unannotated small RNA sequences. doi:10.1371/journal.pone.0065809.g001 June 2013 | Volume 8 | Issue 6 | e65809 miRNA Library Preparation The hearts of 3 male 8 month old Sprague-Dawley rats were rapidly extracted after euthanasia with sodium pentobarbital. A section of the free wall of the left ventricle was dissected into epicardium, mid-myocardium and endocardium by cutting approximately 1 mm from the epicardial and endocardial surfaces. Tissue was weighed, chopped, stored in RNAlater RNA stabilisation reagent (Qiagen, Crawley UK) and then flash frozen in liquid nitrogen before placing in storage at 280uC. Small RNA was extracted (miRNeasy Kit; Qiagen), quantified (Nano- drop; Thermo Scientific) and quality assessed for degradation (RNA Nano Chip, Bioanalyser 2100; Aligent Technologies, Wokingham UK; only samples with a RNA integrity no. (RIN) $8 were carried forward) and retention of small RNA (Small RNA Chip, Bioanalyser 2100). Small RNA was preferentially ligated with adapters, reverse transcribed into cDNA and amplified with 9 individually tagged primer indices (TruSeq Small RNA Sample Preparation Kit; Illumina, Little Chesterford, UK) and a library of small RNA created for each sample. After gel purification the cDNA products were again analysed on the bioanalyser using a High Sensitivity DNA Chip and assessed for the presence and concentration of the peak corresponding to ligated and tagged miRNA (approximately 147 nt). It was PLOS ONE | www.plosone.org June 2013 | Volume 8 | Issue 6 | e65809 2 Figure 2. miRNA detection frequency. Most highly detected miRNAs grouped on mature sequence, normalised to total annotated reads (expressed in reads per million mapped (RPMM) from 3 rat mid-myocardial samples). doi:10.1371/journal.pone.0065809.g002 MicroRNAs in Rat Heart Left Ventricle Figure 2. miRNA detection frequency. Most highly detected miRNAs grouped on mature sequence, normalised to total annotated reads (expressed in reads per million mapped (RPMM) from 3 rat mid-myocardial samples). doi:10.1371/journal.pone.0065809.g002 PhusionH Hot Start II High-Fidelity DNA Polymerase (see Table S1 for primer sequences). observed that the successful presence of an appropriate end product was not wholly dependent on the integrity of the initial RNA sample (RIN; See Fig. S1) but could be better predicted from the presence or relative absence of a range of small RNA species (tRNA, 5S and miRNA) assessed on the Small RNA Chip electropherogram; therefore only samples with suitable RIN values exhibiting good retention of small RNA species were used for library preparation. After pooling, the samples were sequenced by TrinSeq (Trinity Genome Sequencing Lab & Neuropsychiatric Genetics Group, Trinity College Dublin, Ireland (http://www. Dual Fluorescence miRNA Targeting Assay y This assay has been described in detail previously [70]. HEK293 cells were plated into a 24-well plate. The following day, cells were transiently transfected with pmChGSN, in addition to one of pSM30miR-133a, pSM30miR-133a(v), pSM30NTC or pSM30siR-mCh using Turbofect Transfection reagent (Thermo Scientific, Hemel Hempstead, UK). Cells were imaged at 20X magnification using a Nikon Eclipse TE2000-U microscope equipped with a 488 nm filter (red; mCherry fluorescence) and a 550 nm filter (green; EGFP fluorescence). Red and green images were acquired of multiple fields using NIS Elements software (Nikon, Badhoevedorp, Netherlands). Cross-channel bleed- through was found to be negligible by acquiring red images of cells transfected with pSM30miR-133a only, and green images of cells transfected with pmChGSN only. These images were then used for subtraction of background fluorescence. Images in tiff format were analysed by Volocity software (Volocity 5.5.1, PerkinElmer Inc., Waltham, MA, USA). We developed a Volocity Quantitative PCR was carried out using Taqman small RNA assays and TaqMan gene expression assays (Invitrogen – Life Technologies Ltd, Paisley, UK) on RNA samples from rat hearts dissected and extracted as detailed above. REST 2009 software [67,68] was used to determine the expression ratio between epicardial and endocardial samples using multiple endogenous controls which were tested for consistent expression across samples using BestKeeper [69]. PCR Genomic DNA was extracted from rat heart (Direct PCR lysis reagent; Viagen Biotech Inc, Los Angeles, CA, USA) to form the template for confirmation of the stem loop sequence for pre-miR- 486 in a PCR reaction using primers (Table S1) designed from a rat genomic DNA sequence (gnl|ti|1621823597; gnl|ti|1718641223) not present in the most recent build of the rat genome (Baylor 3.4), and DreamTaq polymerase (Fermentas). Cycling parameters were: 95uC for 3 min, 356 (95uC for 30 s, 49uC for 30 s, 72uC for 20 s), 72uC for 15 min. Amplified products were sequenced (Genomics Core, Queen’s University Belfast). Genomic DNA was extracted from rat heart (Direct PCR lysis reagent; Viagen Biotech Inc, Los Angeles, CA, USA) to form the template for confirmation of the stem loop sequence for pre-miR- 486 in a PCR reaction using primers (Table S1) designed from a rat genomic DNA sequence (gnl|ti|1621823597; gnl|ti|1718641223) not present in the most recent build of the rat genome (Baylor 3.4), and DreamTaq polymerase (Fermentas). Cycling parameters were: 95uC for 3 min, 356 (95uC for 30 s, 49uC for 30 s, 72uC for 20 s), 72uC for 15 min. Amplified products were sequenced (Genomics Core, Queen’s University Belfast). Following confirmation of inserts by DNA sequencing (Geno- mics Core, Queen’s University Belfast, UK), plasmids were prepared with a Maxiprep kit (Qiagen, West Sussex, UK) following the manufacturer’s instructions. Custom oligonucleotides were purchased from Eurogentec (Seraing, Belgium). miRNA Library Preparation medicine.tcd.ie/sequencing); using TruSeq SR Cluster Kit v5 (Illumina) and the resultant data trimmed and aligned to miRBase v18 (CLC Genomics Workbench v4.0; CLC bio, Swansea UK). The product was restricted with XhoI and BamHI and ligated into pmR-mCherry (Clontech, Saint-Germain-en-Laye, France; modified as described [70]). The final product (pmChGSN) has the CMV promoter driving transcription of mCherry with the gelsolin fragment as 39 UTR. The plasmid pSM30 (encoding EGFP) was used for expression of miRNAs [71]. Complementary pairs of oligonucleotides (25 mM) encoding artificial miRNA sequences (miR-133a canon- ical isomiR and most common isomiR (miR-133a(v))), a siRNA sequence designed to down regulate mCherry expression (siR- mCh), or a random non-targeting negative control sequence (NTC) were annealed and ligated (T4 DNA ligase; New England Biolabs, Hitchin, UK) into pSM30 restricted with BsmBI (New England Biolabs, Hitchin, UK). Oligonucleotide sequences are shown in Table S2. MicroRNAs in Rat Heart Left Ventricle Plasmid Construction A 102 bp fragment of the human gelsolin sequence (NCBI Reference: Chromosome 9, NC_000009.11 (124030380.124095120)) was amplified from human genomic DNA (Bioline, London, UK) by a two-stage nested PCR using PLOS ONE | www.plosone.org June 2013 | Volume 8 | Issue 6 | e65809 3 pipeline for identifying cells in green images and measuring the total red and green intensities of each identified cell. Data were exported in Microsoft Excel format for further analysis (Prism 4, GraphPad, San Diego, CA, USA). Results Overview of Next Generation Sequencing Data A total of 2,947,762 reads between 12 and 73 nt in leng produced by next generation sequencing of the 3 samples myocardium (Fig. 1). miRNAs were aligned and grouped Figure 3. Frequency of isomiRs with 59 and/or 39 variations. Distributions of 59 (left) and 39 (right) end variants are shown for miRNA from the 59 arm (A) and from the 39 arm (B) of the pre-miR hairpin. Data are mean 6 SEM for three mid-myocardial samples. doi:10.1371/journal.pone.0065809.g003 MicroRNAs in Rat Heart Left MicroRNAs in Rat Heart Left Ventricle Figure 3. Frequency of isomiRs with 59 and/or 39 variations. Distributions of 59 (left) and 39 (right) end variants are shown for miRNAs derived from the 59 arm (A) and from the 39 arm (B) of the pre-miR hairpin. Data are mean 6 SEM for three mid-myocardial samples. doi:10.1371/journal.pone.0065809.g003 Figure 3. Frequency of isomiRs with 59 and/or 39 variations. Distributions of 59 (left) and 39 (right) end variants are shown for miRNAs derived Figure 3. Frequency of isomiRs with 59 and/or 39 variations. Distributions of 59 (left) and 39 (right) end variants are shown for miRNAs derived from the 59 arm (A) and from the 39 arm (B) of the pre-miR hairpin. Data are mean 6 SEM for three mid-myocardial samples. doi:10.1371/journal.pone.0065809.g003 pipeline for identifying cells in green images and measuring the total red and green intensities of each identified cell. Data were exported in Microsoft Excel format for further analysis (Prism 4, GraphPad, San Diego, CA, USA). pipeline for identifying cells in green images and measuring the total red and green intensities of each identified cell. Data were exported in Microsoft Excel format for further analysis (Prism 4, GraphPad, San Diego, CA, USA). pipeline for identifying cells in green images and measuring the total red and green intensities of each identified cell. Data were exported in Microsoft Excel format for further analysis (Prism 4, GraphPad, San Diego, CA, USA). Results Overview of Next Generation Sequencing Data A total of 2,947,762 reads between 12 and 73 nt in length were produced by next generation sequencing of the 3 samples of mid- myocardium (Fig. 1). miRNAs were aligned and grouped on the mature sequence, allowing for up to 2 mismatches within the sequence and/or 3 additions or deletions from either the 59 or 39 PLOS ONE | www.plosone.org June 2013 | Volume 8 | Issue 6 | e65809 4 MicroRNAs in Rat Heart Left Ventricle Figure 4. IsomiRs of selected miRNAs. IsomiR sequences of the 10 most highly detected miRNAs (mature miRNA highlighted in yellow) with expression values in brackets from a mid-myocardial sample aligned to the published pre-miR sequence (boxed in green; miRBase v18). Consensus sequence (boxed in blue) for each miRNA represents the most prevalent nt aligned at each position (nts highlighted in pink represent variations from the miRBase published mature sequence). doi:10.1371/journal.pone.0065809.g004 Figure 4. IsomiRs of selected miRNAs. IsomiR sequences of the 10 most highly detected miRNAs (mature miRNA highlighted in yellow) with expression values in brackets from a mid-myocardial sample aligned to the published pre-miR sequence (boxed in green; miRBase v18). Consensus sequence (boxed in blue) for each miRNA represents the most prevalent nt aligned at each position (nts highlighted in pink represent variations from the miRBase published mature sequence). doi:10.1371/journal.pone.0065809.g004 major mature strand form. By combining the 3 sample groups and normalising to reads per million mapped (RPMM), the most highly detected miRNAs ($1000 RPMM) were identified (Fig. 2). miR-22, miR-486, miR133a and miR-143 were detected at greater than 100,000 RPMM. Twelve miRNAs were detected at greater than 10,000 RPMM and a further 40 miRNAs were detected at greater than 1,000 RPMM. Six of the top 56 most highly detected miRNAs were * sequences. Fifty-three of the top 56 most highly detected miRNAs were previously annotated rat miRNAs, but the second most highly detected miRNA mapped to the human miRNA hsa-miR-486-5p and has yet to be registered in miRBase as a rat miRNA, although Small et al. [72] have shown it to be highly expressed in rat myocardium. Characterisation of the rat miR-486 gene is described below. ends. This identified 1,782,482 reads that were annotated to known miRNA sequences from Rattus norvegicus entries in miRBase v18 and a further 307,672 annotated to miRNA sequences only previously reported in other species (potentially representing novel orthologs). June 2013 | Volume 8 | Issue 6 | e65809 Abundance of isomiR Sequences The frequency of isomiRs with 59 and/or 39 variations from the canonical (miRBase) sequence is summarised in Fig. 3 for a mid- myocardial sample. Of the sequences derived from the 59 arm of the hairpin miRNA, 62.6% varied from the canonical sequence at the 39 end (with approximately half being longer and half being shorter), and 3.6% varied at the 59 end (with approximately two- thirds being shorter). Of the sequences derived from the 39 arm, 50.1% varied from the canonical sequence at the 39 end (with approximately three-fifths being longer), and 13.8% varied at the 59 end (with a strong bias towards shorter variants). This distribution is similar to that found in cardiomyocyte-derived HL-1 cells [61]. The greater 59 end variability in the 3’ arm- derived compared to 5’ arm-derived miRNAs corresponds with the previously reported higher fidelity of Drosha vs Dicer cleavage [78]. Variability at the 3’ end is also influenced by post-cleavage processing [79,80]. Closer analysis of the top 56 most highly detected miRNAs revealed that the mature form (or the mature* sequence) of the miRNA as published in miRBase was not always the most common sequence encountered. For the top 10 most highly detected miRNAs, the reads representing the 10 most common isomiR sequences are illustrated in Fig. 4. For only 4 of these, miR-22, 486, 26a and 181a, is the annotated mature form the most highly detected and even for these miRNAs significant numbers of several other isomiRs were also detected. For example, while the exact mature sequence of miR-22 constituted 93.0460.35% of its total reads, the exact mature sequence of miR-181a only constituted 51.0960.59%. Characteristically for miRNAs, the majority of miR-486 isomiR sequences differed at the 39 end of the sequences and were shortened mature sequences (sub) or mature sequences with additional nts (super) matching the Surprisingly, miR-10b exhibited no exact mature sequences (see Table S3B); on closer analysis sequences aligned to miR-10b were found to exhibit shifting in the mature miRNA cleavage position with 12.3860.77% of the reads including 1 extra nt and 87.3960.91% 2 extra nts from the pre-miR sequence at the 59 end (see Fig. S2). This type of 59 end variation has been attributed to variability in Drosha cleavage position [83]. Results Differential suppression of a gelsolin sequence- tagged reporter gene by miR-133a isomiRs. HEK293 cells were transfected with expression plasmids encoding mCherry with a 39 partial gelsolin sequence and pSM30 containing inserts for miR-133a, miR-133a(v), a random non-targeting sequence (NTC) or siRNA against mCherry (siR-mCh). Cells were imaged and analysed for both green and red fluorescence intensity. A decrease in red/green ratio vs NTC indicates downregulation of target gene expression. Data were log transformed and compared by one-way ANOVA with Bonferroni’s Multiple Comparison Test. All pairwise comparisons were significant (p,.001). Comparison of miR-133a vs miR-133a(v) is indicated (***). Number of cells (n): miR-133a 4640; miR-133a(v) 2843; NTC 4088; siR- mCh 3884. Representative of 3 separate experiments in which miR- 133a(v) was significantly more effective than miR-133a. doi:10.1371/journal.pone.0065809.g005 Figure 6. Relative miRNA abundance by TaqMan assay. Expression of miRNAs relative to rno-miR-1 according to 22DCt where DCt = Ct – CtmiR-1 (mean 6 SEM). Ct values were compared by repeated measures ANOVA and Tukey’s multiple comparison test; **p,.01, ***p,.001. Mid-myocardial samples from n = 6 hearts. doi:10.1371/journal.pone.0065809.g006 pre-miR sequence. However, substantial populations of reads exhibited nt substitutions varying from the pre-miR sequence (compare pre-miR and consensus sequences in Fig. 4B), largely by the addition of nts at the 39 end, most commonly T’s and A’s. the addition of nts at the 3 end, most commonly T s and A s. Analysis of the sequences attributed to miR-143 (Fig. 4D) showed that the mature sequence was only the 4th most common sequence, indeed the most common miR-143 sequence is an isomiR with an unambiguous post-transcriptional modification, that is a non-templated addition of A at nt no. 22. Additional significantly expressed variants include nt no. 21 C to T, and nt no. 23 G to T. As expected, little variation was observed at the 59 end of the majority of the most highly detected miRNAs. However, miR-378 (Fig. 4G) and the cardiac muscle-enriched miR-133a are exceptions. As illustrated in Fig. 4C the most frequently recorded miRNA sequence attributed to miR-133a is missing nt no. 1; indeed 53.9660.46% of all miR-133a reads are missing this nt. This truncation could have a profound effect on target sequence recognition, and therefore the spectrum of target genes regulated. Indeed, DIANA-microT v3.0 (which accepts novel miRNA sequences) [81,82] predicts 602 target genes for canonical miR-133a and 145 for the 59 variant (miR-133a(v)), with 90 genes in common. Results Gelsolin is among the 55 genes predicted to be targeted by miR-133a(v) but not by miR-133a itself. We used the dual-fluorescence miRNA targeting assay [70] to determine the effects of miR-133a and miR-133a(v) on the expression of a reporter gene with a 39 gelsolin fragment incorporating the more conserved of the two predicted miR-133a(v) target sites. Suppres- sion of the target gene is reported as a decrease in red/green fluorescence intensity ratio in this assay. Fig. 5 shows that miR- 133a(v) was more effective at suppressing the mCherry-gelsolin construct than was miR-133a, and was comparable to a siRNA targeted against the mCherry sequence. transcription factor IRX5, which negatively regulates Kv4.2 K+ channel expression [76,77]. transcription factor IRX5, which negatively regulates Kv4.2 K+ channel expression [76,77]. Results The proportions of reads of length .12 representing known rat miRNAs, potential novel orthologs or not matching any known miRNA (unannotated) are shown in Fig. 1. In all 3 samples the median length of reads was 22 nts and 80.7360.20% (mean 6 sem) of the reads at this length were annotated to known rat miRNA sequences. In total, 145 previously characterised rat miRNA sequences had representation of $10 raw reads prior to normalisation (Table S3; S3A compiled from miRNAs with $10 raw ‘exact mature’ reads, S3B compiled from miRNAs with ,10 ‘exact mature’ reads but $10 ‘grouped on mature’ raw reads) in at least one of the 3 samples. Using the same criteria a further 68 potential novel orthologs were identified (Table S4). Additionally all reads of sequences originally reported in miRbase as the minor strand (*) were grouped (upon similar criteria as above; Table S5) resulting in a further 58 miRNAs of interest, 16 of which (including miR-126*, miR-140*, miR-151* and miR-28*) were detected between 1.23 and 99.56 fold higher in the * form than the ‘major’ mature sequence, while miR-501* was not detected in the Individually, miR-22 has been implicated in the phenylephrine- or angiotensin II-induced hypertrophic reduction in phosphatase and tensin homolog (PTEN) [24]; miR-26a and 30d in calcium dependent cardiac dysfunction [73], and miR-27b in heart development and hypertrophy [74,75]. PTEN and Foxo1a are known targets of miR-486 [72]. Of particular note in the context of cardiac physiology, miR-486 is predicted by TargetScan 6.2 (Whitehead Institute for Biomedical Research) to target the June 2013 | Volume 8 | Issue 6 | e65809 5 PLOS ONE | www.plosone.org June 2013 | Volume 8 | Issue 6 | e65809 MicroRNAs in Rat Heart Left Ventricle Figure 5. Differential suppression of a gelsolin sequence- tagged reporter gene by miR-133a isomiRs. HEK293 cells were transfected with expression plasmids encoding mCherry with a 39 partial gelsolin sequence and pSM30 containing inserts for miR-133a, miR-133a(v), a random non-targeting sequence (NTC) or siRNA against mCherry (siR-mCh). Cells were imaged and analysed for both green and red fluorescence intensity. A decrease in red/green ratio vs NTC indicates downregulation of target gene expression. Data were log transformed and compared by one-way ANOVA with Bonferroni’s Multiple Comparison Test. All pairwise comparisons were significant (p,.001). Comparison of miR-133a vs miR-133a(v) is indicated (***). Number of cells (n): miR-133a 4640; miR-133a(v) 2843; NTC 4088; siR- mCh 3884. Results Representative of 3 separate experiments in which miR- 133a(v) was significantly more effective than miR-133a. doi:10.1371/journal.pone.0065809.g005 pre-miR sequence. However, subst exhibited nt substitutions varying f (compare pre-miR and consensus seq the addition of nts at the 39 end, mo Analysis of the sequences attrib showed that the mature sequence wa sequence, indeed the most commo i iR i h bi Figure 5. Differential suppression of a gelsolin sequence- tagged reporter gene by miR-133a isomiRs. HEK293 cells were transfected with expression plasmids encoding mCherry with a 39 partial gelsolin sequence and pSM30 containing inserts for miR-133a, miR-133a(v), a random non-targeting sequence (NTC) or siRNA against mCherry (siR-mCh). Cells were imaged and analysed for both green and red fluorescence intensity. A decrease in red/green ratio vs NTC indicates downregulation of target gene expression. Data were log transformed and compared by one-way ANOVA with Bonferroni’s Multiple Comparison Test. All pairwise comparisons were significant (p,.001). Comparison of miR-133a vs miR-133a(v) is indicated (***). Number of cells (n): miR-133a 4640; miR-133a(v) 2843; NTC 4088; siR- mCh 3884. Representative of 3 separate experiments in which miR- 133a(v) was significantly more effective than miR-133a. doi:10.1371/journal.pone.0065809.g005 Figure 6. Relative miRNA abun Expression of miRNAs relative to rno-m DCt = Ct – CtmiR-1 (mean 6 SEM). Ct valu measures ANOVA and Tukey’s multip ***p,.001. Mid-myocardial samples fro doi:10.1371/journal.pone.0065809.g006 Figure 6. Relative miRNA abundance by TaqMan assay. Expression of miRNAs relative to rno-miR-1 according to 22DCt where DCt = Ct – CtmiR-1 (mean 6 SEM). Ct values were compared by repeated measures ANOVA and Tukey’s multiple comparison test; **p,.01, ***p,.001. Mid-myocardial samples from n = 6 hearts. doi:10.1371/journal.pone.0065809.g006 Figure 5. Differential suppression of a gelsolin sequence- tagged reporter gene by miR-133a isomiRs. HEK293 cells were transfected with expression plasmids encoding mCherry with a 39 partial gelsolin sequence and pSM30 containing inserts for miR-133a, miR-133a(v), a random non-targeting sequence (NTC) or siRNA against mCherry (siR-mCh). Cells were imaged and analysed for both green and red fluorescence intensity. A decrease in red/green ratio vs NTC indicates downregulation of target gene expression. Data were log transformed and compared by one-way ANOVA with Bonferroni’s Multiple Comparison Test. All pairwise comparisons were significant (p,.001). Comparison of miR-133a vs miR-133a(v) is indicated (***). Number of cells (n): miR-133a 4640; miR-133a(v) 2843; NTC 4088; siR- mCh 3884. Representative of 3 separate experiments in which miR- 133a(v) was significantly more effective than miR-133a. doi:10.1371/journal.pone.0065809.g005 Figure 5. Abundance of isomiR Sequences The 2 extra 59 nts give the most abundant isomiR the same seed sequence as rno- miR-10a-5p, although the rest of the isomiR sequence corresponds to rno-miR-10b-5p, which has a single internal mismatch compared to rno-miR-10a-5p. Of 148 target sites in 85 genes predicted for rno-miR-10a-5p by DIANA-microT v3.0, 147 are PLOS ONE | www.plosone.org June 2013 | Volume 8 | Issue 6 | e65809 June 2013 | Volume 8 | Issue 6 | e65809 6 MicroRNAs in Rat Heart Left Ventricle Figure 7. Novel rno-miR-1 sequence as detected by deep sequencing. A: Ten most frequently detected isomiR sequences of miR-1 (rat mature miRNA highlighted in yellow) with expression values in brackets aligned to the published pre-miR sequence (boxed in green; miRBase v18). The consensus sequence (boxed in blue) represents the most prevalent nt aligned at each position (nts highlighted in pink represent variations from the miRBase published mature sequence). B: miR-1 sequences as published in miRBase v18 showing the novel rat miR-1 sequence aligned with the previously reported rat miR-1 and the human sequence (with which it is identical). C: Predicted stem-loop structure (mFold 3.2) of proposed pre-miR deduced from genomic sequence. Mature product highlighted in blue. doi:10.1371/journal.pone.0065809.g007 Figure 7. Novel rno-miR-1 sequence as detected by deep sequencing. A: Ten most frequently detected isomiR sequences of miR-1 (rat mature miRNA highlighted in yellow) with expression values in brackets aligned to the published pre-miR sequence (boxed in green; miRBase v18). The consensus sequence (boxed in blue) represents the most prevalent nt aligned at each position (nts highlighted in pink represent variations from the miRBase published mature sequence). B: miR-1 sequences as published in miRBase v18 showing the novel rat miR-1 sequence aligned with the previously reported rat miR-1 and the human sequence (with which it is identical). C: Predicted stem-loop structure (mFold 3.2) of proposed pre-miR deduced from genomic sequence. Mature product highlighted in blue. doi:10.1371/journal.pone.0065809.g007 data shown in Fig. 6 which suggests that, based on relative Ct values, miR-1 is more abundant than miR-22 and miR-486 which are the two most common miRNAs in the deep sequencing data (Fig. 2). Another consideration when comparing sequencing and TaqMan data is the specificity of TaqMan assays for the canonical mature sequence. For example 57% of miR-486 reads in the sample shown in Fig. Abundance of isomiR Sequences 4 are 39 variants that would not be detected by TaqMan because of the 39 specificity of the looped RT primer used in TaqMan small RNA assays. also predicted for the most frequent miR-10b isomiR, reflecting the dominance of the seed region in target recognition. In comparison, only one of the 10 predicted target genes found for canonical rno-miR-10b is shared with the isomiR. A Novel Rat miRNA Analysis of unannotated reads by the miRanalyser utility [89] yielded a putative novel miRNA. This miRNA has a mature sequence similar to mmu-miR-676-3p and hsa-miR-676-3p (Fig. 8A). The genomic context has a predicted stem-loop structure at chrX:88378185-88378273 (Ensembl genome ID EN- SRNOG00000040353) (Fig. 8B). A Novel Rat miR-1 Analog It was surprising that miR-1 was only the 28th most abundant miRNA in the sequencing data (Fig. 2) because it is generally considered to be one of the most highly expressed in the heart [84]. It has been demonstrated that observed miRNA abundance can vary significantly between experimental methods [85]. It is possible that preferential cloning of certain sequences or differ- ences in PCR amplification may skew the numbers of reads representing different miRNAs. Indeed, the RNA adapters used can introduce sequencing bias through secondary structure preferences of RNA ligases [86]. The possibility that miR-1 is under-represented in our libraries is supported by the RT-qPCR Notwithstanding the potentially anomalous measure of abun- dance, the sequencing data enabled us to observe a variant in the sequence of miR-1; at nt no. 17 an A was frequently present in place of the G in the pre-miR sequence (77.2361.13% of reads; Fig. 7A). The G to A substitution gives a sequence equivalent to hsa-miR-1 (and mmu-miR-1) which has not been reported before in rat (Fig. 7B). It is unlikely that G could be converted to A by post-transcriptional editing; therefore we investigated the possibil- June 2013 | Volume 8 | Issue 6 | e65809 PLOS ONE | www.plosone.org 7 June 2013 | Volume 8 | Issue 6 | e65809 MicroRNAs in Rat Heart Left Ventricle Figure 8. Novel rat miRNA similar to miR-676-3p. A: Alignment of the novel rat miRNA sequence to miR-676-3p sequences as published in miRBase v18. B: Predicted stem-loop structure (mFold 3.2) of proposed pre-miR deduced from genomic sequence. Mature miRNA highlighted in blue. doi:10.1371/journal.pone.0065809.g008 Figure 8. Novel rat miRNA similar to miR-676-3p. A: Alignment of the novel rat miRNA sequence to miR-676-3p sequences as published in miRBase v18. B: Predicted stem-loop structure (mFold 3.2) of proposed pre-miR deduced from genomic sequence. Mature miRNA highlighted in blue. doi:10.1371/journal.pone.0065809.g008 Cloning and Sequencing of the Rat miR-486 Gene ity that the rat harbours a second miR-1 gene. Although the most common sequence carrying the A (59-TGGAATGTAAAGAAG- TATGTAT-39) could not be detected in the latest available build of the Rattus norvegicus genome (Baylor 3.4), the sequence matched two rat whole genome shotgun sequences (gb|AAHX01027125.1|; gb|AABR06028312.1|) which have not been incorporated into this build. Fig. 7C shows the genomic context of this novel miRNA and the predicted stem-loop structure (mFold version 3.2 [87]). This sequence is also present in the rat heart data set of Linsen et al. [88]. The existence of two rat miR-1 genes has implications for understanding how this important miRNA is regulated. No pre-miR sequence has been published for miR-486 in rat and the mature miRNA sequence is not present in the most recent build of the rat genome (Baylor 3.4). However, alignment of the mouse pre-miR-486 (MI0003493) sequence revealed a potential rat pre-miR-486 sequence on chromosome 16:73341164 (reverse strand) which is truncated just before the end of the potential miR- 486 mature sequence at nt 73341114 by a gap in the genome sequence. A BLAST search revealed two whole genome shotgun sequences spanning this gap, allowing the design of PCR primers that were used to amplify the region from rat genomic DNA. This enabled confirmation of the sequence of the putative miR-486 gene, which contains a stem-loop sequence matching both mature miR-486 reads in the 5p arm and observed ‘star’ sequences from the 3p arm (Fig. 9). Transmural Distribution of miRNA Expression Several aspects of cardiomyocyte physiology are known to vary across the left ventricular wall [66,90–111] and the mechanism undoubtedly involves differential gene expression. Transmural expression level is uniform for the vast majority of genes but there Figure 9. The rat miR-486 gene. A: Alignment of the genomic miR-486 sequence across nine species including rat (highlighted in red). Blue shading indicates percentage sequence identity and the mature and ‘star’ sequences represented by the most common reads are indicated with arrows. B: Predicted stem-loop structure (mFold 3.2) of pre-miR-486 deduced from genomic sequence. Mature miRNA highlighted in blue. doi:10.1371/journal.pone.0065809.g009 Figure 9. The rat miR-486 gene. A: Alignment of the genomic miR-486 sequence across nine species including rat (highlighted in red). Blue shading indicates percentage sequence identity and the mature and ‘star’ sequences represented by the most common reads are indicated with arrows. B: Predicted stem-loop structure (mFold 3.2) of pre-miR-486 deduced from genomic sequence. Mature miRNA highlighted in blue. doi:10.1371/journal.pone.0065809.g009 June 2013 | Volume 8 | Issue 6 | e65809 June 2013 | Volume 8 | Issue 6 | e65809 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 8 MicroRNAs in Rat Heart Left Ventricle Table 1. Epicardial/endocardial differential expression of exact mature (or mature*) miRNAs ($ 61.5 fold difference for miRs of $10 raw reads detected in either layer). Table 1. Epicardial/endocardial differential expression of exact mature (or mature*) miRNAs ($ 61.5 fold difference for miRs of $10 raw reads detected in either layer). Transmural Distribution of miRNA Expression This could explain the high Ct values (low abundance) we obtained with the miR-10b TaqMan assay which is designed to be specific for the exact 39 end of the canonical sequence. Additionally, sequencing analysis identified 8 individual sequences within the miR-10a/b family (unique sequences; Table 3) as having .2 fold detected difference (epicardium.endocardium) within the samples tested. are some notable exceptions [112–114]. Given the role of miRNAs in regulating gene expression it would be useful to determine the extent of transmural heterogeneity of the expression of miRNAs themselves. In accordance with other gene expression data [112] we found the transmural distribution of miRNA expression to be generally uniform. Nevertheless, differences in relative abundance were observed between endocardium and epicardium for several miRNAs (Tables 1, 2, and 3). Some of the more abundantly detected miRNAs showed some transmural difference that did not reach statistical significance across the three hearts. We selected some of these miRNAs for further investigation by qPCR (TaqMan miRNA assays) in four additional hearts and found statistically significant (p,.05) transmural expression gradients for miR-10b, miR-21, miR-99b and miR-486 (Fig. 10). Transmural expression gradients for IRX5 and FOXP2 were in agreement with a previous report [112]. Deep sequencing shows a transmural gradient (epicardium.endocardium) for miR-10b (Table 2, ‘‘grouped on mature’’). As discussed above, this group includes Transmural Distribution of miRNA Expression Exact mature/*sequences Epi-cardium Endo-cardium RPMM RPMM miR name Fold change Mean SEM Mean SEM rno-mir-31 3.00 4.45 2.62 13.36 3.49 rno-mir-326 1.92%%% 12.52 0.98 24.06 2.03 rno-mir-872 1.80 11.42 2.54 20.61 5.32 rno-mir-1249 1.66 62.40 12.09 103.28 28.92 rno-mir-503* 1.64 5.02 1.45 8.21 0.65 rno-mir-193 1.54 14.14 2.23 21.82 8.28 rno-mir-872* 1.53 51.11 7.20 78.40 14.18 mmu-mir-1983 1.53 8.16 3.02 12.45 2.99 rno-mir-145 1.50 1038.98 188.02 1558.55 346.49 rno-mir-10a 21.51 1967.41 189.80 1305.65 527.54 rno-mir-9-1//mir-9-3//mir-9-2 21.52 64.65 13.48 42.62 11.51 rno-mir-208 21.52 12.33 3.51 8.09 3.41 rno-mir-132 21.61 18.32 1.35 11.37 2.24 rno-mir-138-2//mir-138-1 21.66 17.24 2.76 10.36 4.83 rno-mir-210* 21.69 7.65 1.69 4.53 1.10 rno-mir-100 21.70 2089.07 827.35 1230.28 597.96 rno-mir-133b 21.70 15.27 4.06 8.98 2.54 rno-mir-133a* 21.72 206.39 59.22 120.26 27.43 rno-mir-17-1//mir-17-2 21.81 10.74 1.68 5.93 0.32 rno-mir-322* 21.98 20.39 8.69 10.31 5.30 mmu-mir-720 22.00 23.31 5.27 11.66 1.00 rno-mir-425 22.25 15.66 3.01 6.96 3.58 hsa-mir-720 22.30 39.25 8.00 17.09 2.93 rno-mir-374 22.49 8.51 3.73 3.42 0.66 rno-mir-410 22.88%% 56.31 2.35 19.58 5.31 hsa-mir-4492 22.92 26.70 10.82 9.14 4.45 rno-mir-200b 25.88 13.39 10.25 2.28 1.23 rno-mir-547* 29.73 6.82 3.21 0.70 0.70 %%%P,0.001, %%P,0.01, Baggerleys multiple comparison test. doi:10.1371/journal.pone.0065809.t001 are some notable exceptions [112–114]. Given the role of miRNAs in regulating gene expression it would be useful to determine the extent of transmural heterogeneity of the expression of miRNAs themselves. In accordance with other gene expression data [112] we found the transmural distribution of miRNA expression to be generally uniform. Nevertheless, differences in relative abundance were observed between endocardium and epicardium for several miRNAs (Tables 1, 2, and 3). Some of the more abundantly detected miRNAs showed some transmural difference that did not reach statistical significance across the three hearts. We selected some of these miRNAs for further investigation by qPCR (TaqMan miRNA assays) in four additional hearts and found statistically significant (p,.05) transmural expression gradients for miR-10b, miR-21, miR-99b and miR-486 (Fig. 10). Transmural expression gradients for IRX5 and FOXP2 were in agreement with a previous report [112]. Deep sequencing shows a transmural gradient (epicardium.endocardium) for miR-10b (Table 2, ‘‘grouped on mature’’). As discussed above, this group includes no canonical miR-10b, with only 5.6% of reads having the canonical 39 end (4th and 6th in the list in Fig. S2). June 2013 | Volume 8 | Issue 6 | e65809 Prediction of Signalling Pathway Targets To reveal the pathways most likely to be significantly regulated by miRNAs, pathway analysis was performed based upon the top 16 most highly detected miRNA sequences (.10,000 RPMM) utilizing DIANA LAB (loose, strict and Beta algorithms as well as TargetScan5 and Pictar [115] combined with TargetScan 6.0 (including poorly conserved sites – Total context score #-0.1) and Micro-org individual predictions (mirSVR score # -0.3). Exclud- ing targets which only appeared on one of the 7 prediction lists, June 2013 | Volume 8 | Issue 6 | e65809 PLOS ONE | www.plosone.org 9 MicroRNAs in Rat Heart Left Ventricle Table 2. Epicardial/endocardial differential expression of ’grouped on mature’ miRNAs ($ 61.5 fold difference for miRs of $10 raw reads detected in either layer). Table 2. Epicardial/endocardial differential expression of ’grouped on mature’ miRNAs ($ 61.5 fold difference for miRs of $10 raw reads detected in either layer). ’Grouped on mature’ sequences Epi-cardium Endo-cardium RPMM RPMM miR name Fold change Mean SEM Mean SEM rno-mir-200b 24.26 25.92 16.66 6.08 3.31 rno-mir-410 22.87%%% 70.10 3.37 24.41 5.32 mmu-mir-690 22.55 12.73 3.72 5.00 1.39 rno-mir-10b 22.51%% 3064.12 431.15 1221.18 441.95 mmu-mir-720 22.20 27.78 6.87 12.63 1.12 mmu-mir-351 22.08 11.37 2.93 5.47 0.60 hsa-mir-4485 22.07 9.81 2.27 4.75 1.81 rno-mir-341 22.05 7.78 2.10 3.80 2.45 mmu-mir-5115 21.99 5823.13 1542.50 2930.00 1218.62 rno-mir-384 21.97 8.73 2.79 4.43 1.46 bta-mir-2478 21.96 358.54 67.41 182.69 21.31 hsa-mir-720 21.96 68.26 12.81 34.90 6.30 hsamir-4492 21.92 43.13 13.29 22.46 9.31 cbr-mir-1 21.91 10.46 4.84 5.47 1.06 rno-mir-10a 21.87 30279.72 3036.38 16169.54 6084.64 hsa-mir-1260a 21.77 278.56 53.99 157.62 42.34 rno-mir-338 21.77 117.08 1.79 66.29 7.61 mmu-mir-5102 21.73 11.49 3.65 6.65 1.16 rno-mir-138-2//mir-138-1 21.71 27.70 5.62 16.18 8.01 rno-mir-100 21.70 5514.57 2226.60 3245.17 1552.80 rno-mir-132 21.63 37.90 2.55 23.21 4.82 rno-mir-9-1//mir-9-3//mir-9-2 21.58 169.45 26.81 107.12 26.08 ssc-mir-186 21.57 16.66 5.25 10.60 1.13 dre-mir-26b 21.54 25.55 7.97 16.64 2.29 cte-mir-1 21.53 6.37 1.86 4.17 1.71 gga-mir-3535 21.52%% 9.46 0.21 6.24 1.83 rno-mir-374 21.52 64.17 18.59 42.34 7.63 rno-mir-411 21.50% 39.14 3.72 26.11 4.25 rno-mir-145 1.50 1537.62 272.14 2300.09 506.26 ola-mir-27d 1.50 6.30 1.14 9.42 3.09 gga-mir-193b 1.51 28.48 5.52 42.86 6.54 mmu-mir-101c 1.56 7.32 2.86 11.39 0.42 rno-mir-326 1.57 37.54 1.72 59.00 5.46 bta-mir-1306 1.63 5.20 1.59 8.45 0.66 rno-mir-877 1.64 8.05 1.21 13.20 1.75 sha-mir-716a 1.75 8.22 0.13 14.37 6.10 rno-mir-674 2.15 5.44 0.81 11.67 1.74 rno-mir-31 2.54 9.37 4.26 23.78 6.64 %%%P,0.001. June 2013 | Volume 8 | Issue 6 | e65809 Prediction of Signalling Pathway Targets Thus it provides the ability RPMM RPMM Sequence miR name (type) Fold change Mean SEM Mean SEM TACCCTGTAGAACCGAATTTG rno-mir-10b(P) 22.99 140.45 22.37 46.95 18.79 CTGGACGCGAGCCGGGCCCTT mmu-mir-5115(MSupV) 22.82 753.60 405.10 267.59 109.66 TACCCTGTAGAACCGAATTTGT rno-mir-10b(MSub/Sup) 22.54 2108.85 325.91 829.98 294.94 ATCCCACTTCTGACACCA bta-mir-2478(MSub) 22.45% 196.81 33.86 80.26 6.23 CCGCGTCGGCGGTTCCC nlo-mir-125(PV) 22.43 138.79 42.98 57.12 25.49 ACCCTGTAGAACCGAATTTGT rno-mir-10b(MSub/Sup) 22.42 119.77 21.71 49.43 15.33 TACCCTGTAGATCCGAATTTGA rno-mir-10a(MSubV) 22.22 325.04 20.21 146.52 55.43 CTGGACGCGAGCCGGGCCCTTCCC mmu-mir-5115(PV) 22.21 147.76 43.14 67.01 26.00 TCGTACCGTGAGTAATAATGC rno-mir-126(MSub) 22.17 821.18 347.35 378.83 99.04 TACCACAGGGTAGAACCACGGAA rno-mir-140(M*SupV) 22.16 348.57 66.62 161.25 22.11 ACCCTGTAGATCCGAATTTG rno-mir-10a(MSub) 22.14 272.77 37.62 127.17 50.55 ACCCTGTAGATCCGAATTTGT rno-mir-10a(MSub) 22.11 1779.84 246.64 842.11 327.87 CTGGACGCGAGCCGGGC mmu-mir-5115(MSubV) 22.10 508.53 119.83 241.60 100.94 TACCCTGTAGAACCGAATTTGTG rno-mir-10b(MSub/Sup) 22.09 192.96 12.82 92.16 32.52 TACCCTGTAGATCCGAATTT rno-mir-10a(P) 22.08 349.57 32.37 168.25 69.42 CTGGACGCGAGCCGGGCCCTTCC mmu-mir-5115(PV) 22.05 188.96 62.04 92.07 34.57 CTGGACGCGAGCCGGGCCCTTC mmu-mir-5115(MSupV) 22.04 775.70 233.89 380.53 157.79 AACCCGTAGATCCGAACTTGTGA rno-mir-100(MSupV) 22.01 768.45 357.07 382.85 189.43 TGTAAACATCCTCGACTGGAAGCG rno-mir-30a(MSupV) 2.06 118.32 23.96 243.52 139.23 TCCTGTACTGAGCTGCCCCGG bta-mir-486(MSubV) 2.39 133.14 28.86 318.16 181.42 GGGGGGCCCAAGTCCTTCTGATCGAGGCCC mmu-mir-5105(P) 2.93 73.61 16.57 215.65 93.91 CATTCAACGCTGTCGGTGAGT rno-mir-181a-2//mir-181a- 1(MSub) 40.70 6.08 0.93 247.35 243.97 CATTCAACGCTGTCGGTGAG rno-mir-181a-2//mir-181a- 1(MSub) 63.29 2.65 0.93 167.78 165.76 doi:10.1371/journal.pone.0065809.t003 Figure 10. Transmural miRNA expression gradients. Taqman small RNA assays and TaqMan gene expression assays were performed on epicardial and endocardial samples from rat hearts. Endocardial/ epicardial expression ratios were determined by analysis of Ct values using REST 2009. The reference genes utilised were miRNAs with stable expression across samples according to BestKeeper: miR-22, miR-30a, miR-30c, miR-30e and miR-100. The reference genes for IRX5 and FOXP2 were GAPDH, HPRT1 and 18s rRNA. Data are mean 6 SEM; n = 4 hearts for miRNAs (n = 3 of these for IRX5 and FOXP2). *P,.05; **P,.01; ***P,.001. doi:10.1371/journal.pone.0065809.g010 j p pathway (MAPKinase; P = 1.8661026; 75 targets; Fig. 11), Regulation of Actin Cytoskeleton (P = 2.2961024; 56 targets), Wnt pathways (P = 3.4561024; 42 targets), Calcium Signaling (P = 5.0161024; 49 targets), Gap junctions (P = 6.4361024; 27 targets) and Arrhythmogenic Right Ventricular Cardiomyopathy pathway (ARVC; P = 0.047; 20 targets). Further analysis of a shortlist of genes important in contraction, electrical activity, Ca2+ signalling, cell communication, ion homeostasis and gene expression showed potential targeting of components contributing to the generation of INa, ICa,L, IKur, Ito, IKATP, INaCa, If, IKp as well as SR Ca2+ re-uptake and contraction (see Table S6). Discussion Figure 10. Transmural miRNA expression gradients. Taqman small RNA assays and TaqMan gene expression assays were performed on epicardial and endocardial samples from rat hearts. Endocardial/ epicardial expression ratios were determined by analysis of Ct values using REST 2009. The reference genes utilised were miRNAs with stable expression across samples according to BestKeeper: miR-22, miR-30a, miR-30c, miR-30e and miR-100. The reference genes for IRX5 and FOXP2 were GAPDH, HPRT1 and 18s rRNA. Data are mean 6 SEM; n = 4 hearts for miRNAs (n = 3 of these for IRX5 and FOXP2). *P,.05; **P,.01; ***P,.001. Prediction of Signalling Pathway Targets %%P,0 01 the resultant 2808 genes were compared with a list of genes expressed in heart (as identified from 9 Sprague-Dawley rat left ventricular myocardium samples from 2 microarray experiments published on GEO (GSE6943; GSM160095-100 & GSE6880; GSM158589-91; http://www.ncbi.nlm.nih.gov/gds)) resulting in 1624 potential targets expressed in and therefore most relevant to heart. Of the significantly targeted pathways the most relevant to cardiac physiology and pathophysiology were the Ubiquitin- mediated Proteolysis pathway (Benjamini corrected P = 7.1261027; 46 targets), Mitogen-Activated Protein Kinase the resultant 2808 genes were compared with a list of genes expressed in heart (as identified from 9 Sprague-Dawley rat left ventricular myocardium samples from 2 microarray experiments published on GEO (GSE6943; GSM160095-100 & GSE6880; GSM158589-91; http://www.ncbi.nlm.nih.gov/gds)) resulting in June 2013 | Volume 8 | Issue 6 | e65809 June 2013 | Volume 8 | Issue 6 | e65809 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 10 MicroRNAs in Rat Heart Left Ventricle Table 3. Epicardial/endocardial differential expression of unique sequences ($ 62 fold difference for miRs of $100 mean normalised reads detected in either layer). %P,0.05 Baggerleys multiple comparison test. Unique sequences Epi-cardium Endo- cardium RPMM RPMM Sequence miR name (type) Fold change Mean SEM Mean SEM TACCCTGTAGAACCGAATTTG rno-mir-10b(P) 22.99 140.45 22.37 46.95 18.79 CTGGACGCGAGCCGGGCCCTT mmu-mir-5115(MSupV) 22.82 753.60 405.10 267.59 109.66 TACCCTGTAGAACCGAATTTGT rno-mir-10b(MSub/Sup) 22.54 2108.85 325.91 829.98 294.94 ATCCCACTTCTGACACCA bta-mir-2478(MSub) 22.45% 196.81 33.86 80.26 6.23 CCGCGTCGGCGGTTCCC nlo-mir-125(PV) 22.43 138.79 42.98 57.12 25.49 ACCCTGTAGAACCGAATTTGT rno-mir-10b(MSub/Sup) 22.42 119.77 21.71 49.43 15.33 TACCCTGTAGATCCGAATTTGA rno-mir-10a(MSubV) 22.22 325.04 20.21 146.52 55.43 CTGGACGCGAGCCGGGCCCTTCCC mmu-mir-5115(PV) 22.21 147.76 43.14 67.01 26.00 TCGTACCGTGAGTAATAATGC rno-mir-126(MSub) 22.17 821.18 347.35 378.83 99.04 TACCACAGGGTAGAACCACGGAA rno-mir-140(M*SupV) 22.16 348.57 66.62 161.25 22.11 ACCCTGTAGATCCGAATTTG rno-mir-10a(MSub) 22.14 272.77 37.62 127.17 50.55 ACCCTGTAGATCCGAATTTGT rno-mir-10a(MSub) 22.11 1779.84 246.64 842.11 327.87 CTGGACGCGAGCCGGGC mmu-mir-5115(MSubV) 22.10 508.53 119.83 241.60 100.94 TACCCTGTAGAACCGAATTTGTG rno-mir-10b(MSub/Sup) 22.09 192.96 12.82 92.16 32.52 TACCCTGTAGATCCGAATTT rno-mir-10a(P) 22.08 349.57 32.37 168.25 69.42 CTGGACGCGAGCCGGGCCCTTCC mmu-mir-5115(PV) 22.05 188.96 62.04 92.07 34.57 CTGGACGCGAGCCGGGCCCTTC mmu-mir-5115(MSupV) 22.04 775.70 233.89 380.53 157.79 AACCCGTAGATCCGAACTTGTGA rno-mir-100(MSupV) 22.01 768.45 357.07 382.85 189.43 TGTAAACATCCTCGACTGGAAGCG rno-mir-30a(MSupV) 2.06 118.32 23.96 243.52 139.23 TCCTGTACTGAGCTGCCCCGG bta-mir-486(MSubV) 2.39 133.14 28.86 318.16 181.42 GGGGGGCCCAAGTCCTTCTGATCGAGGCCC mmu-mir-5105(P) 2.93 73.61 16.57 215.65 93.91 CATTCAACGCTGTCGGTGAGT rno-mir-181a-2//mir-181a- 1(MSub) 40.70 6.08 0.93 247.35 243.97 CATTCAACGCTGTCGGTGAG rno-mir-181a-2//mir-181a- 1(MSub) 63.29 2.65 0.93 167.78 165.76 d i 10 1371/j l 0065809 003 Table 3. Epicardial/endocardial differential expression of unique sequences ($ 62 fold difference for miRs of $100 mean normalised reads detected in either layer). %P,0.05 Baggerleys multiple comparison test. Table 3. Prediction of Signalling Pathway Targets Epicardial/endocardial differential expression of unique sequences ($ 62 fold difference for miRs of $100 mean normalised reads detected in either layer). %P,0.05 Baggerleys multiple comparison test. Table 3. Epicardial/endocardial differential expression of unique sequences ($ 62 fold difference for miRs of $100 mean normalised reads detected in either layer). %P,0.05 Baggerleys multiple comparison test. pathway (MAPKinase; P = 1.8661026; 75 targets; Fig. 11), Regulation of Actin Cytoskeleton (P = 2.2961024; 56 targets), Wnt pathways (P = 3.4561024; 42 targets), Calcium Signaling (P = 5.0161024; 49 targets), Gap junctions (P = 6.4361024; 27 targets) and Arrhythmogenic Right Ventricular Cardiomyopathy pathway (ARVC; P = 0.047; 20 targets). Further analysis of a shortlist of genes important in contraction, electrical activity, Ca2+ signalling, cell communication, ion homeostasis and gene expression showed potential targeting of components contributing to the generation of INa, ICa,L, IKur, Ito, IKATP, INaCa, If, IKp as well as SR Ca2+ re-uptake and contraction (see Table S6). Discussion Value of Deep Sequencing Data The rat is widely used as a model of heart disease and the importance of miRNAs in heart disease is by now well recognised. Therefore it is important to know as much as possible about miRNA expression in the rat heart. Deep sequencing yields information over and above that which can be obtained from microarrays or qPCR, in that the miRNAs that can be detected are not pre-designed into the method. Value of Deep Sequencing Data Value of Deep Sequencing Data The rat is widely used as a model of heart disease and the importance of miRNAs in heart disease is by now well recognised. Therefore it is important to know as much as possible about miRNA expression in the rat heart. Deep sequencing yields information over and above that which can be obtained from microarrays or qPCR, in that the miRNAs that can be detected are not pre-designed into the method. Thus it provides the ability June 2013 | Volume 8 | Issue 6 | e65809 PLOS ONE | www.plosone.org 11 MicroRNAs in Rat Heart Left Ventricle Figure 11. Pathway analysis of MAP Kinase signaling (Kegg pathway). Highlighted genes are targets predicted by 2 or more algorithms of the top 16 highest detected miRNAs (boxed in red; P = 1.8661026; 75 targets) showing overlap with genes expressed in the heart (highlighted; expression data 9 SD rat left ventricular myocardium samples from 2 microarray experiments, (GSE6943; GSM160095–100 & GSE6880; GSM158589–91; http://www.ncbi.nlm.nih.gov/gds). doi:10.1371/journal.pone.0065809.g011 Figure 11. Pathway analysis of MAP Kinase signaling (Kegg pathway). Highlighted genes are targets predicted by 2 or more algorithms of the top 16 highest detected miRNAs (boxed in red; P = 1.8661026; 75 targets) showing overlap with genes expressed in the heart (highlighted; expression data 9 SD rat left ventricular myocardium samples from 2 microarray experiments, (GSE6943; GSM160095–100 & GSE6880; GSM158589–91; http://www.ncbi.nlm.nih.gov/gds). doi:10.1371/journal.pone.0065809.g011 (Illumina, HiSeq 2000 and SOLiDTM platforms respectively). Of the Top 20 miRNAs detected in each mouse study 6 were detected in all 3 (mmu-miR-1, 126, 378, 26a, 125b & 133a). Thirteen of the top 20 most frequently detected miRNAs in our data were also in the top 20 of Rao et al.’s data [117], while 7 were also highly detected in the Humphreys et al. study [61] and 12 were also highly detected by Yang et al. [118]. The differences between studies are likely attributable to the combined effects of different species, tissue types (left ventricular wall vs whole heart), sequencing technologies and data analysis parameters. Addition- ally, given that the myocardium is not composed solely of cardiomyocytes it is unlikely that these profiles are the product of only this cell type; indeed Humphreys et al. identified 143 miRNAs which were expressed in either mouse left ventricular or HL-1 cell samples but not in both [61]. Transmural Expression Gradients TargetScan 6.2 predicts 307 conserved targets for hsa-miR-21, 56 for hsa-miR-99b and 154 for hsa-miR-486-5p. FOXP2 is a predicted target of miR-21; its expression is higher in epicardium than endocardium [112] (Fig. 10), which is the opposite to the gradient for miR-21 expression. This is consistent with miR-21 influencing transmural FOXP2 expression. Calcineurin Aa (PPP3CA) is a predicted target of miR-21 and miR-99b (TargetScan 6.2) and is expressed more in epicardium than endocardium [113]. Calcineurin is a calmodulin-activated serine/threonine protein phosphatase that mediates cardiac hypertrophy progressing to heart failure [133] and electrical remodelling [134]. In relation to left ventricular transmural physiology, calcineurin downregulates expression of the Na+ channel Nav1.5 (SCN5A) and the gap junction proteins Cx40 (GJA5) and Cx43 (GJA1) [135], all of which are expressed in a transmural gradient opposite to that of calcineurin Aa [100,112– 114]. Expression of the K+ channel Kv4.2 (KCND2) [136], the sarcoplasmic reticulum Ca2+ pump SERCA2 (ATP2A2) [137] and the Na+-Ca2+ exchanger NCX1 (SLC8A1) [137] is upregulated by calcineurin, and the transmural expression pattern of these genes parallels that of calcineurin [112,113]. Thus, at the level of gene expression, there are consistencies that point toward a role of calcineurin Aa in determining transmural gradients in cardiac physiology. Nevertheless there may be non-transcriptional effects that make the picture more complicated. Calcineurin phosphatase activity is actually higher in endocardium than epicardium, secondary to higher intracellular [Ca2+] [138]. Calcineurin may have non-transcriptional inhibitory effects on SERCA2 [139] and NCX1 [140,141] activity. The fast transient outward potassium current (Ito,f) was increased by overexpression of constitutively active calcineurin in cultured neonatal rat ventricular myocytes [136] but reduced in calcineurin-overexpressing transgenic (TG) mice [142]. The b isoform of calcineurin A is also expressed in heart, uniformly across the left ventricular wall [138]. The relative importance of the two isoforms is not clear. Inactivation of the calcineurin Ab gene in mice caused an 80% decrease in calcineurin enzymatic activity in the heart [143], suggesting that Ab is the dominant calcineurin A isoform. On the other hand, silencing of calcineurin Aa was more effective at reducing SERCA2 expression than calcineurin Ab silencing in neonatal rat cardiomyocytes [137]. Thus a role for miR-21 and/or miR- 99b in influencing transmural physiology is an intriguing possibility but requires further study. It should also be mentioned that the determination of transmural calcineurin Aa expression was made in myocardial samples containing all cell types [113]. MicroRNAs in Rat Heart Left Ventricle [121,122], especially in disease states [5], the cellular origin of the sequence may be of less importance than where they are functionally active. version of miRBase. This is presumably because the complete miR-486 gene is not in the rat genome database. We used PCR to read into this gap and determined the sequence of the pre-miR (Fig. 9). This information should be useful for future studies of miR-486 in rat models. IsomiR Expression Variability at the 59 end is likely to have a significant effect on targeting because nts 2–8 form the seed region of the miRNA, which has a considerable contribution to target determination [1]. Therefore the ability to detect isomiRs and distinguish them from canonical miRNAs will enhance our understanding of the role of miRNAs in health and disease. A good case in point is miR-133a, of which more than half of the reads in our dataset were of a 59 variant isomiR (miR-133a(v)). Furthermore there is evidence from miRBase for a similar proportion of miR-133a(v) vs miR-133a expression in human. Since miR-133a is enriched in the heart and dysregulated in heart disease [123–128], some changes in gene expression in heart disease may be attributable to changes in miR- 133a(v). Genes predicted by DIANA-microT v3.0 to be targets of miR-133a(v) but not canonical miR-133a include gelsolin and KCNIP2 (KChIP2). Gelsolin is upregulated and promotes apoptosis in cardiomyopathy [129]. If a substantial proportion of all miR-133a isomiRs expressed in normal myocardium is miR- 133a(v) (as suggested by our data) then the downregulation of this variant could contribute to the upregulation of gelsolin in heart disease. A precedent for the possibility of miR-133a isomiR- specific targeting has already been established [61]. Additionally, KCNIP2 (an accessory protein that modulates several cardiac ion channels and is involved in cardiac arrhythmia and heart failure [130]) was downregulated by overexpression of a miR-133a genomic precursor in mouse heart [131]. This was a surprising finding because KCNIP2 is not a predicted target of miR-133a. It is plausible that KCNIP2 was downregulated by miR-133a(v) if a substantial proportion of the genomic precursor were processed to form this isomiR. Transmural Expression Gradients The above discussion pertains to cardiomyocytes but a transmural expression gradient in other cell types cannot be excluded. miR-21 is abundant in cardiac fibroblasts [119], in which calcineurin may be involved in regulating cell proliferation [144,145]. miR-21 is enriched in endothelial cells [54,55,120], in which it mediates the endothelial-to-mesenchymal transition [146] and angiogenesis [147,148]. Enrichment of miR-99b was found in cardiac valves [116], although there is no available evidence for a role of miR- 99b in valve disease. NCX1 (SLC8A1) is important for cardiomyocyte Ca2+ extru- IsomiRs with variations at the 39 end are less likely to differ in terms of genes targeted because they do not alter the seed region. However, 39 variation may affect certain target sites which rely on complementarity at the 39 end, with potentially important physiological consequences. Value of Deep Sequencing Data As the mid-myocardium (as used in the present study) also contains fibroblasts and vascular endothelial cells, the contribution of these cell types to the expression of miRNA needs to be considered; miR-21 (which we detected as 30th highest) has been shown to be expressed up to 4- fold higher in cardiac fibroblasts than myocytes [119] as well as being highly detected in various vascular beds [54,55,120]. However as some miRNAs are known to be highly exported to detect novel sequences, including novel miRNAs and novel isomiRs of known miRNAs. This advantage is well illustrated in the present study. Our data provide some interesting comparisons with the few previous next-generation sequencing studies in the heart. Linsen et al. [88] analysed rat heart using the SOLiDTM platform (Life Technologies) and detected 180 miRNAs in common with our study. There were 206 miRNAs detected in the present study that were not found in rat heart by Linsen et al. and 357 miRNAs reported in heart by Linsen et al. did not appear in our data. However of the top 20 miRs detected by Linsen et al., 10 were also highly detected in the present study. More recently Vacchi- Suzzi et al. [116] also investigated the expression of miRNA in rat left ventricle by next generation sequencing using HiSeq in conjunction with TruSeq Small RNA Sample Preparation Kits (Illumina). Vacchi-Suzzi et al. identified 636 rno- and/or mmu- miRs including expression of mmu-miR-486 as well as co- incidence of 13 of their top 20 highest detected miRNAs with the present study. In the 3 rat studies 5 common miRNAs were highly detected; rno-miR-26a, 30a, 30c, 30d & 30e. In mouse heart 3 groups have investigated the miRNA expression profile ([61,117,118] using next generation technologies PLOS ONE | www.plosone.org June 2013 | Volume 8 | Issue 6 | e65809 June 2013 | Volume 8 | Issue 6 | e65809 12 MicroRNAs in Rat Heart Left Ventricle Signalling Pathways Within the Ubiquitin-mediated Proteolysis pathway the E1 enzyme UBLE1A and 14 of the 22 heart-expressed E2 enzymes are predicted to be targeted by the 16 most highly expressed miRNAs. Six of the 11 heart-expressed HECT type E3 enzymes, 3 out of 5 heart expressed U-box type E3 enzymes, 6 of 13 heart expressed single RING-finger type E3 enzymes and 10 of 36 heart expressed proteins within the multi subunit RING-finger type E3 complex are predicted targets. Recent studies have described how ubiquitination in the heart is upregulated in common cardiac diseases, including cardiac hypertrophy, heart failure, ischemia- reperfusion injury, and diabetes (for review see [150]). The overwhelming nature of the predicted targeting of the Ubiquitin- mediated Proteolysis Pathway suggested by the expression profile of our samples may reflect the importance of keeping this mechanism under tight control in normal tissue. Table S1 Sequences of PCR primers. (PDF) Table S2 Oligonucleotide sequences of pSM30 inserts utilized to express artificial pre-miRs. (PDF) Table S3 MiRNA reads annotated to previously char- acterised rat sequences. (PDF) Table S4 MiRNA annotated to sequences not previously characterised in rat but identified in other species. (PDF) Table S5 *strand miRNA annotated to previously characterised sequences. (PDF) Within the MAP Kinase pathway 55 of the 98 heart expressed gene families (representing a total of 75 targets in 151 heart genes) were predicted as potential targets (Fig. 11). Interestingly the Ras gene family within this pathway was predicted to be targeted by 14 of the top 16 highest detected miRNAs; this family of genes as a part of the Ras/Raf-1/MEK/ERK cascade is generally regarded as playing a highly significant role in cardiac hypertrophy, myocardial cell death and myocardial remodelling [151]. Table S6 Shortlist of genes relevant to cardiac contrac- tile function predicted to be targeted by the top 16 highest detected miRNAs. (PDF) Table S6 Shortlist of genes relevant to cardiac contrac- tile function predicted to be targeted by the top 16 highest detected miRNAs. Another highly targeted pathway, Wnt signalling, which is required for basic developmental processes such as control of asymmetrical cell division, remodelling of the cytoskeleton and cell adhesion [152,153] is likely to be of importance in the heart especially in relation to the Wnt5 receptor, as Frizzled, and downstream proteins PLC, CAMKII and Calcineurin are all implicated in NFAT transcriptional changes associated with hypertrophy [154,155]. Novel Rat miRNAs and miRNA Genes The importance of miR-1 in the heart is by now well recognised [132]. Manipulation of miR-1 expression in the rat heart may be a useful strategy for further investigating the physiological role of miR-1. However, such studies could have been confounded by the unknown existence of a second miR-1 gene. Our discovery of the second gene is therefore critical for miR-1 expression studies in the rat. The putative novel miRNA reported in Fig. 8 has potential implications for the expression of its target genes. DIANA-microT v3.0 predicts 25 target sites in 7 genes for this miRNA sequence. The putative target genes are ADAMTS13 (von Willebrand factor-cleaving protease), SLC6A3 (Sodium-dependent dopamine transporter), PCNT (Pericentrin), MBD3 (Methyl-CpG-binding domain protein 3), ICOSLG (ICOS ligand precursor (B7 homolog 2)), FOXK2 (Forkhead box protein K2), and FOXK1 (Forkhead box protein K1). Small et al. [72] reported enrichment of miR-486 in cardiac and skeletal muscle, and presented evidence for miR-486 as a downstream mediator of the actions of serum response factor, myocardin-related transcription factor-A and MyoD in muscle cells and a potential modulator of PI3K/Akt signalling in rat neonatal cardiomyocytes. Despite the likely importance of this miRNA in rat muscle, rat miR-486 does not appear in the current NCX1 (SLC8A1) is important for cardiomyocyte Ca2+ extru- sion. Its higher expression in epicardium vs endocardium [113] is consistent with high endocardial intracellular [Ca2+] [138]. NCX1 June 2013 | Volume 8 | Issue 6 | e65809 PLOS ONE | www.plosone.org 13 MicroRNAs in Rat Heart Left Ventricle is a predicted target of miR-486 (TargetScan 6.2), which we found to be higher in endocardium than epicardium (Fig. 10). Therefore miR-486 could conceivably influence transmural differences in intracellular Ca2+ handling. highlighted the complexity of the in vivo miRNA profile, as suggested by previous studies of cardiomyocyte cells [61]. Many miRNAs not previously reported in rat were detected and for each miRNA a range of isomiRs was present. Thorough characterisa- tion of the normal pattern of miRNA expression is an important basis from which to study the role of miRNAs in regulating gene expression in the heart. Whilst transmural expression was uniform for most miRNAs, the gradients found for several miRNAs are intriguing and suggest potentially important roles in heart physiology. IRX5 is a predicted target of miR-486, as discussed above. IRX5 negatively regulates Kv4.2 K+ channel expression, and the IRX5 transmural expression gradient (higher in endocardium) [112] (Fig. Acknowledgments We thank Dr G. Du for the pSM30 plasmid and Dr A. O’Connor for the non-targeting control construct and assistance with qPCR. We thank Dr G. Du for the pSM30 plasmid and Dr A. O’Connor for the non-targeting control construct and assistance with qPCR. Signalling Pathways All of our top 16 highest detected miRNAs are predicted to target at least one of the genes encoding proteins in the Wnt5 pathway. Novel Rat miRNAs and miRNA Genes 10) is responsible for the expression gradient (higher in epicardium) of Kv4.2 in the heart [76,77]. miR-486 may be able to influence IRX5 expression but its own expression gradient tends in the opposite direction for it to be responsible for the transmural gradient of IRX5. Author Contributions Conceived and designed the experiments: MKMcG JG-F DAS AC. Performed the experiments: MKMcG JMY AD LJF AC. Analyzed the data: MKMcG JMY LJF DAS AC. Wrote the paper: MKMcG DAS AC. Revising the article critically for important intellectual content: MKMcG JMY AD JG-F LJF DAS AC. Supporting Information DIANA-microT v3.0 predicts 147 targets in 85 genes for the most abundant rno-miR-10b isomiR (Fig. S2). Among these, the transcription factor TBX5 is expressed in a transmural gradient that changes with heart development [149]. Figure S1 miRNA library construction from three rat heart samples assessed using the Bioanalyser 2100. 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In: Barciszewski J, PLOS ONE | www.plosone.org June 2013 | Volume 8 | Issue 6 | e65809 June 2013 | Volume 8 | Issue 6 | e65809 16 MicroRNAs in Rat Heart Left Ventricle (2008) In calcineurin-induced cardiac hypertrophy expression of Nav1.5, PLOS ONE | www.plosone.org June 2013 | Volume 8 | Issue 6 | e65809 17
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ERROR: type should be string, got "https://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\nValidation of a modelling methodology for wind turbine rotor blades\nbased on a full scale blade test\nPablo Noever-Castelos1 , Bernd Haller2 , and Claudio Balzani1\n1\n2\n\nLeibniz University Hannover, Institute for Wind Energy Systems, Appelstr. 9A, Hanover, 30167, Germany\nFraunhofer Institute for Wind Energy Systems IWES, Am Seedeich 45, 27572 Bremerhaven, Germany\n\nCorrespondence: Pablo Noever-Castelos (research@iwes.uni-hannover.de)\nAbstract. Detailed 3D finite element simulations are state of the art for structural analyses of wind turbine rotor blades. It is\nof utmost importance to validate the underlying modelling methodology in order to obtain reliable results. Validation of the\nglobal response can ideally be done by comparing simulations with full scale blade tests. However, there is a lack of test results\nfor which the blade data are completely available.\nThe aim of this paper is to validate one particular blade modelling methodology that is implemented in an in-house model\n\n5\n\ngenerator, and to provide respective test results to the public. A hybrid 3D shell/solid element model is created including the\nrespective boundary conditions. The problem is solved via a commercially available finite element code. A full scale blade\ntest is performed as the validation reference, for which all relevant data are available. Some data have been measured prior to\nor after the test in order to account for manufacturing deviations. The tests comprise classical bending tests in flap-wise and\n10\n\nlead-lag direction as well as torsion tests.\nFor the validation of the modelling methodology, global blade characteristics from measurements and simulation are compared. These include the overall mass and centre of gravity as well as their distributions along the blade, deflections, strain\nlevels, and natural frequencies and modes. Overall, good agreement is obtained, though some improvements might be required\nfor the response in torsion. As a conclusion, the modelling strategy can be rated as validated.\n\n15\n\n1\n\nIntroduction\n\nRotor blades are major components of wind turbines. They are susceptible to damages, which, in case they need repair, can\nresult in severe turbine downtimes (Reder et al., 2016). It is thus crucial to develop a blade design that withstands all designated\nloads without damage. Though a blade prototype is always tested at the full blade scale in the certification process (International\nEletrotechnical Comission, 2014), such tests are very costly and time-consuming, especially for growing blade dimensions (Ha\n20\n\net al., 2020). For this reason, full scale blade tests are executed one time only for the final validation of design assumptions.\nHence, a reliable and fast virtual blade design procedure is required. Full 3D finite element (FE) analysis is accurate but\ncomputationally expensive. A widely used approach for wind turbine blade design is to carry out two-dimensional crosssectional analyses that offer a reduced level of complexity but are a fast and efficient alternative for rotor blade pre-designs\n(Chen et al., 2010). Tools like VABS (Yu et al., 2002) or BECAS (DTU Wind Energy) compute cross-sectional properties based\n\n1\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\n25\n\non a 2D-FE-analysis, which are necessary to feed the aero-elastic models in order to recalculate the design loads on the turbine\nblades and close the design iteration loop. Nevertheless, at a final stage 3D FE analyses have to be performed in order to obtain\na reliable blade design and double check structural details such as adhesive joints, geometric discontinuities, ply drops, etc.\nAutomated model creation is state of the art and a key to enhance the design process significantly by reducing time consumption, increasing the possible number of design loops and avoiding modelling errors. Among a vast selection of common\n\n30\n\nsoftware tools originated from the scientific community, QBlade (D. Marten et al., 2013) for example focuses on the aerodynamic blade design, neglecting details on structural information. Sandia’s NuMAD (Jonathan C. Berg and Brian R. Resor,\n2012) additionally contains a more sophisticated structural description taking into account a proper composite definition for the\nblades’ subcomponents. Same holds for the software package FOCUS developed by WMC Laboratories which is now part of\nLM Wind Power (N. P. Duineveld, 2008), which is a state of the art tool used for blade design in many engineering offices. In\n\n35\n\nFOCUS the user discretizes a few stations in span-wise direction with all necessary geometrical information of these particular\ncross-section and in between the tool interpolates linearly all missing data. Hence a high discretization of stations along the\nblade span is necessary to correctly reproduce non-linear changing geometrical or material information in span-wise direction.\nAnother more advanced tool is the optimization framework CP-Max, see Bottasso et al. (2014). The parametrization is based\non mathematical functions for the blade design description in span-wise direction. This method has the advantage of reducing\n\n40\n\nthe number of stations along the blade without loosing information in between, while enabling the framework to efficiently\nmanipulate the parameters during optimization. The focus of the optimization framework is to find a solution at minimum\ncosts. A similar blade parametrization is used within the FUSED-Wind Framework (Zahle et al., 2020), which contains spline\ndescriptions for each parameter as shown in the prominent example of the DTU 10MW reference blade design (C. Bak et al.,\n2013). An interface to the framework was later incorporated into the python-tool FEPROC and the correct modelling process\n\n45\n\nwas verified against the DTU 10MW reference blade (Rosemeier, 2018). Another blade modelling tool developed at Ghent\nUniversity also relies on function-based descriptions of the blade parameters and focuses on a modular principle of Finite\nElement (FE) constellations for modelling the different blade components and joints in the structure (Peeters et al., 2018). The\nlatter algorithm and CP-Max are able of generating solid element models, while the others rely on more common shell element\nrepresentations.\n\n50\n\nThough some of these model creation frameworks may work with functions or splines describing the blade’s geometrical or\nlayup information, most of them work with a reasonably high number of airfoils/stations that in addition to the blade’s geometry\nyield the outer blade shape by a global linear or higher order interpolation between the airfoils. Similar to Rosemeier (2018),\nwho uses Parametric Geometry Library (PGL, Zahle (2020)) to interpolate additional airfoils, the presented algorithm in this\nwork generates airfoils independent from the other parameters and uses the relative thickness distribution to position these\n\n55\n\nalong the span. Additionally a more detailed adaption of blade and material parameters is implemented to consider changes\nalong the span for all characteristics of the blade’s structure.\nA lot of scientific contributions deal with FE modelling and focus on structural details like trailing edge adhesive joints. Eder\nand Bitsche (2015) for instance use a local model with fracture analysis to deduce the debonding between shell and adhesive\ndue to buckling and validate the behaviour against experimental results. Ji and Han (2014) also apply fracture mechanics and\n2\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\n60\n\nuse a detailed model at the shear web adhesive joint to analyse crack propagation in the bond line. Most of these locally detailed\nmodels are used within a global-local modelling approach like in Chen et al. (2014) to reduce the global model complexity\nwhile keeping a high level of detail at local spots. However, this paper focuses on the global elastic response of wind turbine\nblades, so there is no need for local sub-models.\nDifferent FE modelling procedures can result in different deformation and stress solutions, though based on the same model\n\n65\n\nparameters, see (Lekou et al., 2015). Hence, it is important to validate modelling strategies by comparing simulations with full\nblade tests, which is the aim of this paper. A quasi-static full scale blade test is performed, including not only bending tests in\nflap and lead-lag direction – as are usually executed in the context of blade certification (International Eletrotechnical Comission, 2014) – but also torsion tests. This allows for an exceptionally detailed and thorough validation. Unlike other blade tests\nreported in literature (?), (Chen et al., 2017), (Jensen et al., 2006), (Overgaard and Lund, 2010), (Overgaard et al., 2010), the\n\n70\n\naim of the tests in this work is not the validation of failure models. Hence, the blade is not loaded up to failure. The aim rather\nis to measure the global blade behaviour expressed in terms of deflections, strains, mass distribution, and modal characteristics\nand to validate our own blade modelling technique. The blade under investigation is the SmartBlades DemoBlade (REFERENCE TO COME), a 20 m blade including pre-bend and pre-sweep towards the trailing edge. The blade is modelled with our\nin-house blade model creation tool MoCA (Model Creation and Analysis Tool for Wind Turbine Rotor Blades), taking into\n\n75\n\naccount some major manufacturing-related deviations. The test setup and the load introduction are approximated via a combination of suitable boundary conditions and multiple point constraints. The simulation results are thoroughly compared with\nthe test measurements. Generally good agreement is observed, especially for the bending loads. However, some improvements\nmay be required for accurately modelling the torsional behaviour of the blade.\nThe modelling strategy is addressed in section 2 and section 3. The test setup is described in section 4. The blade was cut into\n\n80\n\nsegments after the tests in order to accurately measure the mass distribution and the locations of the centres of gravity along\nthe blade. These measurements are also described in section 4. The simulation versus test comparison is reported in section 5,\nfollowed by the conclusions in section 6.\n2\n\nModel Creation Framework\n\nA framework to automatically generate fully parameterized 3D FE models of wind turbine rotor blades from a set of parameters\n85\n\nwas developed at the Institute for Wind Energy Systems at Leibniz University Hannover. The purpose of this tool called MoCA\n(Model Creation and Analysis Tool for Wind Turbine Rotor Blades) is to enable users to investigate and analyse different\nblade designs or design parameter variations in an efficient way, including structural details such as e. g. adhesive joints. The\nfollowing section presents a brief description of the framework.\nMoCA is based on a set of input parameters categorised in Geometry, Plybook, Structure, and Material. In general all\n\n90\n\nparameters that describe a distribution along the blade are stored as splines over the blade’s arc length, but even material\nparameters may be varied over the blade arc if necessary by using a spline. The parameter set Geometry contains all information\non the outer geometry of the blade, i. e. the airfoils used and their positions along the blade as well as the distributions of the\n\n3\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\nrelative thickness, chord length, twist angle, threading point location, prebend and presweep. The Structure set is associated\nwith the structural description of the blade. This includes the specification of shear webs, adhesive joints and additional masses\n95\n\nas well as cross-sectional division points that are mainly used to subdivide cross-sections into different regions of interest.\nThe Plybook parameters contain the stacking information of different composite layups used in the blade. The parameter set\nMaterial comprises all material properties assigned for the different materials. These can either be isotropic or anisotropic on\nthe macroscopic scale. The user can also specify a composite material based on microscopic characteristics of the fibre and\nmatrix constituents, which are then transformed to a laminate via the well-known rule of mixtures.\n\n100\n\nThe flowchart in figure 1 depicts the structure of the finite element creation procedure implemented in MoCA on the basis\nof the parameter sets described above. First, the blade segmentation, i. e. the discretization in span-wise direction, is defined.\nFor each blade segment edge, a cross-section of the blade is calculated by evaluating the Geometry data. Then a finite element\ndiscretization of the cross-sections is executed using the information of the Structure, Material, and Plybook parameter blocks.\n\n105\n\nAt this stage, an interface to the BECAS (DTU Wind Energy) software can be utilized to calculate the full 6 × 6 stiffness\n\nand mass matrices of a beam model. However, since our aim is to create a 3D blade model, we continue with the finite\n\nelement discretization in span-wise direction utilising a hybrid shell element/solid element strategy. Therein, we use shell\nelements to model the composite laminates and solid elements for the adhesives. The 3D FE mesh includes the node-toelement connectivity and elemental material assignments. The boundary conditions are added and the FE model is translated to\nan input file for the finite element solver of choice, which in our case is ANSYS Mechanical (ANSYS Inc.). In the following,\n110\n\nwe describe in more detail the different steps of this overall procedure.\n\nStructure\n\nPlanform\n\nLength discretization\n\neval. CS geometry\n\nCSShape\n\nMaterial\n\nPlybook\n\n2D meshing\n\nBECAS interface\n\n3D conectivity\n\n2D 2- & 4-Node Elems\n\nHybrid Shell & Solid 3D Mesh\n\nANSYS interface\n\nFigure 1. Flowchart of the finite element model creation procedure in MoCA.\n\nFigure 2 visualises the process of cross-section geometry calculation. After the blade segmentation, the Geometry data\nsplines are evaluated for the particular blade arc positions of the segment edges. Based on the spline-based interpolation of the\nrelative thickness trel , an airfoil AF is linearly interpolated between the basic input airfoils with the next higher and lower\nrelative thickness. In contrast to a global blade shape interpolation, the use of a blade independent airfoil interpolation enables\n115\n\nthe user to implement an own sub-function and replacing the former. The interpolated airfoils are then scaled by the chord\n4\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\nlength c∗ calculated via the respective spline, shifted along the chord to the correct threading point by the coordinate tp∗ , and\ntwisted by the twist angle θ∗ .\n\nArc Position\n\nrel. Thickness Spl trel\n\nTwist Angle Spl θ\n\nChord Length Spl c\n\nPrebend Spl pb\n\nThreading Point Spl tp\n\nPresweep Spl ps\n\neval. spline\n\nAirfoils AF *\n\ntrel *\n\nc*\n\ntp*\n\nθ*\n\npb*\n\ninterp. AF\n\nscale. AF\n\nthread. AF\n\ntwist. AF\n\n3D pos. AF\n\nps*\n\nCross Sectional Shape CSShape\n\nFigure 2. Flowchart of the calculation of the cross-sectional shapes CSShape .\n\nUntil here, all transformations are performed in a 2D chord coordinate system with its final origin in the threading point. The\ncross-sections are now shifted to the correct 3D position, locating the 2D cross-sectional threading centre on the prebended\n120\n\nand preswept global blade axis. By doing so, the 2D chord coordinate system is still parallel to the blade root plane. Hence, the\ncross-sections are rotated by the slope angles of the prebend and presweep spline functions so that the they are perpendicular\nto the threading axis. These shifted and rotated cross-sections are the final cross-sectional shapes denoted by CSShape .\nAccording to figure 1, the next step is the 2D cross-sectional meshing, which is executed using the cross-sectional shapes\nCSShape and the parameter sets Structure, Material, and Plybook. This process is presented in figure 3. As before, all data is\n\n125\n\nevaluated for the particular arc positions at the blade segment edges. The division points are generated on the cross-sectional\nshapes. They serve to subdivide the cross-sections into regions of different material layups. They are also used to define the\npositions of the shear webs. Then the shapes of the shear web/spar cap and/or trailing edge adhesive joints are computed. The\ncomputation of the blade’s outer geometry and its structural topology is now finished. After inclusion of the Material and\nPlybook information, the FE discretization on 2D cross-section level can be conducted. This yields either a two-dimensional\n\n130\n\nmesh with 4-noded plane elements for the BECAS (DTU Wind Energy) interface or a cross-sectional node map representing a\nhybrid 2D mesh with 2-noded elements for the composite laminates and 4-noded elements for the adhesives.\n\n5\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\nDivision Points dp\n\nArc Position\n\nAdhesive Information adh\n\nWeb Informaiton web\n\nMaterial Information mat\nPlybook Information plyb\n\neval. spline\n\nCSShape\n\ndp*\n\nweb*\n\nadh*\n\nset division points\n\npos. webs on CSShape\n\ndef. adhesive shape\n\ndiscretize shapes\n\nmat*\n\nplyb*\n\n2D 4-Node Elems\n\nBECAS interface\ndef. section layup\n\n2D 2- & 4-Node Elems\n\nFigure 3. Flowchart of the 2D cross-sectional meshing routine in MoCA.\n\nThe last step in the creation of a 3D finite element model is to connect the 2D cross-sectional models, see figure 1. The 2D\nline elements on the cross-sectional level yield 4-noded shell elements on 3D level after the 3D extension, and the 4-noded\nplane elements on cross-sectional level become 3D solid elements, respectively.\n135\n\nAn additional module called TestRig is included in MoCA to model the boundary conditions similar to a full scale blade\ntest. Full clamping of the blade root represents the geometrical boundary conditions, i. e. all degrees of freedom are fixed at\nthe blade root. Figure 4 shows the process of the TestRig module for the introduction of force-like boundary conditions. In the\nreal blade test, a number of load frames introduces loads that approximate the target bending moment distribution (or torsional\nmoment distribution, respectively). The TestRig module approximates the load frames by means of appropriate multiple point\n\n140\n\nconstraints MPC and additional masses. For each load frame, the position along the blade (arc position), the load frame width,\nthe centre of gravity (CoG) and the resulting mass are specified as well as the load and sensor points.\nIn the range where the load frame is located, MoCA searches all elements of the blade shell and defines 2D slave elements\nthat share their nodes. An additional cross-section is created at the desired load frame position according to the procedure\ndepicted in figure 2. In this additional cross-section, the position of the load introduction (load point), the sensor points, and\n\n145\n\nthe centre of gravity of the load frame are given in the blade coordinate system. These points are defined as master nodes.\n\n6\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\nArc Position\n\nLoad Points LP\nSensor Points SP\nCentre of Gravity CoG\n\nLoad Frame Width\n\nShell 3D Model\n\neval. CS geometry\n\nPlanform\n\ncollect Shell Elements\n\nCSShape\n\neval. Point Locations\n\ncreat Slave Nodes\n\ncreat MPC\n\ncreate Master Nodes\n\nLoad\n\nMass\n\nLoad Frame\n\nTest Rig\n\nFigure 4. Flowchart of the procedure to model the boundary conditions in the TestRig module.\n\nMPCs are included that connect the degrees of freedom of the master nodes and the slave nodes by means of a rigid connection,\ni. e. there are no relative displacements between the master and the slave nodes. The additional mass of the load frame is applied\nto the CoG node, while the load is applied to the position where the load is introduced in the real test (load point). In this way,\nwe model solid and quasi-rigid load frames and their effects on the blade response without adding detailed models of the load\n150\n\nframes themselves, which is beneficial in the context of computational costs.\nThe 3D finite element model including the mesh and the boundary conditions is translated to an input file for the finite\nelement solver of choice via an integrated interface.\n3\n\nModelling of the Test Blade\n\nThis section briefly describes the blade under consideration, which is the SmartBlades-DemoBlade, a 20m long blade with\n155\n\nprebend and presweep. It was designed and manufactured in the coordinated research projects Smart Blades (Teßmer et al.,\n2016) and SmartBlades2 (SmartBlades2, 2016-2020). The blade is abbreviated by DemoBlade in the following.\nThe DemoBlade was designed to investigate bend-twist coupling effects in wind turbine rotor blades. Therefore a presweep\nof 1 m towards the trailing edge at the tip is intended to introduce a torsional twist into the blade. The offset between the\naerodynamic centres of the swept airfoils and the pitch axis introduces a torsional moment and thus a torsional deformation, i. e.\n\n7\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\n160\n\na twist in the outer part of the blade. The twist reduces the angle of attack of the respective airfoils and hence the aerodynamic\ncoefficients. In this way the aerodynamic loads can be reduced.\nThe full blade design of the DemoBlade as designed and the manufacturing documentation is available to the authors. In\norder to allow precise modelling of the DemoBlade as built laser scanning of the blade mould was carried out in order to determine the geometry deviations. The derived chord length and absolute thickness distributions for the DemoBlade as designed\n\n165\n\nand as built can be found in Noever-Castelos et al. (2021). Though the manufacturing deviations in the outer geometry are\nnegligibly small, they will be considered in the modelling process.\nAfter the full scale blade tests, the DemoBlade was cut into segments. The masses and the centres of gravity were determined\nfor all blade segments. The respective procedure will be addressed later in this paper, see sections 4.4 and 5.5. Besides the\n\n170\n\nweighing, the geometry was measured thoroughly in each cut cross-section in order to guarantee the correct positioning of the\nshear webs in the FE model and to determine deviations from the design due to manufacturing errors. Especially the dimensions\nof the shear web/spar cap adhesive joints on the pressure side of the blade showed significant deviations to the blade design and\nhad to be adjusted in the FE model. Figure 5 shows the cut at a radial position of 5.2 m. On the suction side we see a nice, thin,\nand over-laminated shear web/spar cap bonding. However, on the pressure side the shear web/spar cap adhesive joint (which\n\n175\n\nwas the blind bond) is much thicker than specified in the design. Moerover, there is a lack of adhesive in large portions of the\nblade, so that the shear web flanges were not covered entirely by adhesive material. Noever-Castelos et al. (2021) contains the\nactual dimensions of the pressure side web adhesive.\nIn the FE model, we apply concentrated and line-distributed additional masses to cover any type of add-ons installed on the\nblade such as the lightning protection cable or reflectors of an optical sensor system. Noever-Castelos et al. (2021) includes\n\n180\n\na table wit all additional masses and the respective modelling methods. MoCA furthermore predefines node positions in the\nblade that correspond to strain gauges installed on the blade. These are documented in Haller and Noever-Castelos (2021).\nThey allow for accurate and easy extraction of strain results at the correct positions.\nIn advance a mesh convergence study based on strain results at different positions was performed to ensure a qualitatively\nsatisfying mesh density. The resulting base model of the DemoBlade consists of 77,693 elements and 71,781 nodes. A total\n\n185\n\nof 71,016 4-noded shell elements (SHELL181 elements in ANSYS) with offset nodes on the outer blade surface represent\nthe composite components and 6,260 8-noded solid elements (SOLID185 elements in ANSYS) the adhesive joints. All other\nelements are used to model additional masses in the blade. The only boundary conditions of the base model are the geometric\nboundary conditions at the blade root (full clamping as described above).\n4\n\n190\n\nTest Description and Virtual Modelling\n\nSeveral test configurations of the full scale blade test were performed to characterize the blade behaviour under different load\nconditions and to prove that the blade design meets all requirements of the certification guidelines(International Eletrotechnical\nComission, 2014). These configurations are than replicated in the virtual test setup and are described in this section.\n\n8\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\nFigure 5. Cut cross-section at a radial position of 5.2 m with a erroneous shear web/spar cap adhesive joint on the pressure side of the blade.\n\n4.1 Mass and Centre of Gravity\nThe first structural characterization considers the blade’s mass and centre of gravity (CoG). An indoor crane equipped with\n195\n\nload cells at every hook lifted two points on each root and tip transport structure as shown in Figure 6. As the blade remained\nstill and horizontally suspended the measurements and radial position of each suspension point was recorded. After weighing\nthe transport structures, loading chains and shackles individually, the weight was subtracted from the total recorded load at the\nmeasurement devices to obtain the total blade mass. Additionally, the weight of the blade bolts was subtracted from the total\nmass.\n\n200\n\nThe CoG is obtained by calculating the moment equilibrium with the measured loads with respect to a pivot point, in this\ncase the blade root centre. This procedure was performed for the z-direction (along the span) and y-direction (along the chord).\nThe mass and CoG of the FE model is calculated during every analysis by default and can be extracted directly from the\nANSYS log-file.\n9\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\nFigure 6. Setup for mass and centre of gravity measurements.\n\n4.2 Modal Analysis\n205\n\nThe experimental modal characterization was carried out by the German Aerospace Center (DLR) for different boundary\nconditions. The methodology is described briefly in the following. For details please refer to Gundlach and Govers (2019).\nFree-free boundary conditions were applied after the blade manufacturing by means of elastic suspensions connected to\nlifting straps. The blade was excited using an impact hammer with soft tip at a total of 8 excitation points. Sensors distributed\nalong the blade recorded the deformations, and the mode frequencies and shapes were extracted from the measurements.\n\n210\n\nThe blade was then transported to Fraunhofer IWES and mounted on the test rig. The aim was a second modal characterization with the boundary conditions of the full scale blade test. Electrodynamic long stroke shakers were employed for the\nexcitation of the blade, and sensor outputs were evaluated for the calculation of the mode frequencies and shapes.\nDuring the FE modal analysis, the boundary conditions are adapted to the different characterization tests. In the free-free\nconfiguration, no boundary conditions are applied at all, partially resulting in zero eigenvalues related to rigid body motions.\n\n215\n\nThese are not considered in the validation process. For the test rig configuration, the blade root is fully clamped, i. e. all 6\ndegrees of freedom of the shell elements are fixed, for the sake of simplicity. Note that we neglect flexibilities of the bolts and\nthe test rig in this way, which we have to keep in mind when evaluating the simulation results.\n\n10\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\n4.3 Static Bending and Torsion Test Configuration\nThe SmartBlades2 DemoBlade was loaded with extreme loads in 4 directions before and after the fatigue test. These four load\n220\n\ncases correspond to maximum and minimum edge-wise loading (MXMAX and MXMIN) as well as maximum and minimum\nflap-wise loading (MYMAX and MYMIN). Furthermore, three static torsion tests were conducted before the fatigue tests,\nin which a torsion moment was applied only at one load frame at a time. The tests are referred to as MZLF2, MZLF3 and\nMZLF4, where LFX indicates the particular load frame, in which the torsion moment was introduced. The static tests provide\nthe necessary information on the structural blade behaviour required to validate the virtual model and test setup.\n\n225\n\nThe tests were performed in the facilities of Fraunhofer IWES, where the blade was mounted almost horizontally on a\ntest rig. The experimental quasi-static loading of the blade is accomplished with a series of horizontally mounted hydraulic\ncylinders. These are connected to the load cells via cables which are attached to the load frames mounted on the rotor blade.\nEach cable runs through pulleys that are mounted on the floor and redirect the forces from a horizontal to a vertical orientation.\nBy attaching the load cells to the load frames (load point), the actual load applied to the rotor blade is measured and friction as\nwell as weight of the loading cables do not affect the measurements. The general test setup is shown in Figure 7.\n\nFigure 7. Photo of a static blade test configuration in flapwise direction.\n230\n\n11\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\nIn the following, some general information is given that is valid for all test setups. The test block angle (cone angle) is 7.5°\nupwards. The coordinate system referred to in this paper has its origin in the centre of the blade root. The y-axis is vertical,\nthe z-axis points horizontally from the origin towards the blade tip (parallel to the floor, not to the pitch axis), and the x-axis\nfollows from the right-hand rule (pointing left watching towards the tip). After turning the blade to the correct position and\n235\n\nwaiting for a static state, the signals of the load cells and the strain gauges are reset to zero. In the virtual test this is achieved\nby activating gravity, extracting the deformed nodal coordinates and taking these as the undeformed and stress-free state for\nthe load tests. Gravity is thus not applied in the further analysis and the nodal displacements are virtually reset to zero so that\nit is easier to postprocess the results. Preliminary verifications showed that the corresponding error is less than 0.5%.\nIn the tests, four steel load frames with wooden inlays that follow the blade shape at the respective span-wise positions are\n\n240\n\nused to introduce the loads, see Haller and Noever-Castelos (2021). In the following, we refer to the load frames (LF) as LF1\n(@ r = 6.7m), LF2 (@ r = 9.7m), LF3 (@ r = 14.0m), and LF4 (@ r = 17.7m), where r denotes the span-wise position along\nthe blade. Depending on the test setup, not all load frames are installed. Please refer to Noever-Castelos et al. (2021) to find an\noverview of all test setups. Each load frame is equipped with two eye-bolts to attach the load cables. These bolts are roughly\npositioned at the shear centre position in the blade’s cross-section to avoid unintended torsion loads. Detailed information on\n\n245\n\nthe load frames, such as mass, centre of gravity, and the corresponding shear centre position in the blade’s cross-section are\ngiven in Haller and Noever-Castelos (2021) .\nThe test setup is equipped with two different kinds of displacement measurements, an optical displacement measurement\nsystem and draw-wire-sensors (DWS). For the model validation in this paper, the DWS signals are considered. Using LINK11Elements in ANSYS provides a simple and exact model of the draw-wires by defining the attachment points only. The defor-\n\n250\n\nmation measured by the DWS is then modeled by the element-length variations of the link elements.\nAll necessary sensor positions (SP) and load introduction points (LP) on the load frames for the different test setups can\nbe found in Haller and Noever-Castelos (2021). At each load frame position, either with or without installed load frame, two\nDWS are attached. One is connected to a point most to the front bottom corner, i. e. negative y-direction and one at the rear\nbottom corner, i. e. positive y-direction, of the load frames or blade shells in case no load frame is installed. These two DWS\n\n255\n\nwill be referred to as front and rear DWS in the following. At the blade tip, one DWS is attached referred to as Tip DWS. Note\nthat during several load cases, one or the other load frame is not applied due to the setup design, thus the respective DWS have\nto be attached directly to the blade shell.\nThe angle between the loading cable and the blade axis can be adjusted in the experiment by changing the pulley block\nlocation within a discrete set of fixing points on the floor. Prior to the test setup, the optimal position for each pulley was\n\n260\n\ndetermined based on the predicted blade deformation and the desired loading cable angle. The applied loads should be aligned\nto the load frame planes in the deformed configuration. The DWS floor attachment and pulley block positions are specified for\neach test setup individually.\nAdditional to the DWS and the optical measurement system, several cross-sections along the blade are equipped with strain\ngauges, see Haller and Noever-Castelos (2021). The cross-sections at r = 5 m and r = 8 m are instrumented with strain gauge\n\n265\n\nrosettes (bi-axial strain gauges) with 0°/90° and ±45° orientations. The angles 0° and 90° denote the span-wise and the cross12\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\nsection-wise direction, wheres ±45° is defined accordingly. The 0°/90° rosettes are positioned every approx. 250-300 mm\n\nalong the shell circumference. The ±45° rosettes are located at each web position as well as the leading and trailing edges.\nDetails on strain gauge positions can be found in Haller and Noever-Castelos (2021).\n\nAll load cases have the same basic experimental procedure. They were designed to ensure that the actual test matches the\n270\n\nspecification requirements as closely as possible. Prior to each load case, the rotor blade is rotated to the desired position and\nmounted to the test stand (with the aforementioned 7.5° cone angle). The load cable pulley blocks are fixed to the appropriate\nfixation points on the floor. The load cells are installed between the load frames and the loading cables and are then connected\nto the data acquisition system. Each of the DWS is attached to the blade. The DWS base is positioned so that the wires run\nperpendicular to the floor. Finally, the loading cables are connected to the hydraulic cylinders.\n\n275\n\nThe tests are then executed in the following order:\n1. Functionality check of load cells and displacement sensors.\n2. Compensation of load cell and strain gauge measurements (reset to zero).\n3. Start data acquisition.\n4. Ramp up loads until 100% of the target load, pausing at 40%, 60% and 80% partial loads for 10s each.\n\n280\n\n5. Ramp down loads, pausing at same load fractions as at ramp up.\n6. Stop data acquisition and save measurement data to log file.\nThe process is similar in the simulation. Starting from the base model, which does not have a cone angle and the blade is\npositioned with the trailing edge pointing upwards, the steps are as follows:\n1. Install necessary load frames.\n\n285\n\n2. Rotate blade around z-axis to desired position.\n3. Include cone angle of test rig (incline the blade by 7.5° upwards around x-axis).\n4. Apply gravity and extract new nodal coordinates.\n5. Replace old nodal coordinates by the extracted new nodal coordinates (equal to resetting sensors to zero).\n6. Apply and ramp up loads onto the LINK11 elements acting as loading cables.\n\n290\n\n7. Extract element length variation of the LINK11 elements acting as DWS for 40%, 60%, 80% and 100% of the target\nload.\nAll individual setups for the simulation with modifications to the base model, all necessary load frames, load points, sensor\npositions, and forces as well as the corresponding ground positions of the pulley blocks and the DWS attachments are summarized in Haller and Noever-Castelos (2021). The ground position coordinates are given in the blade coordinate system of the\n\n295\n\nbase model (no cone angle, or rotation) described above at the beginning of this subsection.\nIn contrast to the bending tests, the torsion tests have a pair of forces pulling vertically upwards and downwards as shown in\nfigure 8. Because the blade is still mounted at a block angle of 7.5° the torsional moment is not parallel to the pitch axis. The\nload cable oriented upwards was attached to a ceiling crane and to the load frame at approximately the shear centre position.\nAs the ceiling crane location is hard to record, but the load rope is perpendicular to the ground it was assumed that the location\n13\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\n300\n\nis 30m above (y-direction) the corresponding load point. The force facing downwards was applied onto the load frame corner\non the trailing edge side in order to introduce a torsional moment in that load frame location.\n\nFigure 8. Configuration example of a static torsional loading on the blade with marked up and downwards facing forces.\n\n4.4 Blade Segment Measurement\nAfter finishing the full blade tests, the blade was cut into 17 segments for further characterization. Figure 5 shows a cut surface\nof the 7th segment at a span-wise position of r = 5.2 m. To determine the 3D centre of gravity (CoG), the segment was suspended\n305\n\nat one point with a flexible rope, so that the CoG settled exactly underneath this point (like a pendulum). Hence, the vector in\ndirection of the suspension rope defines an axis on which the CoG must be located (CoG axis). This procedure was repeated\nwith different suspension points at least 2 times. The CoG was then found in the intersection point of the different CoG axes.\nThe measurement setup can be seen in Figure 9 as well as a digital representation of the intersection of different CoG axes.\nTo measure the vectors and analyse the data an optical measurement system (photogrammetry) was used. Every segment\n\n310\n\nwas equipped with several coded and uncoded reflecting marks to obtain the shape of the segment, the suspension points and\na plummet that was used to get the CoG axes. All the point clouds were analysed in Autodesk Inventor and Siemens NX. All\nsegments were aligned in CAD and the CoG was extracted for each segment with regard to the blade coordinate system. In this\nway we obtained the distribution of the segment CoGs along the blade.\n\n14\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\nFigure 9. Measurement setup (left) and extracted vectors in CAD with intersection point defining the centre of gravity (right).\n\nConsidering the model validation, MoCA is able to generate the respective segments at their correct positions in the blade,\n315\n\nso the segment masses and CoGs are a natural output of ANSYS.\n5\n\nComparison of Experimental and Simulation Results\n\nIn this section, we compare the experimental results with the simulations. The observation scale will continuously decrease\nfrom a global to a more local scale. We start with the global blade characteristics such as eigenfrequencies, total mass, and total\ncentre of gravity. These give a rough estimate of the modeling correctness. Then the blade deformations by means of bending\n320\n\nand twist distributions during the static extreme load tests will be analysed. Finally the strain levels in two cross-sections during\nthe extreme load tests and the masses and centres of gravity of the cut blade segments are compared, which give a more detailed\nview on a local scale.\n5.1 Blade Mass, Centre of Gravity, and Eigenfrequencies\nTable 1 lists the total blade mass and the location of the centre of gravity in longitudinal (z) and chord direction (y) as well as\n\n325\n\nthe measurement uncertainties and the deviation of the numerical model. We see that the model from MoCA is 115.5 kg lighter\nthan the real blade, which corresponds to 6.44% relative difference related to the measurement. In contrast the measurement\nuncertainty is 45 kg. The mass difference is likely due to manufacturing deviations and/or additional masses (e.g. sensor wires\nand installations) that have not been considered in the numerical model. The location of the CoG matches perfectly in the\n\n15\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\nchord direction. There is only little deviation of 230 mm in the span-wise direction, which is almost within the measurement\n330\n\nuncertainty range of ± 200 mm.\nTable 1. Comparison of the total mass and the centre of gravity (CoG).\nExperiment\n\nUncertainty\n\nMoCA\n\nDifference\n\n(in kg)\n\n(in kg)\n\n(in kg)\n\n(in kg)\n\nMass\n\n1793\n\n45\n\n1673.5\n\n-115.5\n\nCoG\n\nExperiment\n\nUncertainty\n\nMoCA\n\nDifference\n\n(in m)\n\n(in m)\n\n(in m)\n\n(in m)\n\ny\n\n0.10\n\n0.04\n\n0.10\n\n0.00\n\nz\n\n6.58\n\n0.20\n\n6.35\n\n0.23\n\nThe results of the modal analysis, both experimental and numerical, are listed in Table 2. The experimental results are\ntaken from Gundlach and Govers (2019). The flapwise frequencies are in acceptable agreement with deviations of less than\n8%. The largest deviation in flapwise modes is found for the 2nd edgewise mode in the test rig configuration (7.94%, which\ncorresponds to an absolute deviation of 0.54 Hz). The smallest deviation can be observed for the 1st flapwise mode in the\n335\n\nfree-free configuration, which is 5.83% or 0.28 Hz, respectively. In edgewise direction, the approximation is even better. The\nlargest relative deviation is seen for the 1st edgewise mode in the test rig configuration, which is 4.84% (or 0.15 Hz in absolute\nnumbers). The 2nd edgewise mode is only 0.83% (or 0.09 Hz in absolute numbers) smaller in the simulation compared to\nthe experiment in the test rig configuration, which is an excellent agreement. The largest absolute deviation is present in the\nfree-free configuration, where the 1st edgewise mode is 0.36 Hz lower than the measured value. Anyways, the deviation of the\n\n340\n\nedgewise modes is less than 5% in all cases, which is a very good agreement. The 1st torsion mode is quite well approximated\nin the free-free configuration, where the simulation is 5.62% lower than the experiment. However, in the test rig configuration\nthe deviation is -11.76% (more than 2 Hz less compared to the test), which is relatively high. In general, the simulations\nagree better with the test results in the free-free configuration than in the test rig configuration. This is likely due to the rigid\nrepresentation of the test rig and the connection bolts, as already mentioned in section 4.2. Especially in torsion, the flexibility\n\n345\n\nof the test rig may not be negligible.\n5.2 Static Bending Tests\nThe results of the static bending tests will be illustrated by means of deflection lines. For each test setup, two lines exist, one\nfor the front and one for the rear DWS. The deflections in the front DWS are plotted in Figures 10 for each pausing load during\nramp-up (40 %, 60 %, 80 % and 100 % of the target load as described in section 4.3). The plots for the rear DWS are added in\n\n350\n\nappendix A. A table is added in each of the figures that show the differences between the simulations and the tests (in absolute\n16\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\nTable 2. Comparison of the modal analyses for the free-free (top) and the test rig (bottom) configuration. Experimental results are taken from\n(Gundlach and Govers, 2019).\n\nMode\n\nExperiment\n\nMoCA\n\n(in Hz)\n\n(in Hz)\n\n1st Flap-wise\n\n4.8\n\n5.08\n\n0.28\n\n5.83%\n\n1st Edge-wise\n\n10.1\n\n9.74\n\n-0.36\n\n-3.56%\n\n1st Torsion\n\n16.9\n\n15.95\n\n-0.95\n\n-5.62%\n\nMode\n\nExperiment\n\nMoCA\n\ntest rig\n\n(in Hz)\n\n(in Hz)\n\n(in Hz)\n\n(in %)\n\n1st Flap-wise\n\n2.2\n\n2.37\n\n0.17\n\n7.73%\n\n2nd Flap-wise\n\n6.8\n\n7.34\n\n0.54\n\n7.94%\n\nst\n\n3.1\n\n3.25\n\n0.15\n\n4.84%\n\nnd\n\n10.9\n\n10.81\n\n-0.09\n\n-0.83%\n\nst\n\n18.7\n\n16.50\n\n-2.20\n\n-11.76%\n\nfree-free\n\n1 Edge-wise\n2 Edge-wise\n1 Torsion\n\nDifference\n(in Hz)\n\n(in %)\n\nDifference\n\nand relative numbers). The tip DWS values are the same for the rear and the front DWS, as only one DWS is installed at the\nblade tip.\nFigure 10 (a) shows the result of the front DWS during the MXMAX load case. For this scenario a maximum deflection of\n180 mm at the blade tip is reached. The simulation shows excellent agreement for the front DWS sensors, with a maximum\n355\n\nabsolute difference of -2.3 mm at the tip for 100 % load and a maximum relative difference of -4.0 % at LF1, whereas the\ndeviations in all other positions are well below 2.0 %. The rear DWS results shown in figure A1 (a) in appendix A have slightly\nhigher errors with a maximum of -5.5 % at LF1 for full load.\nFor load case MXMIN, Figure 10 (b) illustrates the front DWS results. Except for LF1 the results are in very good agreement\nwith a maximum deflection error of -1.6 % at LF2 at full load. However, the results in LF1 return maximum errors of 3.8 % at\n\n360\n\n40 % load, which decreases to 1.8 % at full load. Similar behaviour is found for the rear DWS (Figure A1 (b)); excluding LF1\nthe maximum error is 1.7 % in LF3 and the tip during 40 % load.\nThe results of the front DWS during the maximum flap-wise setup (MYMAX, Figure 10 (c)) are in very good agreement,\nwhen excluding the LF1 data. The LF1 results tend to show the highest errors. This might probably be due to the smallest\nabsolute deflection values, as a systematic sensor/measurement inaccuracy will have a higher impact on relative errors. Con-\n\n365\n\ncerning the other load frames the maximum error is found to be -2.6 % for the LF4 DWS at full load, which corresponds to\n-22.4 mm deflection error at a maximum deflection of 875 mm in the experiment. All other values range between -0.9 % and\n-2.4 %. The excluded LF1 results show higher errors of up to 9.0 % for 60 % load. For the rear DWS (Figure A1 (c)) though\n\n17\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\n40\n0\n\nMXMIN-Front\n160\n\nDifference\n\n120\n80\n40\n0\n\n800\n600\n400\n200\n0\n\n600\n400\n200\n0\n0\n\n6\n\n8\n\n10\n\n12\n\n14\n\n16\n\n18\n\nabsolute\nabsolute\n\nCase Load\n40%\n60%\n80%\n100%\n40%\n60%\n80%\n100%\n\nMYMIN-Front\n800\n\nDifference\n\n(d)\n\nDisplacement [mm]\n\n1000\n\nabsolute\n\nCase Load\n40%\n60%\n80%\n100%\n40%\n60%\n80%\n100%\n\nMYMAX-Front\n\n1000\n\nDifference\n\nDisplacement [mm]\n\n1200\n\n(c)\n\nUnit LF1 LF2 LF3 LF4 Tip\nmm\n0.4 0.0 0.2 0.2 1.2\nmm\n0.4 -0.4 -0.2 -0.3 0.6\nmm\n0.5 -0.3 0.3 0.6 2.0\n0.4 -0.8 -0.2 -0.1 1.5\nmm\n%\n3.8 -0.1 0.5 0.3 1.7\n2.7 -1.4 -0.4 -0.3 0.5\n%\n%\n2.6 -0.8 0.4 0.5 1.3\n1.8 -1.6 -0.2 -0.1 0.8\n%\n\nrelative\n\nDisplacement [mm]\n\n200\n\n(b)\n\nCase Load\n40%\n60%\n80%\n100%\n40%\n60%\n80%\n100%\n\nDifference\n\n80\n\nrelative\n\n120\n\nUnit\nmm\nmm\nmm\nmm\n%\n%\n%\n%\n\nabsolute\n\n160\n\nCase Load\n40%\n60%\n80%\n100%\n40%\n60%\n80%\n100%\n\nrelative\n\nMXMAX-Front\n\nrelative\n\n(a)\n\nDisplacement [mm]\n\n200\n\nUnit\nmm\nmm\nmm\nmm\n%\n%\n%\n%\n\nUnit\nmm\nmm\nmm\nmm\n%\n%\n%\n%\n\nLF1\n-0.1\n-0.4\n-0.7\n-0.9\n-1.5\n-2.8\n-3.5\n-4.0\n\nLF1\n-2.2\n-3.5\n-4.5\n-5.6\n-8.8\n-9.0\n-8.8\n-8.7\n\nLF1\n-0.9\n-1.6\n-2.7\n-3.7\n-4.6\n-5.5\n-6.8\n-7.3\n\nLF2\n0.4\n0.0\n-0.1\n-0.2\n2.5\n0.2\n-0.2\n-0.5\n\nLF2\n-1.4\n-2.3\n-3.2\n-4.0\n-2.2\n-2.3\n-2.4\n-2.4\n\nLF3\n0.5\n-0.1\n-0.4\n-0.5\n1.3\n-0.2\n-0.5\n-0.5\n\nLF3\n-2.4\n-3.7\n-5.7\n-7.3\n-1.3\n-1.4\n-1.6\n-1.6\n\nLF4\n0.1\n-1.1\n-1.5\n-1.9\n0.1\n-1.2\n-1.2\n-1.3\n\nTip\n0.5\n-0.7\n-1.4\n-2.3\n0.7\n-0.7\n-1.0\n-1.2\n\nLF4 Tip\n-6.9 -4.3\n-11.7 -8.4\n-16.9 -14.5\n-22.4 -19.8\n-2.0 -0.9\n-2.2 -1.2\n-2.4 -1.6\n-2.6 -1.7\n\nLF2 LF3 LF4 Tip\n-0.3 2.3 4.4 -1.8\n-0.8 3.5 7.1 -1.6\n-1.9 2.6 6.0 -5.8\n-2.8 2.6 6.3 -8.3\n-0.6 1.6 1.7 -0.5\n-1.0 1.6 1.8 -0.3\n-1.8 0.9 1.1 -0.8\n-2.2 0.7 1.0 -1.0\n\n20\n\nRadial Position [m]\nExp: 40% L.\n\nExp: 60% L.\n\nExp: 80% L.\n\nExp: 100% L.\n\nSim: 40% L.\n\nSim: 60% L.\n\nSim: 80% L.\n\nSim: 100% L.\n\nFigure 10. Bending lines extracted from the front draw wire sensor for the (a) MXMAX; (b) MXMIN; (c) MYMAX; (d) MYMIN experiment\nand simulation. Results are shown for 40%, 60%, 80% and 100% of the target load. The table on the right shows the differences between the\nsimulation and the test.\n\n18\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\nexcluding LF1 (max. error -17.6 %) the LF2 results show errors above 6.7 % with the highest reaching -8.8 % during full load.\nFor the other two load frames the errors are low again and are between -0.9 % and -2.4 %. If taking a closer look at the LF2\n370\n\nfull load deflection d in the test and experiment the front DWS shows dExp,f = 165 mm and dSim,f = 161 mm, whereas the\nrear DWS returns dExp,r = 175 mm and dSim,r = 160 mm. That means the overall deflection of the simulation is less than in\nthe experiment but the difference between rear and front is ∆dExp = dExp,r − dExp,f = 10 mm and ∆dSim = −1 mm, i. e.\n\nthe simulation shows a positive twist while the experiment returns a much higher negative twist. The twist angle Θ can be\n\ncalculated by the relationship\n\u0012\n\u0013\n∆d\n375 Θ = arcsin\n,\nlSP\n\n(1)\n\nwhere lSP is the distance of both front and rear DWS attachment points on the load frame. The twist angles becomes\nΘExp,LF 2 = −0.268° in the experiment and ΘSim,LF 2 = 0.042° in the simulation. Assuming the pivot point is at the shear\n\ncentre (SC), a correction could be calculated to see if the bad results of the rear DWS at LF2 is due to the wrong twist. All\nnecessary geometric data can be found in Haller and Noever-Castelos (2021). Following Equation (1) and using the distance\n380\n\nof the front or rear DWS attachment to the shear centre, the front absolute difference is increased to -7.87 mm which results\nin an error of -4.8 % and the rear deflection is reduced to -7.7 mm, respective -4.4 % during 100 % loading. By this correction\ndue to a wrong predicted twist angle the rear DWS approximation improves by 4 %, while the accuracy of the front sensor\ndecreases by only 2.4 %. This correction is introduced to evaluate the accuracy for the bending prediction and only holds for\nthe LF2 position, as the other positions have different twist angle deviations. Additionally the major influence on the flap-wise\n\n385\n\nloading has to be noted as the DWS attachment distances to the shear centre are much higher than for the edge-wise loading,\ni.e. the influence from twist angle deviations is amplified significantly.\nFigure 10 (d) shows the front DWS results comparison during the minimum flap-wise loading scenario (MYMIN). All load\nframes are installed so can be evaluated and the results show a very good agreement with errors below 2.2 % for all DWS\nexcept LF1. At this first load frame again the results have significantly higher errors of up to -7.3 % at full load. Figure A1 (d)\n\n390\n\ncontains the rear DWS result of the MYMIN load case and lists throughout higher deviations of up to -13 % for the LF1 sensor.\nHere again, by analysing the twist behaviour of the blade all load frames show significant twist differences and after estimating\na correction, e.g. the accuracy of the LF1 front sensor would decrease to a deviation of -11 %, while that of the rear sensor\nincreases to -10.4 %. This is the worst approximation of the simulation for the static extreme load bending setups. Anyways,\nthe other load frames are in very good agreement.\n\n395\n\n5.3 Static Torsion Tests\nFull scale blade tests in pure torsion are usually not included in certification processes according to (International Eletrotechnical Comission, 2014) and are thus rarely available. As described in section 4.3 the blade is twisted during three different setups\nsuccessively at the load frames LF2, LF3 and LF4. The results of the tests and the simulations are plotted in Figure 11. The\nstructural behaviour behind the actual loaded frame position to the tip will not be addressed in this paper and is highlighted as\n\n400\n\ngrey-coloured areas, as the areas loaded in torsion are located between the root and the respective load frame. Though the raw\n19\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\nresults similar to the static bending experiments are the DWS length variation, for these torsional experiments the more relevant\ntwist angles are calculated and plotted according to Equation 1. Figure 11 (a) shows the first torsional test loaded at LF2. The\nabsolute angle deviation from experiment to simulation are in between -0.06° and -0.15° but yields high relative deviation up\nto 30 % due to the small twist angles of -0.55° at LF1 and -1.72° at LF2 during 100 % load.\n\nUnit\ndeg\ndeg\ndeg\ndeg\n%\n%\n%\n%\n\nLF1\n-0.06\n-0.07\n-0.08\n-0.09\n44.0\n32.9\n27.0\n24.9\n\nLF2\n-0.16\n-0.20\n-0.24\n-0.28\n44.8\n35.6\n32.0\n30.0\n\nLF3\n-0.53\n-0.60\n-0.59\n-0.50\n35.4\n25.0\n17.7\n11.7\n\nLF4\n-0.75\n-1.01\n-1.25\n-1.46\n59.3\n51.5\n47.1\n43.7\n\n−3\n−4\n\nMZLF3\n\n0\n−2\n\nCase Load\n40%\n60%\n80%\n100%\n40%\n60%\n80%\n100%\n\nUnit\ndeg\ndeg\ndeg\ndeg\n%\n%\n%\n%\n\nLF1 LF2\n-0.04 -0.03\n-0.04 -0.01\n-0.05 0.01\n-0.05 0.03\n36.1\n9.4\n24.1\n2.0\n19.4 -1.5\n15.8 -3.7\n\nLF3\n-0.18\n-0.14\n-0.10\n-0.05\n17.0\n9.1\n4.7\n1.8\n\nLF4\n-1.26\n-1.50\n-1.63\n-1.68\n45.5\n34.7\n27.9\n22.8\n\nDifference\n\n−4\n−6\n−8\n\nMZLF4\n0\n\n6\n\n8\n\n10\n\n12\n\n14\n\n16\n\n18\n\nabsolute\n\nCase Load\n40%\n60%\n80%\n100%\n40%\n60%\n80%\n100%\n\nDifference\n\nLF4\n-0.03\n-0.14\n-0.21\n-0.30\n3.9\n15.0\n16.4\n19.4\n\nDifference\n\nDisplacement [mm]\n\n−2\n\n−5\n\nDisplacement [mm]\n\nLF3\n-0.23\n-0.32\n-0.41\n-0.51\n43.8\n41.4\n39.0\n38.7\n\nrelative\n\nMZLF2\n\n−1\n\n−10\n\nLF2\n-0.12\n-0.15\n-0.13\n-0.11\n20.2\n15.6\n9.8\n6.6\n\nabsolut\n\n−1.5\n\n0\n\n(c)\n\nLF1\n-0.06\n-0.08\n-0.10\n-0.13\n30.5\n26.1\n22.9\n22.7\n\nrelative\n\n−1\n\n−2\n\n(b)\n\nUnit\ndeg\ndeg\ndeg\ndeg\n%\n%\n%\n%\n\nabsolut\n\n−0.5\n\nCase Load\n40%\n60%\n80%\n100%\n40%\n60%\n80%\n100%\n\nrelative\n\n(a)\n\nDisplacement [mm]\n\n0\n\n20\n\nRadial Position [m]\nExp: 40% L.\n\nExp: 60% L.\n\nExp: 80% L.\n\nExp: 100% L.\n\nSim: 40% L.\n\nSim: 60% L.\n\nSim: 80% L.\n\nSim: 100% L.\n\nLF Position\n\nexcluded Region\n\nFigure 11. Twist angles calculated from the draw wire sensors results for the (a) LF2; (b) LF3; (c) LF4 torsional loading experiment and\nsimulation. Results are shown for 40%, 60%, 80% and 100% of the target load. The table on the right shows the differences between the\nsimulation and the test.\n405\n\nMoving the load application to LF3 (Figure 11 (b)) does not change the situation. At the load application position the\nabsolute error is high with up to -0.6° at a maximum twisting of -4.3°. All errors exceed -10 % dramatically. However, the\n20\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\nexperiment with torsional loading on LF4, see Figure 11 (c), shows reasonably good results for the twist angle at LF2 and LF3\nwith angle deviations of 3.7 % and 1.8 %, respectively. The results at LF4, where the load is applied and which shows the\nhighest twist angle keeps high deviations of about 20 % for full load. Such high errors during torsional loading may base on the\n410\n\nshell element with a node offset to the exterior surface used for this model. Pardo and Branner (2005) and especially Laird et al.\n(2005) already stressed the high inaccuracy of shell elements with node offsets to predict the structural behaviour of hollow\nstructures subjected to torsional loading. However, the twisting is generally overestimated throughout the three torsional tests,\nwhich is inline with the aforementioned references.\n5.4 Local Strain Comparison\n\n415\n\nAs stated in section 4.3 the highly instrumented cross-sections at r = 5 m and r = 8 m offer a more detailed view on the local\nstrain levels in the rotor blade. The strain results are used to compare the simulations with the tests and to verify that local\neffects are correctly reproduced. We have selected a few representative load cases in this section. The remaining load cases can\nbe found in appendix B.\nIn Figure 12 (a) the strain in 0° (span-wise direction, in blue) and 90° (cross-wise, in yellow) directions for the MXMIN\n\n420\n\nsimulation (solid lines) and experiment (circles) are plotted over the normalized airfoil circumference (denoted by S) for\nthe 5 m cross-section, starting at the suction side trailing edge (S = 0), moving along the suction side to the leading edge\n(S ≈ 0.5), and then along the pressure side to the pressure side trailing edge (S = 1). This cross-section shows some general\ncharacteristics in all load cases, which are:\n\n– In the simulation at S = 0, there is a strain peak in the 90° direction, because the sandwich core material vanishes towards\n425\n\nthe trailing edge.\n– In the simulation at S = 0 − 0.25, there is an excessive or wrong curvature in the 90° strain curve, for which we do not\nhave a feasible explanation.\n\n– In the simulation at S = 0.25 − 0.35, there is a stepped dip or raise of the 90° strain, because the sandwich core material\nis substituted by core and UD layers and then completely by the UD spar cap and vice versa.\n\n430\n\n– In the simulation at S = 0.5, there is a strain peak in the 90° direction, because the sandwich core material vanishes\naround the leading edge.\n– In the experiment at S = 0.5 − 0.65, there is a strain dip in the 0° direction, for which we do not have a feasible explanation. The structure should be symmetric next to the leading edge.\n\n– In the simulation at S = 1, there is a strain peak in the 90° direction, because the sandwich core material vanishes towards\n435\n\nthe trailing edge.\nApart from the unclear dip around the suction side leading edge panel (S = 0.5 − 0.65), the longitudinal strain (in 0° direction)\n\ndiffers along the circumference only about ± 150 µm/m. This is about 15 % related to the maximum ± 1000 µm/m at the\nleading or trailing edge. However, the cross-wise strains (in 90° direction) reach deviations of up to ± 200 µm/m, which\n\ncorresponds to about 50 % related to its maximum. The MXMAX results (Figure C1 (a)) are very similar concerning maximum\n440\n\nvalues and strain errors.\n21\n\n\f(c)\n\nµ-Strains [µm/m]\n\n(b)\n\nµ-Strains [µm/m]\n\n(a)\n\nµ-Strains [µm/m]\n\nhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\n1000\nMXMIN\n500\n0\n−500\n−1000\n2000\nMYMAX\n1000\n0\n−1000\n−2000\n400\nMZLF3\n\n200\n0\n−200\n−400\n\n0\n\n0.1\n\n0.2\n\n0.3\n\n0.4\n\n0.5\n\n0.6\n\n0.7\n\n0.8\n\n0.9\n\n1\n\nNormalized Profile Circumference [-]\nExp: 0◦\n\nExp: 90◦\n\nSim: 0◦\n\nSim: 90◦\n\nFigure 12. Span-wise and cross-wise strains of the simulation and the test, plotted against the normalized profile circumference of the\ncross-section at r = 5 m for the (a) MXMIN; (b) MYMAX; (c) MZLF3 load case.\n\nFigure 12 (b) shows the MYMAX load case. Unlike the edge-wise case a wrong calibration or malfunction of the strain\ngauge at S = 0.3 was recorded in the experiment. The flap-wise bending of the blade in general is more excessive compared\nto the edge-wise bending and provokes the highest longitudinal strains in the spar cap positions reaching maximum values of\n445\n\nup to ± 2000 µm/m in the outer shell layer. Consequently the cross-wise strain also increases with maxima of ± 500 µm/m,\n\nboth approximately twice as much as in the edge-wise load case. All other aforementioned issues are also present here, some\nmore and some less pronounced. The same conclusion also holds for the MYMIN case in figure C1 (b), though the maximum\nvalues are slightly lower, due to smaller load sets.\nTaking a look at the torsion tests, in particular for the MZLF3 load case plotted in Figure 12 (c), the longitudinal strain shows\n\n450\n\na relatively good agreement with the test, except for S = 0.5 − 0.65 and at the pressure side trailing edge panel (S = 0.85 − 1).\n\nThe cross-wise strain shows partially good agreement with the experiments, except for the aforementioned characteristics\nwhich are more dominant than in the bending tests. E. g. the peaks at the trailing edge is more pronounced. As for the longitudinal strain, the cross-wise strain shows a disagreement between simulation and experimental results, which is even stronger\ndue to a shifted curvature in the plot. These can also be seen during the remaining two torsion tests. The MZLF4 load case in\n\n22\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\nFigure C1 (d) is very similar to the MZLF3 load case, whereas the MZLF2 load case (Figure C1 (c)) shows all of the stated\n455\n\ncharacteristics in a more pronounced manner as the load introduction is shifted closer to the evaluated section at r = 5 m.\nThe next highly equipped cross-section is at r = 8 m. While the previous cross-section was located at maximum chord,\nthis one is already in a region where geometric curvatures are smoother. For direct comparison the same three load cases were\nselected for this cross-section. As depicted in Figure 13 (a) the longitudinal as well as the cross-wise strains during the MXMIN\ntest follow very well the experimental results, both qualitatively and quantitatively. Strain levels are similar to the cross-section\n\n460\n\nat r = 5 m, but the strain errors of the simulation compared to the experiments are much lower (between ± 75 µm/m). Same\n\nholds for the MXMAX loading, see Figure D1 (a), where the strain error is even between ± 50 µm/m most of the time. Though\nthese are not very pronounced, the peaks at the trailing and leading edges as well as the stepped dips or raises can be identified\n\n(c)\n\nµ-Strains [µm/m]\n\n(b)\n\nµ-Strains [µm/m]\n\n(a)\n\nµ-Strains [µm/m]\n\nas consistent characteristics throughout all test.\n1000\n\nMXMIN @ r=8 m\n\n500\n0\n−500\n\n−1000\n−1500\n2500\n\nMYMAX @ r=8 m\n\n1250\n0\n−1250\n−2500\n200\n\nMZLF3 @ r=8 m\n\n100\n0\n−100\n−200\n\n0\n\n0.1\n\n0.2\n\n0.3\n\n0.4\n\n0.5\n\n0.6\n\n0.7\n\n0.8\n\n0.9\n\n1\n\nNormalized Profile Circumference [-]\nExp: 0◦\n\nExp: 90◦\n\nSim: 0◦\n\nSim: 90◦\n\nFigure 13. Span-wise and cross-wise strains of the simulation and the test, plotted against the normalized profile circumference of the\ncross-section at r = 8 m for the (a) MXMIN; (b) MYMAX; (c) MZLF3 load case.\n\nComparing the results of the MYMAX test depicted in Figure 13 (b), the good agreement between the simulation and the\n465\n\ntest are evident. Even the stepped raise at the two spar caps (S = 0.3 and S = 0.67) exist in the experimental results. The strain\nerror range is approximately between ± 100 µm/m, which is less than for the other cross-section, while having slightly higher\n\nstrain levels. This excellent agreement is also found in figure D1 (b) for the MYMIN load case.\n23\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\nHowever, the results from the torsional tests do not agree. As seen in figure D1 (c) the simulation results of the longitudinal\nstrain during the MZLF3 test may follow some correct trend of the experiments, but has significant variations. The same applies\n470\n\nto the cross-wise strains. Though the strain errors are in the same range as the bending test results, compared to the absolute\nstrain levels these have the same magnitude as the error. The remaining torsional test results (Figure D1 (c) and (d)) show\nsimilar problems.\n5.5 Segment mass and CoG comparison\nIn this subsection, we compare the experimental mass and CoG measurement of each segment with the respective simulation\n\n475\n\nresults. Table 3 contains the segment numbers, the segment locations along the blade defined by the span-wise positions of the\nleft and the right cutting sections r1 and r2 , respectively, and the differences of the segment masses and the CoG locations (in\nabsolute and relative numbers).\nTable 3. Segment mass and centre of gravity (CoG) differences between experiment and simulation. The relative distances of the CoG are\ngiven with respect to their corresponding geometrical cross-section parameter, i. e. absolute thickness (X), chord length (Y), and span-wise\nsegment length (Z).\n\nr2\n\n(in m)\n\n(in m)\n\n(in kg)\n\n1\n\n0.0\n\n0.9\n\nSection No.\n\nCentre of Gravity\n\nMass\n\nr1\n\nX\n\nY\n\nZ\n\nX\n\nY\n\nZ\n\n(in %)\n\n(in m)\n\n(in m)\n\n(in m)\n\n(in %)\n\n(in %)\n\n(in %)\n\n34.6\n\n9.8%\n\n-0.030\n\n0.000\n\n0.003\n\n-2%\n\n0%\n\n0%\n\n2\n\n0.9\n\n2.0\n\n-7.36\n\n-5.1%\n\n-0.003\n\n0.009\n\n0.035\n\n0%\n\n1%\n\n3%\n\n3\n\n2.0\n\n3.0\n\n-10.96\n\n-9.3%\n\n-0.031\n\n-0.004\n\n0.065\n\n-3%\n\n0%\n\n6%\n\n4\n\n3.0\n\n3.5\n\n-4.74\n\n-8.0%\n\n-0.066\n\n0.000\n\n-0.007\n\n-6%\n\n0%\n\n-1%\n\n5\n\n3.5\n\n4.0\n\n-3.419\n\n-6.1%\n\n-0.076\n\n-0.005\n\n0.021\n\n-8%\n\n0%\n\n4%\n\n6\n\n4.0\n\n5.2\n\n-7.39\n\n-5.9%\n\n-0.094\n\n-0.060\n\n0.055\n\n-10%\n\n-3%\n\n5%\n\n7\n\n5.2\n\n6.5\n\n-6.07\n\n-4.9%\n\n-0.102\n\n-0.036\n\n0.054\n\n-13%\n\n-2%\n\n4%\n\n8\n\n6.5\n\n8.5\n\n-9.81\n\n-5.8%\n\n-0.074\n\n-0.008\n\n0.071\n\n-12%\n\n0%\n\n4%\n\n9\n\n8.5\n\n9.5\n\n-3.572\n\n-4.8%\n\n-0.050\n\n0.007\n\n0.040\n\n-10%\n\n0%\n\n4%\n\n10\n\n9.5\n\n10.5\n\n-5.236\n\n-7.3%\n\n-0.049\n\n0.004\n\n0.132\n\n-11%\n\n0%\n\n13%\n\n11\n\n10.5\n\n11.5\n\n-3.685\n\n-5.4%\n\n-0.041\n\n-0.005\n\n0.108\n\n-10%\n\n0%\n\n11%\n\n12\n\n11.5\n\n12.5\n\n-4.087\n\n-6.6%\n\n-0.031\n\n0.003\n\n0.090\n\n-9%\n\n0%\n\n9%\n\n13\n\n12.5\n\n16.0\n\n-18.59\n\n-9.9%\n\n-0.036\n\n0.007\n\n0.091\n\n-13%\n\n1%\n\n3%\n\n14\n\n16.0\n\n16.5\n\n4.007\n\n16.3%\n\n-0.003\n\n-0.048\n\n0.128\n\n-1%\n\n-4%\n\n26%\n\n15\n\n16.5\n\n17.5\n\n16\n\n17.5\n\n19.0\n\n-4.405\n\n-9.1%\n\n-0.025\n\n0.094\n\n0.195\n\n-15%\n\n11%\n\n13%\n\n17\n\n19.0\n\n20.0\n\n1.104\n\n9.5%\n\n0.010\n\n0.023\n\n0.041\n\n10%\n\n4%\n\n4%\n\nThe relative difference of the mass is related to the measured segment mass and the CoG positions are with respect to the\ncorresponding geometrical mid cross-sectional dimensions, i. e. absolute thickness (X), chord length (Y) and radial segment\n480\n\nlength(Z). It was not possible to measure segment 15. The mass differs from -4.8 % to -9.9 % except for segment 1,14, and 17,\n24\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\nwhere the mass was overestimated. Unfortunately it was not possible to calculate an overall blade mass as one segment result\nwas missing. Concerning the CoG differences, the coordinate in cross-section thickness direction (X) varied up to -15 % but\nwas most of the time predicted closer to the suction side. The CoG location in chord direction (Y) agreed very well with the\n485\n\nmeasurement, except for segment 16, the variation were below ±4 %. The radial locations match well for most of the segments\n\n(≤ 6 %). However, the sections 10, 11, 12, 14, and 16 resulted in higher variations, predicting the CoG position closer to the\ntip by more than 10 % of the segment length.\n6\n\nConclusions\n\nThe aim of this paper was the validation of a parametrization and modelling methodology for wind turbine rotor blades. This\nmethodology was implemented in the in-house 3D finite element model generator MoCA (Model creation and analysis tool),\n490\n\nwhich creates hybrid shell/solid finite element models.\nFull-scale blade tests were performed on the SmartBlades DemoBlade as an experimental reference. The blade has a length\nof 20 m and is designed with pre-bend and pre-sweep. The following magnitudes were determined experimentally: The total\nmass and the centre of gravity of the full blade, the mass and centre of gravity distributions along the blade by weighing of blade\nsegments, the natural frequencies in a free-free and a clamped cantilever configuration, the deflection curves along the blade\n\n495\n\nfor both flap-wise and edge-wise bending as well as torsion, and the strains in the cross-sectional and longitudinal direction\nclose to the maximum chord position. The governing parameters such as geometry, material layup, manufacturing deviations,\nadditional sensor and load frame masses were extracted from the blade and test documentations. These were fed into MoCA.\nFinite element models for all test setups were created and the simulations were executed in the commercially available finite\nelement code ANSYS. Then, the simulations were compared with the experimental results.\n\n500\n\nThe mass and centre of gravity of the full blade compared very well (error of -6%). The masses and centres of gravity of the\nblade segments, i. e. the mass and centre of gravity distributions along the blade, were also in good agreement (error of 5-10%).\nModal analysis concluded for th natural frequencies with free-free boundary conditions also well (error <6%) matching results,\nthose for the clamped cantilever configuration matched reasonably well (error <8% for bending, 11.7% for torsion).\nThe deflections for bending in edge-wise direction was in excellent agreement (error <4%). While the deflection curve for\n\n505\n\nbending in flap-wise direction showed a comparably large deviation of 13% at the root, which decreased substantially towards\nthe tip (error at the tip <4%). A reason for that was an elastic twist during the test that was not replicated in the simulations.\nDuring torsion, the authors identified quite large deviations in the elastic twist distributions along the blade, because shell\nmodels cannot properly replicate torsional behaviour, as is also reported in literature.\nFor both flap-wise and edge-wise bending the strains in span-wise direction were in very good agreement. Strain gauges\n\n510\n\nwere distributed along the circumference of the cross-sections at span-wise positions of 5 m and 8 m, respectively, in order\nto measure the cross-sectional deformations. There, the authors observed good agreement between the simulations and the\nexperiments, especially at a span of 8 m. However, some local effects close to the spar-caps could not be resolved in the\n\n25\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\nsimulations. In the torsion test, the strains showed quite large deviations. Though, the longitudinal strains agreed sufficiently\nwell, at least qualitatively.\n515\n\nGenerally speaking, the authors observed good agreement between the simulations and the experiments in almost all situations. The parametrization and modelling methodology can thus be rated as validated. However, there were significant deviations in torsion, which need to be investigated further. The authors currently work on evaluating blade modelling by means of\nsolid elements and/or solid shell elements. In this way, the performance in torsion might be improved significantly.\n\nCode and data availability. The code of MoCA is not publicly available, but may be made available on request at conditions that need to be\n520\n\nagreed upon. All experimental and simulation data that support the results of this research as well as the baseline finite element model of the\nblade as an ANSYS mechanical input file are uploaded in Noever-Castelos et al. (2021)\n\n26\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\nAppendix A: Static bending test results\n\n40\n\nMXMIN-Rear\n160\n\n800\n600\n400\n200\n0\n\nMYMIN-Rear\n800\n600\n400\n200\n0\n0\n\n6\n\n8\n\n10\n\n12\n\n14\n\n16\n\n18\n\nLF2\n-0.2\n-0.7\n-1.1\n-1.3\n-1.0\n-2.8\n-3.2\n-3.1\n\nLF3\n-0.3\n-1.2\n-2.0\n-2.6\n-0.8\n-2.3\n-2.8\n-3.0\n\nLF4\n-0.5\n-2.1\n-3.0\n-3.9\n-0.8\n-2.4\n-2.6\n-2.7\n\nTip\n0.5\n-0.7\n-1.4\n-2.3\n0.7\n-0.7\n-1.0\n-1.2\n\nUnit\nmm\nmm\nmm\nmm\n%\n%\n%\n%\n\nLF1 LF2 LF3 LF4 Tip\n-4.1 -4.6 -2.7 -0.7 -4.3\n-6.6 -7.9 -4.7 -3.0 -8.4\n-9.3 -11.6 -8.5 -6.7 -14.5\n-12.2 -15.4 -12.2 -10.9 -19.8\n-15.1 -6.7 -1.4 -0.2 -0.9\n-16.0 -7.5 -1.7 -0.6 -1.2\n-16.9 -8.3 -2.3 -0.9 -1.6\n-17.6 -8.8 -2.6 -1.2 -1.7\n\nCase Load\n40%\n60%\n80%\n100%\n40%\n60%\n80%\n100%\n\nUnit\nmm\nmm\nmm\nmm\n%\n%\n%\n%\n\nLF1\n-2.2\n-3.6\n-5.1\n-6.8\n-10.7\n-11.8\n-12.4\n-13.0\n\nDifference\n\nDisplacement [mm]\n\n1000\n\nLF1\n-0.3\n-0.7\n-0.9\n-1.2\n-3.7\n-5.2\n-5.4\n-5.5\n\nCase Load\n40%\n60%\n80%\n100%\n40%\n60%\n80%\n100%\n\nDifference\n\nDisplacement [mm]\n\nMYMAX-Rear\n\n1000\n\nabsolute\n\n40\n\nrelative\n\n80\n\n1200\n\n(d)\n\nabsolute\n\nDifference\n\n120\n\n0\n\n(c)\n\nUnit LF1 LF2 LF3 LF4 Tip\nmm\n0.9 0.3 0.7 0.9 1.2\n1.1 0.1 0.4 0.5 0.6\nmm\nmm\n1.6 0.5 1.1 1.8 2.0\nmm\n1.8 0.3 0.6 1.3 1.5\n%\n8.5 1.3 1.7 1.4 1.7\n7.2 0.4 0.6 0.5 0.5\n%\n%\n7.8 1.3 1.3 1.5 1.3\n%\n6.9 0.6 0.6 0.8 0.8\n\nabsolute\n\nDisplacement [mm]\n\n200\n\nrelative\n\n80\n\n0\n\n(b)\n\nCase Load\n40%\n60%\n80%\n100%\n40%\n60%\n80%\n100%\n\nDifference\n\n120\n\nUnit\nmm\nmm\nmm\nmm\n%\n%\n%\n%\n\nabsolute\n\n160\n\nCase Load\n40%\n60%\n80%\n100%\n40%\n60%\n80%\n100%\n\nrelative\n\nMXMAX-Rear\n\nrelative\n\n(a)\n\nDisplacement [mm]\n\n200\n\nLF2 LF3 LF4\n-2.8 -3.2 -6.5\n-4.4 -5.2 -9.2\n-6.8 -8.9 -15.4\n-9.2 -12.3 -20.3\n-5.3 -2.2 -2.5\n-5.7 -2.5 -2.4\n-6.5 -3.2 -2.9\n-7.0 -3.5 -3.1\n\nTip\n-1.8\n-1.6\n-5.8\n-8.3\n-0.5\n-0.3\n-0.8\n-1.0\n\n20\n\nRadial Position [m]\nExp: 40% L.\n\nExp: 60% L.\n\nExp: 80% L.\n\nExp: 100% L.\n\nSim: 40% L.\n\nSim: 60% L.\n\nSim: 80% L.\n\nSim: 100% L.\n\nFigure A1. Bending lines extracted from the rear draw wire sensor for the (a) MXMAX; (b) MXMIN; (c) MYMAX; (d) MYMIN experiment\nand simulation. Results are shown for 40%, 60%, 80% and 100% of the target load. The table on the right shows the differences between the\nsimulation and the test.\n\n27\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\n(c)\n\n(d)\n\nµ-Strains [µm/m]\nµ-Strains [µm/m]\n\n(b)\n\nµ-Strains [µm/m]\n\n(a)\n\nµ-Strains [µm/m]\n\nAppendix B: Local strain comparison\n\n1000\n500\n0\n−500\n\nMXMAX @ r=5 m\n\n−1000\n2000\n1000\n0\n−1000\n\nMYMIN @ r=5 m\n\n−2000\n400\n\nMZLF2 @ r=5 m\n\n200\n0\n−200\n−400\n400\n\nMZLF4 @ r=5 m\n\n200\n0\n−200\n−400\n\n0\n\n0.1\n\n0.2\n\n0.3\n\n0.4\n\n0.5\n\n0.6\n\n0.7\n\n0.8\n\n0.9\n\n1\n\nNormalized Profile Circumference [-]\nExp: 0◦\n\nExp: 90◦\n\nSim: 0◦\n\nSim: 90◦\n\nFigure C1. Span-wise and cross-wise strains of the simulation and the test, plotted against the normalized profile circumference of the\ncross-section at r = 5 m for the (a) MXMAX; (b) MYMIN; (c) MZLF2; (d) MZLF4 load case.\n\n28\n\n\f(c)\n\n(d)\n\nµ-Strains [µm/m]\nµ-Strains [µm/m]\n\n(b)\n\nµ-Strains [µm/m]\n\n(a)\n\nµ-Strains [µm/m]\n\nhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\n1000\n500\n0\n−500\n\nMXMAX @ r=8 m\n\n−1000\n2000\n1000\n0\n−1000\n\nMYMIN @ r=8 m\n\n−2000\n300\n\nMZLF2 @ r=8 m\n\n150\n0\n−150\n−300\n200\n\nMZLF4 @ r=8 m\n\n100\n0\n−100\n−200\n\n0\n\n0.1\n\n0.2\n\n0.3\n\n0.4\n\n0.5\n\n0.6\n\n0.7\n\n0.8\n\n0.9\n\n1\n\nNormalized Profile Circumference [-]\nExp: 0◦\n\nExp: 90◦\n\nSim: 0◦\n\nSim: 90◦\n\nFigure D1. Span-wise and cross-wise strains of the simulation and the test, plotted against the normalized profile circumference of the\ncross-section at r = 8 m for the (a) MXMAX; (b) MYMIN; (c) MZLF2; (d) MZLF4 load case.\nAuthor contributions. Pablo Noever-Castelos implemented the parametrization and modeling methodology in MoCA, conducted the numer525\n\nical simulations, compared the simulations with the tests, and wrote the paper. Bernd Haller planned, executed, and documented the tests.\nClaudio Balzani is the supervisor and guided Pablo Noever-Castelos in the conception of the ideas and participated in the specification of\nstrain gauge positions as well as in writing, structuring, and reviewing the paper.\n\nCompeting interests. The authors declare that they do not have any conflicts of interests.\n\n29\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\nDisclaimer. The information in this paper is provided as is and no guarantee or warranty is given that the information is fit for any particular\n530\n\npurpose. The user thereof uses the information at its sole risk and liability.\n\nAcknowledgements. The authors acknowledge the financial support by the Federal Ministry for Economic Affairs and Energy of Germany in\nthe project SmartBlades2 (project numbers 0324032B/C). The authors further acknowledge the coordination effort of the German Aerospace\nCenter (DLR), the very good cooperation with the project partners and the fruitful discussions within the project consortium.\n\n30\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\nReferences\n535\n\nANSYS Inc.: Ansysr Academic Research Mechanical, Release 19.2.\nBottasso, C. L., Campagnolo, F., Croce, A., Dilli, S., Gualdoni, F., and Nielsen, M. 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D.: A Critical\nEvaluation of Structural Analysis Tools used for the Design of Large Composite Wind Turbine Rotor Blades under Ultimate and Cycle\n\n31\n\n\fhttps://doi.org/10.5194/wes-2021-24\nPreprint. Discussion started: 29 April 2021\nc Author(s) 2021. CC BY 4.0 License.\n\n570\n\nLoading, A Critical Evaluation of Structural Analysis Tools used for the Design of Large Composite Wind Turbine Rotor Blades under\nUltimate and Cycle Loading, 2015.\nN. P. Duineveld: Structure and Possibilities of the FOCUS Desgin Package, 2008.\nNoever-Castelos, P., Haller, B., and Balzani, C.: Supplement to: Validation of a modelling methodology for wind turbine rotor blades by\nmeans of a full scale blade test, https://seafile.cloud.uni-hannover.de/d/d0f144c6b65042cbb060/, 2021.\n\n575\n\nOvergaard, L. and Lund, E.: Structural collapse of a wind turbine blade. 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J.: Wind Turbine Failures - Tackling current Problems in Failure Data Analysis, Journal of Physics:\nConference Series, 753, https://doi.org/10.1088/1742-6596/753/7/072027, 2016.\n\n585\n\nRosemeier, M.: FEPROC Blade Model Verification - 3D Shell and Beam Model, https://doi.org/10.5281/ZENODO.1493936, 2018.\nSmartBlades2: Fabrication, Testing, and Further Development of Smart Rotor Blades, coordinated research project (project numbers\n0324032A-H), supported by the Federal Ministry for Economic Affairs and Energy of Germany due to a decision of the German Bundestag, 2016-2020.\nTeßmer, J., Icpinar, C., Sevinc, A., Daniele, E., Riemschneider, J., Hölling, M., and Balzani, C.: Schlussbericht Smart Blades: Technical\n\n590\n\nReport, 2016.\nYu, W., Volovoi, V. V., Hodges, D. H., and Hong, X.: Validation of the Variational Asymptotic Beam Sectional Analysis, AIAA Journal, 40,\n2105–2112, https://doi.org/10.2514/2.1545, 2002.\nZahle, F.: PGL - Parametric Geometry Library, https://gitlab.windenergy.dtu.dk/frza/PGL, 2020.\nZahle, F., Réthoré, P.-E., Graf, P., Dykes, K., and Ning, A.: FUSED-Wind dev, https://github.com/FUSED-Wind, 2020.\n\n32\n\n\f"
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The implication of autoantibodies in early diagnosis and monitoring of plasmonic photothermal therapy in the treatment of feline mammary carcinoma
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The implication of autoantibodies in early diagnosis and monitoring of plasmonic photothermal therapy in the treatment of feline mammary carcinoma OPEN Asmaa M. El‑Rasikh1, Haithem A. M. Farghali2*, Hisham A. Abdelrahman3, Mostafa Elgaffary4, Shaymaa Abdelmalek1, Ibrahim A. Emam2, Magdy A. Ghoneim5 & Salah A. Selim1* Asmaa M. El‑Rasikh1, Haithem A. M. Farghali2*, Hisham A. Abdelrahman3, Mostafa Elgaffary4, Shaymaa Abdelmalek1, Ibrahim A. Emam2, Magdy A. Ghoneim5 & Salah A. Selim1* Feline mammary carcinoma (FMC) shows great similarities to human breast cancer in the cellular and molecular levels. So, in cats as in humans, the role of immune responses is indicated to detect and follow up the development of tumors. As a new breast cancer therapeutic approach, Plasmonic Photothermal Therapy (PPTT) is an effective localized treatment for canine and feline mammary- carcinoma. Its systemic effect has not been inquired yet and needs many studies to hypothesis how the PPTT eradicates tumor cells. In this study, it is the first time to detect (P53, PCNA, MUC-1, and C-MYC) feline autoantibodies (AAbs), study the relationship between PCNA AAbs and mammary- tumors, and investigate the effect of PPTT on the humoral immune response of cats with mammary- carcinoma through detection of AAbs level before, during, and after the treatment. The four-AAbs panel was evaluated in serum of normal and clinically diagnosed cats with mammary tumors using Enzyme-Linked Immunosorbent Assay. The panel showed 100% specificity and 93.7% sensitivity to mammary tumors. The panel was evaluated in PPTT monotherapy, mastectomy monotherapy, and combination therapy. PPTT monotherapy decreased AAbs level significantly while mastectomy monotherapy and combination therapy had a nonsignificant effect on AAbs level. Cats, as companion animals, share similar environmental risk factors as humans. The feline mammary tumor is the counterpart of human breast cancer and this is due to the great resemblance in the late age of onset, incidence, histopathologic features, biological behavior, and pattern of metastasis. This provides an essential conceptual tool for basic and clinical research, grantees a better understanding of breast cancer biology, and consequently establishing an animal patient model for studying breast cancer microenvironments, systemic reflections, and targeted ­therapies1.h g p The interaction between the immune system and cancer cells proceeds in three phases: elimination; equi- librium; and escape, which are referred to as the “three Es” of cancer immunoediting. During the ‘elimination’ phase, the immune system may succeed in destroying all tumor cells. If that does not happen, it may still be able to control tumor growth but not eradicating it. This phase is referred to as the ‘equilibrium’ phase. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ tumor antigens are rarely observed in healthy individuals, making the response itself a biomarker that betrays the presence of underlying cancer. It is not known if the antibodies to tumor-derived proteins reflect underlying immunosurveillance of cancer or have an impact on the clinical outcome of the ­disease3. The real usefulness of the TAAs as circulating tumor markers in the management of breast cancer has been questioned because of the low diagnostic sensitivity for the early disease before the appearance of clinical manifestation and prior recur- rence or even metastasis. Moreover, their role in monitoring treatment responses through different stages of tumorigeneses, particularly after completion of therapy, remained ­unavailable4. g p yt p py Circulating antitumor markers (AAbs) exhibit increased levels in very early cancer stages in sera prior TAAs can be detected as their production precede clinical confirmation of a tumor by several months or ­years5. AAbs that arise against the TAAs are present in the circulation of people with various forms of a solid tumor before TAAs can be detected, and these molecules can be measured up to 5 years before symptomatic ­disease6. Among 196 specific tumor-associated AAbs, the most frequently studied with a diagnostic value was the P53 antibody followed by autoantibodies against MUC-1, HER2, cyclin B1, NY-ESO-1, HSP60, and C-MYC. Combinations of tumor-associated AAbs showed higher diagnostic sensitivity than individual autoantibodies but single autoanti- bodies usually showed higher ­specificity7. Until now, a wide range of AAbs has been identified. Although several studies present hopeful preliminary results, there is a need to validate AAbs’ signature for biomarker researches in the ­clinic8.h The p53 protein is involved in several critical pathways including cell cycle arrest, apoptosis, DNA repair, and cellular senescence, which are essential for normal cellular homeostasis and genome integrity maintenance. Alteration of the TP53 gene or posttranslational modification in p53 protein can alter its response to cellular stress. The molecular archaeology of the TP53 mutation spectrum generates hypotheses concerning the etiology and molecular pathogenesis of human ­cancer9. Mucin 1, cell surface-associated (MUC-1) also called polymorphic epithelial mucin (PEM) and epithelial membrane antigen (EMA) is a member of the mucin family which is a large, heavily transmembrane glycoprotein. The MUC-1 is generally expressed at low levels by normal simple secretory epithelial tissues, its overexpression is often associated with most carcinomas and in particular by breast cancers, and correlates with high metastatic and poor ­survival10. www.nature.com/scientificreports/ The MYC is a family (C-MYC, L-MYC, and N-MYC) of regulator genes and proto-oncogenes that code for transcription factors as a nuclear phosphoprotein that plays a role in cell cycle progression, apoptosis, and cellular transformation which regulate up to 15% of all human genes. Amplification of these genes is frequently observed in numerous human and animal ­cancers11. The C-MYC function is associated with specific molecular subtypes of breast cancer, its overexpression confers resistance to therapy and its activation has been widely reported in breast cancer ­progression12. Proliferating cell nuclear antigen (PCNA) is a non-histone nuclear protein associated with mitotic activity and tumor ­grade13.h g p y g Plasmonic Photothermal Therapy (PPTT) is a cancer therapy in which gold nanorods are locally injected into the tumor before exposure to near-infrared light causing localized cell death (apoptosis) which applied in dogs and cats suffering from naturally occurring mammary gland carcinoma by professor Mostafa A El-Sayed group and the treated cases showed no recurrence or metastasis for one year after ­treatment14,15. However, the mechanism by which PPTT induces complete ablation of small tumors and immune mechanisms underlying the prevention of in situ recurrence and distant metastasis induced by local PPTT therapy are unknown also the complex interaction between PPTT, breast cancer, and the immune system. Usually innate and adaptive immu- nity play a key role in the elimination of breast cancer. Adaptive immunity exerts its anticancer activity through the production of AAbs against the TAAs and cytotoxic effector CD8 + lymphocyte triggered by cell-mediated immune ­response2. However the role of AAbs in protecting against breast cancer remains elusive, but their valid- ity in diagnosis, early diagnosis, prognosis, predictions, and monitoring of treatment response acquire raising interest and numerous publications reported the validity of AAbs assay in early diagnosis of breast ­cancer16.h p p y y y g The main target of the present investigation was to investigate the validity of AAbs assay in early diagnosis, prognosis, prediction, and monitoring of different treatments (PPTT monotherapy, surgical intervention only (Radical mastectomy), and combination treatment of surgery and PPTT. The implication of autoantibodies in early diagnosis and monitoring of plasmonic photothermal therapy in the treatment of feline mammary carcinoma OPEN Finally, due to selection pressure from the immune system, some cancer cells develop enough resistance that they can escape the immune system, leading to a failure of immune-mediated cancer control which is called the ‘escape’ ­phase2. The release of proteins from tumors triggers an immune response in cancer patients. These tumor anti- gens or also called tumor-associated antigens (TAAs) arise from several mechanisms including tumor-specific alterations in protein expression, mutation, folding, degradation, or intracellular localization. Responses to most 1Department of Microbiology, Immunology, and Mycology, Faculty of Veterinary Medicine, Cairo University, Giza  12211, Egypt. 2Department of Surgery, Anesthesiology, and Radiology, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt. 3Department of Veterinary Hygiene and Management, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt. 4Department of Clinical Pathology, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt. 5Department of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt. *email: dr_haithem0@yahoo.com; dr.salahselim@hotmail.com | https://doi.org/10.1038/s41598-021-89894-x Scientific Reports | (2021) 11:10441 www.nature.com/scientificreports/ Methods i l 18 (DNAStar, Madison, WI) analysis to evaluate their identity % with feline antigens (Uni- ProtKB/ Swiss- Prot: P41685.1, NCBI Reference Sequence: XP_023103255.1, UniProtKB/ Swiss- Prot: P68271.1, and NCBI Reference Sequence: XP_003983789.1. The checkerboard titration was conducted to optimize antigen concentration and serum dilution. Antigen concentrations were 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 ng/well and serum dilutions were 1/40, 1/80, 1/160, 1/320, 1/640, 1/1280, 1/2560, 1/5120, 1/10,240, 1/20,480. ELISA. The optimal antigen concentration and serum dilution were then used for all subsequent ELISA tests and applied according to previously described ­in23–32. Coating of 96 well Immulon 4HBX Microtiter plates (cat. no. 3855; Thermo Fisher Scientific, USA) with antigen diluted in carbonate bicarbonate (SERVA Electrophoresis GmbH, Germany) buffer for 2 h at 37 °C then overnight at 4 °C. Washing four times with Phosphate Buffered Saline with 0.05% Tween 20, pH 7.4 (PBST) (SERVA Electrophoresis GmbH, Germany). Blocking the wells with 100 µl/well by 5% skimmed milk for 2 h at 37 °C. Washing then adding 100 µl of diluted serum samples in a duplicate manner and incubated for 90 min at 37 °C with shaking. Washing and adding of 100 µl per well of diluted Goat anti-feline IgG (H + L) secondary antibody horseradish peroxidase (HRP) conjugated (cat. no. PA1- 84,673; Thermo Fisher Scientific, USA) according to manufacture instruction (1/10,000) incubated for 90 min at room temperature with shaking. Washing seven times with PBST and adding 50 µl/well of UltraTMB (3, 3’, 5, 5’-tetramethylbenzidine) coloring reagent (cat. no.34028; Thermo Fisher Scientific, USA) according to manufac- ture instruction. Stopping the reaction after 15 min with 50 µl/well of 2 M Sulfuric acid (ADWIC, Egypt) and the absorbance was read immediately at 450 nm BioTek ELX808IU ™ microtiter ELISA reader (BioTek, Winooski, VT, USA). Statistical analyses. The receiver operating characteristic (ROC) analysis was performed in SigmaPlot v14.0 (Systat Software, San Jose, CA, USA) using mammary tumor status (with tumor vs not tumor) as the binary state classification variable and AAbs values on a continuous scale as the test variable to determine the area under the curve (AUC) which is an important measure of the accuracy of the diagnostic marker. Pairwise comparisons of AUC among the four studied markers were performed using Chi-square (χ2) tests. Methods i l Group no Group name Animal groups Cases no Lung metastasis 1 H Apparently healthy cats 11 0 2 TN cats with variable sizes of the mammary tumor and did not receive any treat- ment for the tumor 15 5 3 TP cats bearing mammary tumors smaller than 10 ­cm2 treated with PPTT monotherapy 10 2 4 TS cats bearing mammary tumors larger than 10 ­cm2 treated with surgery only 3 1 5 TSP cats bearing mammary tumors larger than 10 ­cm2 treated with the combina- tion of surgery and PPTT 6 2 Total animal population 45 cat 10 cats Table 1. Animal groups. (TN) tumor-no treatment, (TP) tumor-PPTT alone, (TS) tumor-surgery alone, (TSP) tumor-surgery, and PPTT. Table 1. Animal groups. (TN) tumor-no treatment, (TP) tumor-PPTT alone, (TS) tumor-surgery alone, (TSP) tumor-surgery, and PPTT. pre-medicated with atropine sulfate (1%, 0.05–0.1 mg/kg b. wt.; ADWIA Co. S.A.E., Cairo, Egypt) and xylazine HCl (1 mg/kg b. wt.; Xyla-Ject 2%, ADWIA Co. S.A.E.), and then anesthesia was induced using ketamine HCl (10–15 mg/kg b. wt.; Ketalar, Sigma-Aldrich Co.) and maintained by ketamine ­HCl18. In the TSP group, the treatment was performed according to previously ­described15. pre-medicated with atropine sulfate (1%, 0.05–0.1 mg/kg b. wt.; ADWIA Co. S.A.E., Cairo, Egypt) and xylazine HCl (1 mg/kg b. wt.; Xyla-Ject 2%, ADWIA Co. S.A.E.), and then anesthesia was induced using ketamine HCl (10–15 mg/kg b. wt.; Ketalar, Sigma-Aldrich Co.) and maintained by ketamine ­HCl18. In the TSP group, the treatment was performed according to previously ­described15. Samples. Serum samples were collected from cats treated with PPTT monotherapy (TP) before, during, and after the course of treatment with two weeks intervals between each sample. While the serum samples from cats treated with mastectomy only (TS) or combination therapy (TSP) were collected before surgery and within one month and a half after the surgery. All collected serum samples were immediately stored at − 40 °C until use. Checkerboard titration method. The first step was the standardization of indirect Enzyme-Linked Immunosorbent Assay (ELISA) by checkerboard titration as described in previous ­studies19–22 using the fol- lowing recombinant proteins as coating antigens: TP53 (ABIN1046804), MUC1 (ABIN1877158), MYC (ABIN2130698), and PCNA (ABIN622005). All the antigens were purchased from Antibodies Online (https://​ www.​antib​odies-​online.​com/) (Germany) and they are of human origin. Antigens were subjugated to Lasergene software version 3. Methods i l Animals. The animals were admitted to the clinic of the Department of Surgery, Anesthesiology, and Radiol- ogy, Faculty of Veterinary Medicine, Cairo University, Egypt from August 2018 to February 2020. All cats did not receive any treatment for mammary tumors before. Mammary tumors were diagnosed through physical examination of cats. The initial tumor dimensions were measured in all animal groups using calipers. Additional measurements of tumor dimensions were made with each session of PPTT treatment. Cats were also examined by X-ray (Fisher, Berlin, Germany). The radiographic setting factors were 58–70 kVp, 10 mAs, and a 90 cm focal spot film distance. The radiographic exposures were conducted dorsoventrally and right laterally. The cats were divided into 5 groups which included group 1 (H: clinically healthy cats), group 2 (TN: tumor without treat- ment), group 3 (TP: tumor treated with PPTT monotherapy), group 4 (TS: tumor treated with mastectomy only) and group 5 (TSP: tumor treated with a combination of surgery and PPTT.) (Table 1). Treatments. In the TP group, the number of PPTT cycles was correlated to the size of the mammary tumor with two weeks interval between each ­session14. Gold nanorods (AuNRs) with an average size of 27 (± 5) × 6 (± 1) nm (length × width) coated with Methoxy polyethylene glycol thiol (mPEG-SH) and Arg − Gly − Asp (RGD) peptides and 808 nm diode laser with a power of 0.5 W/cm2 and a spot size of around 5.6 ­mm2 were kindly gifted by prof. Mostafa A. El-Sayed; Laser Dynamics Laboratory, School of Chemistry and Biochemistry, Georgia Insti- tute of Technology, USA. In the TS group, the mastectomy operations were performed as previously ­described17. Mastectomy operations were conducted by members of the surgery, anesthesiology, and radiology department of the Faculty of Veterinary Medicine, Cairo University. Under general injectable anesthesia, each animal was https://doi.org/10.1038/s41598-021-89894-x Scientific Reports | (2021) 11:10441 | www.nature.com/scientificreports/ Table 1. Animal groups. (TN) tumor-no treatment, (TP) tumor-PPTT alone, (TS) tumor-surgery alone, (TSP) tumor-surgery, and PPTT. Methods i l Mean ± standard error (SE) of AAbs values measured for normal cats (n = 6) and cats diagnosed with mammary tumor (n = 32), the area under the receiver operating characteristic curve (AUC) ± SE for the four studied AAbs and their 95% confidence intervals (C.I.), and p-value. Table 2. Mean ± standard error (SE) of AAbs values measured for normal cats (n = 6) and cats diagnosed with mammary tumor (n = 32), the area under the receiver operating characteristic curve (AUC) ± SE for the four studied AAbs and their 95% confidence intervals (C.I.), and p-value. Figure 1. AAbs mean values measured for normal cats (n = 6, blue) and cats diagnosed with mammary tumor (n = 32, orange). Figure 1. AAbs mean values measured for normal cats (n = 6, blue) and cats diagnosed with mammary tum (n = 32, orange). test was used to compare all measured values for each marker among treatment groups while accounting for the effect of total tumor area as a covariate.fi To evaluate the efficiency of studied markers in predicting the occurrence of tumor progressive events, a two-way repeated-measures ANOVA test was used for each marker to compare the predictive and pre-predictive values among treatment groups. The evaluated tumor progressive events were secondary, recurrent tumor, and metastasis. The term “predictive” value refers to the last marker value before the appearance of the tested event while the “pre-predictive” refers to the last marker value before the predictive value.h p p p For the repeated measures tests, data were blocked by cat ID. The Shapiro–Wilk test was utilized for normality analysis of the variables. The Tukey’s Studentized Range (HSD) test was used for posthoc analysis. All p values less than 0.05 were considered statistically significant. All data were presented as the mean ± standard error of the mean (SE). Analyses were performed with SAS version 9.434. Ethical approval. The study was carried out in compliance with the ARRIVE ­guidelines35,36. All animals were handled following the Association for Assessment and Accreditation of Laboratory Animal Care and Office of Laboratory Animal Welfare guidelines. All animal experiments were approved by the Institutional Animal Care and Use Committee, Cairo University, Egypt (CU-IACUC) (code: CU-IACUC-II-F-9–16). The animal experiments were carried out after the owner’s permission. Written informed consent was provided by each cat owner for the treatments. Treatment was applied with a high standard of veterinary care. Methods i l On ROC curves, sensitivity, specificity, positive likelihood ratio (LR +), negative likelihood ratios (LR −), and Youden’s index (YI) were used to evaluate the diagnostic performance of all AAbs, and these parameters were based on the methodology provided in the Epidemiology ­textbook33. The optimal cut-off values on the ROC curves were determined from YI. Kaplan–Meier survival analyses were performed for estimation of median survival times and survival prob- abilities of cats in each treatment group and cats with or without metastasis. Log-rank tests were used to compare the survival probability among treatments and to evaluate the effect of metastasis on survival probability. f For each marker, a two-way repeated-measures analysis of variance (ANOVA) test was used to compare between first and last marker values within each treatment group (TS, TP, and TSP). At any sampling event, the total tumor area ­(mm2) per cat was calculated. A one-way repeated-measures analysis of covariance (ANCOVA) Scientific Reports | (2021) 11:10441 | https://doi.org/10.1038/s41598-021-89894-x www.nature.com/scientificreports/ Table 2. Mean ± standard error (SE) of AAbs values measured for normal cats (n = 6) and cats diagnosed with mammary tumor (n = 32), the area under the receiver operating characteristic curve (AUC) ± SE for the four studied AAbs and their 95% confidence intervals (C.I.), and p-value. Variable Markers PCNA P53 MUC-1 C-MYC Mean ± SE Normal 0.2133 ± 0.02 0.2458 ± 0.01 0.2303 ± 0.01 0.2625 ± 0.01 With tumor 0.9643 ± 0.08 1.1858 ± 0.09 1.3937 ± 0.10 0.9917 ± 0.05 AUC​ Estimate ± SE 0.9583 ± 0.03 0.9740 ± 0.03 0.9688 ± 0.03 1.00 ± 0.00 95% C.I 0.8931–1.024 0.9219–1.026 0.9075–1.030 1.000–1.000 p-value 0.0004 0.0003 0.0003 0.0001 Table 2. Mean ± standard error (SE) of AAbs values measured for normal cats (n = 6) and cats diagnosed with mammary tumor (n = 32), the area under the receiver operating characteristic curve (AUC) ± SE for the four studied AAbs and their 95% confidence intervals (C.I.), and p-value. Estimate ± SE 0.9583 ± 0.03 0.9740 ± 0.03 0.9688 ± 0.03 1.00 ± 0.00 95% C.I 0.8931–1.024 0.9219–1.026 0.9075–1.030 1.000–1.000 p-value 0.0004 0.0003 0.0003 0.0001 Figure 1. AAbs mean values measured for normal cats (n = 6, blue) and cats diagnosed with mammary tumor (n = 32, orange). Table 2. Results No positive likelihood ratio (LR +) values because all specificity values were 100%. 95%C.I. = 95% confidence intervals. Table 3. Optimal cut off values of the four studied AAbs and their corresponding for sensitivity (%), specificity (%), negative likelihood ratio (LR-), and Youden’s index (YI). No positive likelihood ratio (LR +) values because all specificity values were 100%. 95%C.I. = 95% confidence intervals. As shown in Fig. 2, all 34 cats revealed positive ELISA for C-MYC, 33 cats were positive for MUC-1, and 32 ats were positive for PCNA and P53.fh Areas under the ROC curves were not different among the four studied AAbs (p > 0.05). The highest YI values indicated the optimal cut-off values for the studied AAbs with the corresponding specificity of 100% and sensi- tivity above 93.7% (Table 3) (Fig. 3). The results shown in Fig. 4 indicated the high specificity and sensitivity of each TAA in our panel for detection of AAbs using ELISA. The effect of different treatments on AAbs level. The efficiency of studied markers in monitoring treatment response before, during, and after treatment was evaluated. Values of PCNA, P53, and MUC-1 signifi- cantly decreased after treatment of mammary tumor using PPTT alone (p = 0.0078, 0.0022, and 0.0325 respec- tively) while no significant changes in their values in surgery only or combination therapy (Table 4) (Fig. 5). The effect of different treatments on AAbs level. The efficiency of studied markers in monitoring treatment response before, during, and after treatment was evaluated. Values of PCNA, P53, and MUC-1 signifi- cantly decreased after treatment of mammary tumor using PPTT alone (p = 0.0078, 0.0022, and 0.0325 respec- tively) while no significant changes in their values in surgery only or combination therapy (Table 4) (Fig. 5). The relationship between AAbs level and total tumor area. At any marker reading, regardless treat- ment group, total tumors area in any given cat had no significant effect on the corresponding PCNA (F1,58 = 0.60, p = 0.4407), P53 (F1,57 = 0.07, p = 0.7877), MUC-1 (F1,61 = 0.37, p = 0.5468), and C-MYC (F1,58 = 0.25, p = 0.6201) values. After controlling for total tumors area, there were no significant differences among treatments in all meas- ured values of PCNA (F2,28 = 0.07, p = 0.9362), P53 (F2,22 = 3.13, p = 0.0632), MUC-1 (F2,29 = 1.04, p = 0.3673), and C-MYC (F2,23 = 1.01, p = 0.3790). Results Results Treatment. Animals’ responses to different treatments were described in supplementary data 1 and 2 which show the full treatment follow-up data of all cases in groups (TP, TS, and TSP) before and after the treatments and their figures. Checkerboard titration. The Laser-gene software analysis results were 76.65%, 60%, 88.8%, and 99.22% for TP53, MUC-1, MYC, and PCNA respectively. The optimal antigen concentration was (50 ng/well) and the optimum serum dilution was (1/160) which was applied in subsequent ELISA tests. The specificity and sensitivity of the AAbs to mammary tumors. Results of ROC analysis (Table 2) showed that values of any of the four studied diagnostic markers before application of any treatment could significantly differentiate between the normal group (H; n = 6) and the cats diagnosed with mammary tumor (TN + TP + TS + TSP; n = 32) (Fig. 1). Scientific Reports | (2021) 11:10441 | https://doi.org/10.1038/s41598-021-89894-x www.nature.com/scientificreports/ Figure 2. Measured values of four studied AAbs for six normal cats (No; white-filled symbols) and 32 cats diagnosed with mammary tumor (Yes; black-filled symbols) before application of any treatment. Figure was prepared using SigmaPlot v14.0 (Systat Software, San Jose, CA, USA). Figure 2. Measured values of four studied AAbs for six normal cats (No; white-filled symbols) and 32 cats diagnosed with mammary tumor (Yes; black-filled symbols) before application of any treatment. Figure was prepared using SigmaPlot v14.0 (Systat Software, San Jose, CA, USA). Table 3. Optimal cut-off values of the four studied AAbs and their corresponding for sensitivity (%), specificity (%), negative likelihood ratio (LR-), and Youden’s index (YI). No positive likelihood ratio (LR +) values because all specificity values were 100%. 95%C.I. = 95% confidence intervals. Marker Optimal cut-off Specificity (%) Sensitivity (%) LR- YI Estimate 95% C.I Estimate 95% C.I PCNA 0.314 100 54.07–100 93.75 79.19–99.23 0.063 93.75 P53 0.352 100 54.07–100 96.88 83.79–99.92 0.031 96.88 MUC-1 0.390 100 54.07–100 96.88 83.79–99.92 0.031 96.88 C-MYC 0.389 100 54.07–100 100 89.11–100 0.0 100.0 f Marker Optimal cut-off Specificity (%) Sensitivity (%) LR- YI Estimate 95% C.I Estimate 95% C.I PCNA 0.314 100 54.07–100 93.75 79.19–99.23 0.063 93.75 P53 0.352 100 54.07–100 96.88 83.79–99.92 0.031 96.88 MUC-1 0.390 100 54.07–100 96.88 83.79–99.92 0.031 96.88 C-MYC 0.389 100 54.07–100 100 89.11–100 0.0 100.0 Table 3. Optimal cut-off values of the four studied AAbs and their corresponding for sensitivity (%), specificity (%), negative likelihood ratio (LR-), and Youden’s index (YI). Results The overall prevalence rate (PR). The overall prevalence rate (PR) of developing metastasis after start- ing the treatment course was 17.7%. This PR was 0.0% in TS, 12.5% in TP, and 33.3% in TSP. The overall PR of the appearance of the secondary tumor(s) after starting the course of treatment was 35.3%. This PR was 0.0% in TS, 50.0% in TP, and 33.3% in TSP. The overall PR of the appearance of the recurrent tumor(s) after starting the course of treatment was 35.3%. This PR was 66.7% in TS, 25.0% in TP, and 33.3% in TSP. Scientific Reports | (2021) 11:10441 | https://doi.org/10.1038/s41598-021-89894-x www.nature.com/scientificreports/ p /fihfi Figure 3. Optimal cut-off values of the four studied AAbs and their corresponding for sensitivity (%), specificity (%), negative likelihood ratio (LR-), and Youden’s index (YI). Figure 3. Optimal cut-off values of the four studied AAbs and their corresponding for sensitivity (%), specificity (%), negative likelihood ratio (LR-), and Youden’s index (YI). Figure 3. Optimal cut-off values of the four studied AAbs and their corresponding for sensitivity (%), specificity (%), negative likelihood ratio (LR-), and Youden’s index (YI). The efficiency of studied AAbs panel in early diagnosis. The efficiency of studied markers in the early diagnosis of secondary tumors and metastasis during the PPTT treatment course was evaluated. Within a month before the clinical appearance of secondary tumors, there was an increase in C-MYC values (Pre- predictive value n = 10, Mean ± SE = 0.86 ± 0.05 & predictive value n = 12 Mean ± SE = 0.90 ± 0.06). However, this increase was not statistically significant (F1,8 = 1.11, p = 0.3225). Also, the increases in “predictive” values of stud- ied markers did not appear to be statistically significant (Table 5).hfit i The efficiency of studied markers in the early diagnosis of recurrent tumors after starting the course of treatment was evaluated. Although there were increases in some AAbs values within a month before the clinical appearance of recurrent tumors, however, the increases were not statistically significant (Table 6).hfi i The efficiency of studied markers in the early diagnosis of primary tumors was evaluated. Five apparently healthy (H; n = 5) cats showed positive AAbs values of 1.11 ± 0.10 for PCNA, 1.08 ± 0.12 for P53, 1.15 ± 0.10 for MUC-1, and 1.04 ± 0.07 for C-MYC. Results By following two of them for 11 months, they did not show any clinical manifestation of mammary carcinoma. Therefore, we could not validate AAbs’ efficiency in the early diagnosis of feline mammary carcinoma as in human breast cancer (Fig. 6). The median survival time. In our study, across all treatments, the median survival time ± SE for cats with- out metastasis (530.0 ± 162.79 d; n = 11; 64% censored) was significantly higher (Fig. 7; Log-rank test: χ2 (1) = 8.38, p = 0.004) than that for cats with metastasis (112.0 ± 66.14 d; n = 6; 17% censored). While the median survival times ± SE for TS (90.0 ± 12.25 d; n = 3; 33% censored), TSP (307 ± 213.61 d; n = 5; 20.0% censored), and TP (530 ± 0.0 d; n = 9; 67% censored) were not significantly different (Fig. 8; Log-rank test: χ2 (2) = 3.13, p = 0.209). Scientific Reports | (2021) 11:10441 | https://doi.org/10.1038/s41598-021-89894-x www.nature.com/scientificreports/ Figure 4. Comparison of receiver operating characteristic (ROC) curves of four different AAbs in mammary tumor detection. Figure was prepared using SigmaPlot v14.0 (Systat Software, San Jose, CA, USA). Figure 4. Comparison of receiver operating characteristic (ROC) curves of four different AAbs in mammar tumor detection. Figure was prepared using SigmaPlot v14.0 (Systat Software, San Jose, CA, USA). Table 4. Comparison between AAbs values measured before treatment (first) and after treatment (last). Significant results at p < 0.05 if (bold). Results Marker Sample PPTT Surgery Surgery + PPTT Mean ± SE t(df), p Mean ± SE t(df), p Mean ± SE t(df), p PCNA First 1.20 ± 014 t(14) = 4.29 p = 0.0078 1.25 ± 0.29 t(14) = 0.49 p = 0.9959 1.04 ± 0.29 t(14) = 0.71 p = 0.9776 Last 0.79 ± 0.11 1.17 ± 0.21 0.95 ± 0.26 P53 First 1.52 ± 0.13 t(14) = 4.98 p = 0.0022 1.50 ± 0.29 t(14) = 0.85 p = 0.9522 1.11 ± 0.23 t(14) = 0.31 p = 0.9995 Last 1.06 ± 0.14 1.36 ± 0.25 1.07 ± 0.20 MUC-1 First 1.59 ± 0.15 t(14) = 3.52 p = 0.0325 1.89 ± 0.33 t(14) = 0.73 p = 0.9753 1.48 ± 0.26 t(14) = 1.11 p = 0.8702 Last 1.04 ± 0.17 1.69 ± 0.31 1.71 ± 0.25 C-MYC First 0.92 ± 0.07 t(14) = 1.99 p = 0.3960 1.22 ± 0.25 t(14) = 0.44 p = 0.9975 0.98 ± 0.16 t(14) = 0.06 p = 0.9529 Last 1.09 ± 0.07 1.15 ± 0.14 0.98 ± 0.09 Table 4. Comparison between AAbs values measured before treatment (first) and after treatment (last). Significant results at p < 0.05 if (bold). Discussion Until now no confirmed model of TAAs for detection of AAbs performed with a sensitivity that would be accept- able for use in screening of breast cancer. In the present investigation, we tailored a panel of four TAAs, three of which (P53, MUC-1, and C-MYC) are the top among all breast cancer-associated tumor antigens used for detection of autoantibodies in terms of the frequency studies in addition to their close structural and function in BC. While the fourth TAAs (PCNA), is the first time to study the relationship between its AAbs and mammary tumors concerning that PCNA TAAs were detected in sera of diseased animals with various types of cancers such as duodenal, and in pulmonary cancers and considered as an important prognostic indicator of cancer. Authors added that PCNA TAA’s high levels of expression were closely related to the occurrence, development, and prognosis of ­BC37. Although the detection of the autoantibodies accepted a raise of interest as a tool for diagnosis, early prediction, and monitoring of treatment response of different therapeutic modalities in cancers including breast cancer, PCNA autoantibodies have been seldom reported. In the present article, detection of PCNA AAbs in cats with mammary gland carcinoma revealed 93.75% sensitivity and 100% specificity confirmed the conclusions provided before about the usefulness of PCNA antibodies as a marker of BC and that these antibodies could serve as a highly effective detector of ­malignancy38.f g yf g y While the negative control animal cut-off values for PCNA, P53, MUC-1, and C-MYC were 0.314, 0.351, 0.39, and 0.389, respectively. The obtained results indicated that the high specificity and sensitivity of each TAA in our panel for detection of AAbs using ELISA, however, a single TAA cannot be used alone as the single TAA can detect AAbs in other noncancerous ­diseases39.h Five of the 11 clinically normal cats showed positive levels of AAbs. These positive AAbs permit the sugges- tion that they were asymptomatically diseased animals (negative for clinical manifestation), but they are in the early stage of tumorigenesis and they are at the risk of mammary tumor ­appearance8. If so, our panel of TAA Scientific Reports | (2021) 11:10441 | https://doi.org/10.1038/s41598-021-89894-x www.nature.com/scientificreports/ Figure 5. Comparison between AAbs values measured before treatment (first) and after treatment (last). Figure 5. Comparison between AAbs values measured before treatment (first) and after treatment (last). Table 5. Discussion Comparison between AAbs values measured just before the appearance of secondary tumors and metastasis (predictive), and last values measured before the predictive values (pre-predictive). The overall values were calculated regardless of treatments. Significant results at p < 0.05 if (bold). Discussion Event Marker Sampling time Overall PPTT Surgery + PPTT (n = 2) N Mean ± SE Fdf, p n Mean ± SE t(df) , p Mean ± SE t(df) , p Secondary tumor PCNA Pre-predictive 10 1.16 ± 0.13 F1,11 = 1.01 p = 0.3338 8 1.18 ± 0.11 t(12) = 0.33 p = 0.9864 1.08 ± 0.60 t(11) = 1.28 p = 0.5927 Predictive 12 1.16 ± 0.13 10 1.13 ± 0.10 1.29 ± 0.73 P53 Pre-predictive 10 1.26 ± 0.13 F1,11 = 0.11 p = 0.7420 8 1.38 ± 0.12 t(12) = 0.14 p = 0.9990 0.78 ± 0.09 t(11) = 0.31 p = 0.9894 Predictive 12 1.24 ± 0.12 10 1.34 ± 0.12 0.73 ± 0.11 MUC-1 Pre-predictive 10 1.52 ± 0.12 F1,11 = 0.07 p = 0.7903 8 1.53 ± 0.07 t(12) = 0.01 p = 0.9908 1.52 ± 0.72 t(11) = 0.31 p = 0.9892 Predictive 12 1.48 ± 0.12 10 1.48 ± 0.11 1.46 ± 0.61 C-MYC Pre-predictive 10 0.86 ± 0.05 F1,8 = 1.11 p = 0.3225 8 0.85 ± 0.06 t(8) = 0.87 p = 0.8196 0.91 ± 0.05 t(7) = 0.74 p = 0.8775 Predictive 12 0.90 ± 0.06 10 0.89 ± 0.07 0.98 ± 0.05 Metastasis PCNA Pre-predictive 5 1.20 ± 0.24 F1,6 = 0.04 p = 0.8540 3 1.29 ± 0.23 t(6) = 0.09 p = 0.9997 1.08 ± 0.60 t(6) = 0.32 p = 0.9873 Predictive 5 1.26 ± 0.28 3 1.24 ± 0.30 1.29 ± 0.73 P53 Pre-predictive 5 1.12 ± 0.18 F1,6 = 0.21 p = 0.6628 3 1.34 ± 0.20 t(6) = 0.89 p = 0.8111 0.78 ± 0.09 t(6) = 0.13 p = 0.9990 Predictive 5 1.26 ± 0.27 3 1.61 ± 0.29 0.73 ± 0.11 MUC-1 Pre-predictive 5 1.60 ± 0.31 F1,6 = 0.01 p = 0.9913 3 1.66 ± 0.37 t(6) = 0.07 p = 0.9998 1.52 ± 0.72 t(6) = 0.07 p = 0.9998 Predictive 5 1.61 ± 0.32 3 1.71 ± 0.44 1.46 ± 0.61 C-MYC Pre-predictive 5 0.87 ± 0.06 F1,6 = 3.12 p = 0.1278 3 0.84 ± 0.11 t(6) = 2.08 p = 0.2596 0.91 ± 0.05 t(6) = 0.58 p = 0.9340 Predictive 5 1.02 ± 0.04 3 1.05 ± 0.05 0.98 ± 0.05 Table 5. Discussion The overall values were calculated regardless of treatments. Significant results at p < 0.05 if (bold). Figure 6. Measured and predicted values of P53 in a cat with a mammary tumor. The intersection between measured and predicted values marks the end of the PPTT treatment course. Figure was prepared using SigmaPlot v14.0 (Systat Software, San Jose, CA, USA). Figure 6. Measured and predicted values of P53 in a cat with a mammary tumor. The intersection between measured and predicted values marks the end of the PPTT treatment course. Figure was prepared using SigmaPlot v14.0 (Systat Software, San Jose, CA, USA). can be recommended for early diagnosis of BC, but this suggestion will be confirmed after a follow-up of these five cats until the appearance of primary mammary tumors as we follow them clinically for only 11 months. In women breast cancer, AAbs could be detected in sera of patients between seven and twenty-seven months before the cancer was diagnosed on the screening mammograms, and in a large cohort, AAbs could be detected up to four years before mammographic ­detection40. In the group treated with PPTT monotherapy, the AAbs panel values were non-significantly decreased during different cycles of PPTT. While the AAbs panel values after the end of treatment (after three months) were significantly decreased than the first values before initiation of PPTT therapy. We can indicate that the high level of AAbs before the treatment with PPTT correlated with tumor burden. After three months of the end of treatment, there was a significant decrease in AAbs level associated with a large reduction or complete disappearance of the tumor mass which means a decrease of the TAAs. The reduction of large tumor mass associated with eradication of TAAs hence the immune system would be exposed to fewer cancer antigens reducing the level of ­AAbs41. The group treated with PPTT monotherapy included two cases that were diagnosed with lung metastasis. One of them showed a significant decrease in AAbs values after three months from the end of local treatment of the mammary carcinoma with PPTT monotherapy and exhibited progression-free survival (PFS) and overall survival (OS) similar to the whole group cases. Discussion Comparison between AAbs values measured just before the appearance of secondary tumors and metastasis (predictive), and last values measured before the predictive values (pre-predictive). The overall values were calculated regardless of treatments. Significant results at p < 0.05 if (bold). https://doi.org/10.1038/s41598-021-89894-x Scientific Reports | (2021) 11:10441 | www.nature.com/scientificreports/ Table 6. Comparison between AAbs values measured just before the appearance of recurrent tumors (predictive), and last values measured before the predictive values (pre-predictive). The overall values were calculated regardless of treatments. Significant results at p < 0.05 if (bold). Marker Sampling time Overall (n = 6) Surgery (n = 2) PPTT (n = 3) Surgery + PPTT (n = 1) Mean ± SE Fdf, p Mean ± SE t(df) , p Mean ± SE t(df) , p Mean ± SE t(df) , p PCNA Pre-predictive 1.23 ± 0.18 F1,3 = 2.40 p = 0.2192 0.81 ± 0.24 t(3) = 3.23 p = 0.1777 1.36 ± 0.18 t(3) = 2.70 p = 0.2703 1.68 ± 0.0 t(3) = 1.37 p = 0.7437 Predictive 1.28 ± 0.22 1.37 ± 0.03 0.97 ± 0.31 2.02 ± 0.0 P53 Pre-predictive 1.01 ± 0.09 F1,3 = 0.41 p = 0.5687 1.05 ± 0.06 t(3) = 1.1 p = 0.8537 1.09 ± 0.13 t(3) = 0.35 p = 0.9986 0.69 ± 0.0 t(3) = 0.13 p = 0.9069 Predictive 1.21 ± 0.23 1.51 ± 0.34 1.21 ± 0.36 0.61 ± 0.0 MUC-1 Pre-predictive 1.74 ± 0.14 F1,3 = 0.08 p = 0.7905 1.53 ± 0.02 t(3) = 1.28 p = 0.7848 1.72 ± 0.21 t(3) = 1.65 p = 0.6199 2.24 ± 0.0 t(3) = 0.35 p = 0.9987 Predictive 1.63 ± 0.29 1.98 ± 0.02 1.25 ± 0.50 2.07 ± 0.0 C-MYC Pre-predictive 0.96 ± 0.04 F1,3 = 4.66 p = 0.1198 0.99 ± 0.05 t(3) = 2.33 p = 0.3638 0.94 ± 0.07 t(3) = 2.44 p = 0.3317 0.96 ± 0.0 t(3) = 0.14 p = 0.8989 Predictive 1.17 ± 0.06 1.27 ± 0.14 1.18 ± 0.03 0.93 ± 0.0 Table 6. Comparison between AAbs values measured just before the appearance of recurrent tumors (predictive), and last values measured before the predictive values (pre-predictive). The overall values were calculated regardless of treatments. Significant results at p < 0.05 if (bold). Table 6. Comparison between AAbs values measured just before the appearance of recurrent tumors (predictive), and last values measured before the predictive values (pre-predictive). Discussion We can explain this by the abscopal phenomenon described before as a systemic anti-tumor immune response that reflects the regression of non-irradiated metastatic lesions at a distance from the primary site of irradiation which means a decrease tumor burden all over the body not only the local treated ­site42. Although there was a significant decrease in the AAbs panel values after the end of the treatment, these values did not reach values lower than the cut-off value (do not convert to normal value) which indicated AAbs persist in serum for long period. This data agrees with https://doi.org/10.1038/s41598-021-89894-x Scientific Reports | (2021) 11:10441 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 7. Effect of metastasis on survival probabilities of cats with mammary tumors (Log-rank test: χ2 (1) = 8.38, p = 0.004). Figure was prepared using SigmaPlot v14.0 (Systat Software, San Jose, CA, USA). Figure 7. Effect of metastasis on survival probabilities of cats with mammary tumors (Log-rank test: χ2 (1) = 8.38, p = 0.004). Figure was prepared using SigmaPlot v14.0 (Systat Software, San Jose, CA, USA). Figure 8. Effect of treatment on survival probabilities of cats with mammary tumors (Log-rank test: χ2 (2) = 3.13, p = 0.209). Figure was prepared using SigmaPlot v14.0 (Systat Software, San Jose, CA, USA). Figure 8. Effect of treatment on survival probabilities of cats with mammary tumors (Log-rank test: χ2 (2) = 3.13, p = 0.209). Figure was prepared using SigmaPlot v14.0 (Systat Software, San Jose, CA, USA). the previous study which recorded that patients whose serum is positive for P53 AAbs at diagnosis do not convert to negative even after the cancer is completely excised. It seems that once the patient’s immune system has been primed, there is sufficient P53 antigen available to maintain a long-term anti-p53 humoral ­response43. Depend- ing on this data, we can suggest that any elevation of our AAbs panel values post decrease means the appearance of TAAs subsequent early tumorigenesis process before the appearance of clinical manifestation and paved us to suggest that this elevation can be used as an early prediction for secondary tumor, recurrence, or metastasis. Also, in the group treated with PPTT monotherapy during the treatment course, there is a non-significance increase in AAbs level within a month before the clinical appearance of secondary tumors, recurrence, or metas- tasis. Conclusion Th b f The AAbs profile detected by ELISA in sera of cats has distinct features reflecting a unique autoantibody reper- toire. To enhance the sensitivity and specificity of ELISA detection of AAbs, we selected the most sensitive and specific tumor-associated BC antigens such as P53, MUC-1, PCNA, and C-MYC as coating antigens for ELISA plates. Our findings indicated that our tailored panel revealed high sensitivity and specificity in the diagnostic value of the four AAbs which may be used as potential biomarkers for the early detection of mammary carci- noma. Also, our tailored panel of AAbs can be used for tracking mammary carcinoma response to PPTT therapy. www.nature.com/scientificreports/ PPTT and surgery, there was no significant difference in AAbs panel values before and after the treatment with one month and a half (it is the available follow-up period). The persistence of AAbs after only one month and a half post-surgery confirmed that AAbs persist high for three months after the end of PPTT therapy. So, we can lay the non-significant changes in AAbs values in the TS and TSP groups on the short follow-up period which must be more than three months after the end of treatment. It was reported that AAbs persisted in the serum of colorectal cancer patients for at least six months after surgical removal of ­cancer45. This persistence of AAbs for three months after the end of PPTT therapy and after six months of surgical removal of cancer may be attributed to the long-lived population of memory plasma cells that can survive for months or years in circulation in a quiescent ­state46. The long overall survival of cats treated with PPTT monotherapy can be explained, as reported ­before47, by the abscopal effect of radiation therapy that does not only exert direct cytotoxic effects on tumor cells but also initiates immunogenic cell death causing production and release of the cytokines and chemokines into the tumor microenvironment. This causes chemoattraction and infiltration of dendritic cells (Dcs) to the site of the tumor. Activation of Dcs, which are essential antigen-presenting cells, up-regulation of cytotoxic T lymphocytes, induce a systemic antitumor response outside the irradiation field, and finally significantly delayed tumor growth and prolonged median survival time. But this abscopal effect caused by radiation monotherapy is quite rare and has not been extensively investigated. Data availability y All the relevant data about the study will be made available upon request. Received: 11 February 2021; Accepted: 23 April 2021 References Neoplas. 6, 339–353 (2001). Mammalian MYC proteins and cancer. New J. Sci. 2014, 1–27 (20 . Tansey, W. P. Mammalian MYC proteins and cancer. New J. Sci. y p ( ) 2. Green, A. R. et al. MYC functions are specific in biological subtypes of breast cancer and confers resistance to endocrine therapy in luminal tumours. Br. J. Cancer 114, 917–928 (2016).h 3. Elston, C. W. & Ellis, I. O. Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer experience from a large study with long-term follow-up. Histopathology 19, 403–410 (1991). 13. Elston, C. W. & Ellis, I. O. Pathological prognostic factors in breast cancer. I. 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Depend- ing on this data, we can suggest that any elevation of our AAbs panel values post decrease means the appearance of TAAs subsequent early tumorigenesis process before the appearance of clinical manifestation and paved us to suggest that this elevation can be used as an early prediction for secondary tumor, recurrence, or metastasis. Also, in the group treated with PPTT monotherapy during the treatment course, there is a non-significance increase in AAbs level within a month before the clinical appearance of secondary tumors, recurrence, or metas- tasis. A previous study for the detection of P53 AAbs in colon cancer recorded that any temporal changes in the level of P53-AAbs could be closely correlated with disease progression or ­regression44. The reappearance of such antibodies may indicate a relapse of the disease. 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El-Sayed (Julius Brown Chair and Regents Professor Director, Laser Dynamics Lab, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, USA, E-mail: melsayed@ gatech.edu) for providing the Gold nanorods (AuNRs) and the diode laser used in the present work. We also thank the JESOR program of the Egyptian Academy for Scientific Researches and Technology (Address: 101 Kasr Al-Ainist, Cairo, Egypt, Postal Code: 11516, E-mail: ASRT@ASRT.SCI.EG) for the great support of this work. Author contributions S.A.S. conceived and designed the study. A.M.R., H.A.M.F., and I.A.E. collected the data. H.A.M.F. and I.A.E. diagnosed, performed the surgical interventions (mastectomy), and applied PPTT of mammary carcinoma feline cases. A.M.R., M.G., S.A., and M.A.G. performed Enzyme-Linked Immunosorbent Assay (ELISA), data analysis, and interpretation. H.A.A. performed the statistical analysis. S.A.S., H.A.M.F., H.A.A., and A.M.R. wrote the paper. 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Monitoring of p53 autoantibodies after resection of colorectal cancer: relationship to operative curability. Eur. J. Surg. 167, 50–53 (2001). Fundingh Funding This research was funded by the joint collaborative Efforts of the Egyptian Expatriates and Scientific Organiza- tion toward Tackling National R&D challenges (JESOR). tion toward Tackling National R&D challenges (JESOR). Competing interests  The authors declare no competing interests. Additional information Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​021-​89894-x. Correspondence and requests for materials should be addressed to H.A.M.F. or S.A.S. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Competing interests  The authors declare no competing interests. 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Real time Analysis of Virtual Reality Art Design Based on Immersive communication
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Real time Analysis of Virtual Reality Art Design Based on Immersive communication Liping Liu (  liuliping@henetc.edu.cn ) Henan Economics and Trade Vocational College https://orcid.org/0000-0003-3078-6617 Research Article Keywords: Real time, Immersive communication, Virtual reality technology, Art design Posted Date: August 2nd, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3134273/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Real time Analysis of Virtual Reality Art Design Based on Immersive communication Liping Liu Henan Economic and Trade Vocational College,Computer Engineering College, Zhengzhou 450000, China Correspondence should be addressed to Liping Liu: liuliping@henetc.edu.cn Abstract: The traditional indoor virtual reality art design is quite different from the actual placement of items. Based on the perspective of immersive communication, this paper analyzes the specific ways of applying virtual reality technology in art design from the two dimensions of physiological immersion and psychological immersion, so as to analyze the process of Virtual Reality Art Design Based on Immersive communication. The Kinect device is used to collect the indoor spatial feature depth data. After repair and filtering, the processed data is converted into the data in the spatial coordinate system to build the geometric model of the spatial feature data. This paper analyzes the indoor virtual reality art design by using virtual reality technology. The experiment proves that the placement error of the indoor virtual reality artistic achievements is small. Key words: Real time, Immersive communication; Virtual reality technology; Art design Introduction Virtual reality technology [1] has a clear development direction: high-quality, fast graphics and excellent processing capacity. Through the continuous improvement of these aspects, it can promote the development of output equipment and input equipment, strengthen the processing capacity, and finally strengthen the development of virtual reality technology in environmental art design, reform the essence and methods of environmental art design, and fully express the potential of environmental art design. However, virtual reality technology also has the shortcomings of imperfect technology, and there is still a lot of room for development in the field of design [2-3]. We should improve the demand for interpersonal activities and human activities, implement the idea of serving the people into the whole process of design, and put people's spiritual and material needs, that is, people's needs for the environment, in an important position. Due to the complexity of the specific design process, Designers should put customers' feelings first [4-5]. Virtual reality technology can intuitively display environmental art design [6]. With the continuous acceleration of China's urbanization process and the rapid development of the real estate industry, it is necessary to plan and design more and more public environmental spaces. Taking urban planning as an example, in the reporting stage of urban planning, the development process of environmental art expression means from hand-painted drawings to computer-based rendering to architectural animation, Human beings are constantly looking for new technologies and high-quality performance means to improve the effect of virtual reality technology [7]. Virtual reality technology has great advantages in displaying environmental art design [8]. So that customers can strengthen their senses' acceptance of stimulating information from the perspective of tactile, visual and auditory aspects, so that customers can have an immersive feeling. After realizing sensory stimulation, they can continuously remember the contents of the whole display under the condition of high concentration and extremely active thinking, so that customers' attention can be continuously focused, and finally keep the contents of memory developing in the direction of persistence and profundity. Reference [9] based on the theory of children's intelligence development, combined augmented reality technology (AR) with virtual reality technology (VR), designed on the unity3D platform, developed a children's intelligence development system with game style. Reference [10] in order to explore the impact of immersive virtual environment on learners' art learning, experimental research methods are used to measure the relevant data of learners' art learning by using brain wave equipment and questionnaires. The research finds that: after the art learning based on immersive virtual environment, the learners' art attitude and understanding of art works have significantly improved; In the process of art learning, there is a significant negative correlation between learners' flow and cognitive load; There is a significant negative correlation between learners' meditation and cognitive load in the process of art learning and their scores of painting art works. Therefore, this paper takes interior art design as the research object. On the basis of clarifying the immersive theory of virtual reality technology, combined with the existing research results of "analysis of characteristics of Chinese folk pattern art design based on Immersive communication" [11], this paper discusses the process of Virtual Reality Art Design Based on Immersive communication. 1 Immersion theory of virtual reality technology 1.1 Overview of virtual reality technology Virtual reality technology is a technical mode based on simulation system. On the basis of simulating the real world, it injects the design elements of the three-dimensional world into it to form a virtual world. With the support of virtual reality technology, such a virtual world materializes abstract objects, so as to improve people's experience in observation or use. As a technology implementation method of diversified information integration, virtual reality technology interacts three-dimensional dynamic information with entity behavior, so as to guide users to immerse in the environment simulated by their technology simulation. Based on the two dimensions of virtual and reality, the real-time dynamic information generated Seeks People's visual effects from perception, weakens the differences between the real world and the virtual world, and then the sensory effects on the virtual world will naturally arise, thus forming an art form of poor integration of virtual and reality. 1.2 Experience mode of virtual reality technology on immersion effect Before studying how to apply virtual reality technology to art design, we must analyze the immersion effect. The immersion effect mentioned here is a special definition of immersion mode. From a psychological and physiological point of view, the reason why virtual reality technology can be attractive and bring people's thoughts and consciousness into the virtual world created by artists completely depends on the immersion effect that users can achieve in the virtual world [12]. If we rationally perceive the artistic atmosphere of the virtual world from the time and space dimensions of the objective world, its immersion effect is also difficult to achieve. The real attraction of users' thinking consciousness in the virtual world is the attraction of art design itself to users and the artistic atmosphere created by virtual reality technology [13]. Even if it is infinitely close to the real world, how to guide users to create art that has not really existed has become a fundamental problem. Then we should think about the artistic expression of virtual reality technology from the perspective of users, and users perceive the artistic expression of virtual reality technology from the psychological and physiological dimensions. Therefore, from the perspective of the use of virtual reality technology, the more immersed in the virtual world, the higher the degree of experience, and the more obvious the effect of artistic expression. 2 Immersion dimension of virtual reality technology in art design 2.1 Physiological immersion Physiological immersion is a visual effect that traditional art design cannot achieve. Even though 3D painting style in modern design extremely renders the artistic effect of the three-dimensional world in the plane, the observer can still distinguish its plane subject from objective vision in essence. However, the virtual reality technology is different. Users can form a zero degree experience in perception through the virtual reality technology. This way of zero experience is the artistic communication effect that is infinitely close to the real world. For example, in 3D film and television works, users experience the same visual infection as the real world when the real space is compressed to an extremely real environment, thus forming a virtual immersion of visual senses. The visual effect achieved through technical means is the immersion mode provided by virtual reality technology for people. After ignoring the media transmission speed, the simulation mode provided by virtual reality technology is very close to the real world, which blurs the boundary between the two dimensions of reality and virtual, allowing users to immerse themselves from a physiological point of view. 2.2 Psychological immersion From the above design dimensions, psychological immersion is far higher than the artistic meaning that physiological immersion can reach. In the design of early opera works, its stage setting and other design elements are not only a necessary way to attract the audience, but also a medium to spread artistic information. These media give high psychological hints to the observers, guide people to pay attention to the story environment and scenes, and form a psychological immersion in the art of drama. In virtual reality technology, this medium also exists. An excellent work of art lies in whether it can attract the psychological resonance of the viewer when appreciating the work of art. If this form of resonance occurs, it will also take the virtual reality technology as the carrier to observe the virtual world depicted by the artist. This psychological immersion is more obvious through the stimulation of physiological immersion [14]. Then it provides the experiencer with artistic value beyond the work itself, forming a kind of psychological tacit understanding and sublimation. Just like the different artistic effects shown in the comparison between 3D films and 2D films, the key of virtual reality technology is to guide the audience into the virtual environment and improve the functionality and applicability of the media, so as to form a psychological immersion state in the works of art on the basis of media functions, and then be able to perceive the artistic atmosphere or ideas expressed in the works of art. 2.3 Immersive communication The three characteristics of virtual reality technology are imagination, interaction and immersion. The three are relatively independent and harmonious, forming different immersion dimensions [15-16]. In art design, only by giving full play to the imagination space and improving the interaction effect can we increase the immersion effect of art works. This immersion effect is also the most attractive expression of art works in virtual reality technology. The emergence of VR video technology in virtual reality technology has improved the immersion experience of users. The fundamental factor is that physiological immersion brings users into a specific virtual scene in terms of visual effects, and produces psychological resonance with the virtual scene, thus producing corresponding psychological immersion. Most studies have analyzed immersive communication. This immersive mode based on user experience is an unprecedented means of expression in art design. If it is applied to different fields, it will also enhance the internal expressiveness and value of art design. Under the influence of immersive communication, virtual reality technology extends to different immersion dimensions, and then stimulates the immersion effect of art works from the physical and psychological dimensions. 3 Design and production of Virtual Reality Art Design Before designing the virtual reality art design based on Immersive communication, they collect relevant data and determine the user needs of interior art design [17]. For designers, they want to improve the rendering speed of interior art design, while for users, they want to increase the interactivity of interior art design, they want to be able to view the renderings in real time [18], and have high requirements for the computer rendering results and the actual furniture placement. Therefore, the virtual reality art design of this design will take the furniture placement error as the design goal. 3.1 Virtual reality art design framework based on Immersive communication The virtual reality art design based on Immersive communication mainly includes three types of objects, space objects [19], object objects and scene objects. Space objects mainly include lights, walls, aisles and columns, as well as materials and textures. These objects allow users and designers to change the properties of space objects; Object objects mainly include doors, windows, furniture, etc. users and designers can change them at will and make corresponding choices from the database. Objects in the software are managed by the object management module, and its structure is shown in Figure 1: House type A House type B Room objects Scene objects Spatial objects Item object Triggered object Substitute object Background and explanation Operant object Figure 1 Object oriented data structure At the same time, the indoor environment established by the software can realize the interactive roaming of users, including the following contents: First, the user interface module accepts user requests and displays the three-dimensional space of interior art design according to actual needs [20]; Secondly, the scene management module creates and manages three-dimensional space; Thirdly, the object management module receives the user's request information and receives or returns the reconstruction information; Fourth, communication can be realized through the network structure. The menu and function introduction of the software are shown in Table 1: Table 1 Software menu and function introduction Serial number Menu Function introduction 1 2 3 4 Open Adjust DEM interval Create data File information 5 Orthographic projection 6 Perspective projection 7 8 9 Modeling with points Modeling with lines Modeling with faces Horizontal clockwise rotation Horizontal counterclockwise rotation Clockwise rotation in vertical direction Vertical counterclockwise rotation Open design content Resize grid Convert 2d image format Read data information Display data information in orthographic projection mode Display information in transmission projection mode Display by point Display by line Display by face 10 11 12 13 14 Horizontal propulsion 15 16 17 18 19 20 Pull out horizontally Zoom Move up Move down Material settings Effect settings Rotate 3D terrain clockwise Rotate 3D terrain counterclockwise Rotate 3D terrain vertically clockwise Rotate 3D terrain vertically counterclockwise Recommended in horizontal direction Pull out horizontally Scale to Scale up Scale down Select material Select effect The overall virtual reality art design framework based on Immersive communication is shown in Figure 2: Customer User interface module Scene management module Object management module Knowledge Engine Knowledge base Object management module Server Server Server Network interface module Network interface module Figure 2 Virtual reality art design framework based on Immersive communication After starting the software, the user enters the main interface of the software. The user can click the menu according to his own needs and call the corresponding response functions in the software to meet his own functional needs, because the functions in the software are written in real time according to the actual needs of the user. 3.2 Construction of spatial feature geometric model 3.2.1 Spatial point cloud data acquisition The theoretical basis for using Kinect to obtain spatial point cloud data is: During Kinect scene scanning, there may be missing data due to occlusion or light. Generally, some positions that cannot be scanned or scanning angles are incorrect. Therefore, during the scanning process, it is necessary to adjust the position and angle of the Kinect device, establish a local coordinate system with the Kinect as the center, and collect indoor spatial data with the support of this coordinate system. In the data acquisition process of Kinect device, the main device is the depth camera, which adopts optical coding technology [21], mainly uses the depth sensor to collect indoor spatial position information, and internally represents the depth distance through 13 bit binary numbers. Assume that the color information parameters of a single pixel in the depth map collected from the Kinect device are 16 bit data. According to the description of pixel information in the SDK document, in the depth image pixel information, the first 13 bits in the data contain the depth information, and the last 3 bits in the data contain the user index. When the original depth data of Kinect is obtained [22], the obtained color information and user ID are divided to ensure that the first 13 bits of the data remain unchanged and the last 3 bits return to 0. After internal processing in the SDK, the depth data between 0-4095 can be obtained through the Kinect device. Generally, moving the original Kinect data to the right by three bits is to obtain the depth information of the current pixel, which reflects the real-world distance of the pixel. There are some areas to be repaired in the data collected by Kinect, which are hollow areas, undetectable areas and shadow areas. For different areas, corresponding repair measures are taken to obtain more complete depth data. The repaired data cannot be directly used in the establishment of simulation model, because some deep continuous noise will appear at the edge of the image during the repair process. Therefore, a filter is used to remove the noise. After filtering, the indoor spatial feature set model is established using point cloud data. 3.2.2 Establishment of spatial feature geometric model The spatial data obtained through the above process is mainly displayed in the form of depth image, which is a two-dimensional map. Considering that the spatial characteristics are mainly composed of three-dimensional data, when obtaining the indoor spatial depth image, it is necessary to convert the depth map into the relationship of three-dimensional spatial points in the real world. Kinect has an automatic correction function [23], which can correct the depth and color camera in real time. Therefore, the process of establishing a geometric simulation model of spatial features is actually to complete the projection from spatial points to the image plane. In order to quantitatively describe the establishment process of geometric model, the world coordinate system, camera coordinate system, image pixel coordinate system and physical coordinate system are established respectively. As shown in Figure 3. W ( X s , Ys , Zs ) Zs O' b wb x O1 O' a y Os Xs Ys Figure 3 Camera imaging model In Figure 3, ( Os − X sYs Z s ) represents the camera coordinate system, represents the physical coordinate system, system, and ( O '− ab ) ( O0 − X 0Y0 Z0 ) ( O1 − xy ) represents the world coordinate represents the image pixel coordinate system. In the image pixel coordinate system, the coordinates of each point are represented by ( a, b ) in pixels. The W ( X s , Ys , Z s ) coordinate of object point W in the world coordinate system is in the camera coordinate system, and the physical coordinate system in the simulation model is wb ( xb , yb ) . Assuming that the physical dimensions of the image pixels obtained by Kinect in the a direction are lx , and the physical dimensions in the b direction are l y respectively, it can be seen that the conversion relationship between the image pixel coordinate system and the physical coordinate system is as shown in formula (1): y dy (1) x b = b0 − dx a = a0 − The conversion relationship between the world coordinate system and the camera coordinate system is shown in formula (2): T T Xs  X0   Y  = Q T  Y  + hT (2)  s  0  Z s   Z 0  In the formula, Q represents the rotation matrix and h represents the translation vector. In an ideal situation, the perspective projection transformation under the simulation model is the change from the camera coordinate system to the physical coordinate system, and its transformation relationship is formula (3): x  x  xb = g  b0 −   s dx  zs  (3) x  ys  yb = g  b0 −   dx  zs  In the formula, g represents the conversion coefficient. According to the corresponding relationship between the coordinate systems, the conversion relationship between the world coordinate system and the image pixel coordinate system of the object point w is obtained. In the calculation process, the camera coordinate system is taken as the world coordinate system [24], and the value on the world coordinate system corresponding to the target point in the Kinect data is obtained. The final value of X s , Ys , Z s is formula (4): l   X s = xb ( b − b0 )    l  Ys = yb ( a − a0 )  (4)   Z s = g  l   In the formula,  ,  , a0 and b0 represent the internal parameters of the camera. Equation 4 can be used to complete the conversion from the pixel coordinates of the depth image to the three-dimensional coordinates of the spatial points. The Kinect sensor is used as the origin of the world coordinate system. Through this transformation, the simulation model corresponding to the indoor space depth image can be obtained. 3.4 Realization of indoor virtual reality art design function Based on the simulation model constructed above, combined with VR technology, it is used as the data management platform in the indoor virtual reality art design, as the exchange between virtual reality and indoor article information, to analyze the indoor articles to be designed, and to reflect the positions of the articles to be designed into VR technology. The combination process is shown in Figure 4: Virtual reality environment User Event processing Virtual Reality Technology Query production Data reception Spatial database Virtual reality environment Indoor items database Virtual reality system Figure 4 Schematic diagram of virtual reality technology For the scene construction in the indoor virtual reality art design, the resources to be designed should be imported first, and the panorama of indoor objects should be reconstructed [25-26], and the full angle shooting should be carried out from the effective perspective of human eye observation. In the process of digital generation of building information, the indoor areas are divided by spatial coordinates, and the column and column numbers in the fixed matrix are used to represent the geographical and spatial positions of the indoor areas. The reconstruction process is shown in Figure 5: Start End Pictures of indoor objects Indoor articles fisheye pictures Import computer Running software Open image N Parameter adjustment Y Image mosaic and Preview Figure 5 Establishment process of panorama of indoor articles In the process of indoor scene map reconstruction, there are two variables in the terrain model and the reconstructed panorama. At this time: H ( t , s ) = A ( an ) (5) In the formula, an represents reconstruction parameters and H ( t , s ) represents reconstruction panorama coordinates. On this basis, the interior art design model is established to edit the terrain, and the parameter expression of the 3D surface in the interior virtual reality art design is expressed as follows:  x ( i, j )  m ( i, j ) =  y ( i, j ) (6)   z ( i, j ) In the formula, i and j represent the parameter variables respectively, and x , y and z represent the building location in the indoor virtual reality art design. The finite sequence of three-dimensional vectors is expressed by digital elevation model, and the expression is: M = ( X u , Yu , Zu ) (7) In the formula, X u , Yu and Zu represent the three-dimensional plane coordinates, and M represents the elevation value parameters of the model. Based on the above analysis, the design of indoor virtual reality art design is completed. The overall software flow is shown in Figure 6: Start Program initialization Main interface Menu functions Terrain display mode Texture map Scene and environment settings 3D terrain matching Roaming control Format conversion End Figure 6 Overall flow chart of indoor Virtual Reality Art Design The scene constructed with this software has good rendering speed, scale and rendering quality. After rendering all scenes, panoramic roaming can be generated, as shown in Figure 7: Figure 7 Panoramic view The rendered rendering of Virtual Reality Art Design Based on Immersive communication is shown in Figure 7. In the practical application of the software, users can select maps from the database according to their own needs, and can freely adjust the location of objects in the scene to complete the interior art design. 4 Experimental comparison In order to ensure the practical application effect of the proposed indoor Virtual Reality Art Design Based on Immersive communication, experiments were carried out and the proposed method was compared with the design method (comparison method) of the unity3d platform in reference [8]. 4.1 Experiment contents Taking a residential area as the experimental object, in order to reduce the experimental time, only the indoor refrigerators, beds, washing machines, televisions and water heaters were made. Before production, ask the user the actual position he wants to place in the house, compare the position he wants to place with the placement position of the articles designed by the two software, and compare the furniture placement errors of the two software. The experimental parameter setting table is shown in Table 2: Table 2 Location parameters of experimental objects Furniture name X Y Z Refrigerator Bed Washing machine Television Heater 2.05 1.07 3.00 1.92 6.34 2.33 1.42 0.42 2.83 3.38 1.39 0.95 1.29 1.73 5.52 In Table 2, X , Y and Z respectively represent the location index components of indoor articles. The parameters in the experiment are shown in Table 3: Table 3 Experimental parameters Serial number Equipment Parameter 1 2 Processor Dominant frequency Intel 2.00Hz 3 4 5 Memory Operating system Compilation environment Writing language 6 2.00GB Windows10 Visual 2010 Vc9.0 4.2 Analysis of experimental results The comparison results of indoor Virtual Reality Art Design under the application of the comparison method and the proposed method are shown in Figure 8: 100 Proposed method Comparison method Placement error /% 80 60 40 20 Refrigerator Bed Washing machine Television Heater Placement items / piece Figure 8 Comparison of placement errors By analyzing the results in Figure 8, it can be seen that the placement error of the proposed method is small, less than 20%. The difference between the method proposed by the representative and the actual user needs is small, and it can meet the user needs. The main reason is that the proposed method extracts the indoor space features, optimizes the layout vector, and reduces the placement error. In the spatial feature data clustering experiment, the main purpose is to verify the accuracy of the data of different simulation methods. The better the data clustering effect is, the higher the data accuracy is, the less the burden on the simulation process is, and the more reliable the simulation results are; On the contrary, the worse the data clustering effect and the lower the data accuracy, the less reliable the simulation results are. The third-party software is used to output the clustering results of simulation data, and different simulation methods are compared and analyzed according to the results. The specific results are shown in Figure 9-10. Height direction /mm 100 80 60 40 20 0 20 0 0 40 30 60 60 90 120 80 150 100 Figure 9 Experimental results of comparison method 100 Height direction /mm 80 60 40 20 0 0 20 0 40 30 60 60 90 80 120 150 100 Figure 10 Experimental results of the proposed method Comparing the results in the observation chart, it can be seen that different characteristic data based on the comparison method are mixed together, and there is no obvious segmentation boundary; In the proposed method, the clustering of data with different characteristics is obvious, and there is no hybridity. Combined with the experimental results of the overall deviation of the simulation model, the proposed method has small overall deviation of the simulation model, good data clustering effect, and high data accuracy. This method is superior to the comparison method. The performance of the proposed method for feature extraction of indoor space layout is tested, and the comparison results are shown in Table 4. Table 4 Feature extraction performance of indoor space layout Number of tests Feature extraction accuracy/% Visual fusion Spatial resolution 10 20 94.60 97.32 93.05 96.50 4.76 4.34 30 40 50 60 70 80 90 100 110 120 93.96 98.28 92.68 93.32 90.92 92.20 93.80 90.76 89.48 88.52 92.23 97.72 90.61 91.42 88.37 90.00 92.03 88.16 86.54 85.32 4.40 4.49 4.55 4.31 4.67 4.54 4.05 4.40 4.58 4.40 The simulation results show that this method has high identification and resolution, and improves the display and expression ability of indoor space layout. 5 Conclusion An indoor Virtual Reality Art Design Based on Immersive communication is designed, which solves the problem of large error in the placement of items. The indoor Virtual Reality Art Design Based on virtual reality technology is transparent to users in the production process, easy to operate, simple for users, fast rendering speed, synchronized with online products, etc., which can meet the design requirements of indoor virtual reality art design from the perspective of the Internet. In addition, the software designed this time can operate the software through artistic means according to the needs of users, and integrate technology and art well. Users can construct, edit, rotate, move, zoom and other operations in any space in the software, and observe the design effect of users from any angle. However, the software designed this time has a shortcoming, which is highly dependent on the network environment. Therefore, in the follow-up research, the research will focus on this part. It is hoped that the indoor Virtual Reality Art Design Based on virtual reality technology can provide some help in the field of architectural design, so as to promote the development of the construction industry and user satisfaction. Data Availability The raw data supporting the conclusions of this article will be made available by the authors, without u ndue reservation." Conflicts of Interest The authors declared that they have no conflicts of interest regarding this work." Funding The work is not supported by any funding." Ethical approval This article does not contain any research conducted by any author on human participants or animals. Reference [1] An Ning, Dang Wanping. Application of Virtual Reality Technology in Court Trial[J]. Journal of Tianjin University (Social Sciences), 2021, 23(2):151-155. [2] SUN Zhe, KONG Xiangzheng, LIU Junmin. Construction of interior space design system based on virtual reality technology[J]. Modern Electronics Technique, 2021, 44(22):101-105. 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https://figshare.com/articles/journal_contribution/Microencapsulation_and_storage_stability_of_polyphenols_from_Vitis_vinifera_grape_wastes/5028350/1/files/8493416.pdf
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Microencapsulation and storage stability of polyphenols from Vitis vinifera grape wastes
Food chemistry
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http://dx.doi.org/10.1016/j.foodchem.2015.05.117 0308-8146/ 2015 Elsevier Ltd. All rights reserved. Microencapsulation and storage stability of polyphenols from Vitis vinifera grape wastes Oier Aizpurua-Olaizola ⇑, Patricia Navarro, Asier Vallejo, Maitane Olivares, Nestor Etxebarri Aresatz Usobiaga Analytical Chemistry Department, University of the Basque Country (UPV/EHU), Barrio Sarriena s/n, 48940 Leioa, Spain a r t i c l e i n f o Wine production wastes are an interesting source of natural polyphenols. In this work, wine wastes extracts were encapsulated through vibration nozzle microencapsulation using sodium alginate as poly- mer and calcium chloride as hardening reagent. An experimental design approach was used to obtain calcium-alginate microbeads with high polyphenol content and good morphological features. In this way, the effect of pressure, frequency, voltage and the distance to the gelling bath were optimized for two nozzles of 150 and 300 lm. Long-term stability of the microbeads was studied for 6 months taking into account different storage conditions: temperatures (4 C and room temperature), in darkness and in presence of light, and the addition of chitosan to the gelling bath. Encapsulated polyphenols were found to be much more stable compared to free polyphenols regardless the encapsulation procedure and stor- age conditions. Moreover, slightly lower degradation rates were obtained when chitosan was added to the gelling bath. Article history: Received 11 March 2015 Received in revised form 27 May 2015 Accepted 29 May 2015 Available online 5 June 2015 Keywords: Wine wastes Vibration nozzle methodology (+)Catechin ()Epicatechin Long-term stability diseases (Mandel & Youdim, 2004; Papandreou et al., 2009; Vauzour, Kerr, & Czank, 2014). ⇑Corresponding author. E-mail address: oier.aizpurua@ehu.es (O. Aizpurua-Olaizola). Food Chemistry 190 (2016) 614–621 Food Chemistry 190 (2016) 614–621  2015 Elsevier Ltd. All rights reserved. ⇑Corresponding author. 1. Introduction / Food Chemistry 190 (2016) 614–621 615 (Ostatu winery and Tempranillo red grapes) and the second ones were collected from Getaria, Gipuzkoa (Mokoroaga winery and Hondarribi zuri white grapes), both in the Basque Country (North of Spain). Gasperlin, 2010). In this way, the shelf-life of encapsulated com- pounds can be considerably extended (Bakowska-Barczak & Kolodziejczyk, 2011; Robert et al., 2010) thanks to the coating material which protects the bioactive compounds against degrada- tion reactions and controls release of the compounds. The wine wastes were air dried at room temperature for two days and then mixed to obtain a representative waste sample of the wine production in the Basque Country. Air dried wastes were ground in a cryogenic mill (SPEX SamplePrep 6770 Freezer/Mill) under liquid nitrogen for 10 min at 11 cps. Grounded wastes were mixed with 1:1 ethanol/water (v/v) solution and sonicated in an ultrasonic bath for 1 h. Afterwards, ethanol was evaporated in a rotary evaporator and the remaining water extract was stored in the refrigerator until analysis and encapsulation. Among the different microencapsulation techniques, vibration nozzle microencapsulation (VNM) technology is a simple, able to scale-up industrially and suitable technique for the encapsulation of hydrophilic drugs (Dorati, Genta, Modena, & Conti, 2013). When this microencapsulation technique is used for alimentary or medical purposes, alginate is often used as wall material for bead formation (De Prisco, Maresca, Ongeng, & Mauriello, 2015; Dorati et al., 2013). This encapsulation agent is obtained from brown seaweed and it has free carboxylic groups which react with calcium cations forming stable gels (King, 1983, chap. 3). Moreover, the addition of stabilizing agents such as chitosan could improve the stability of the formed microcapsules (Krasaekoop, Bhandari, & Deeth, 2003). Chitosan is obtained from crab exoes- quelets and it has many AOH and ANH2 groups that act as electron donors. Among several encapsulation agents alginate and chitosan are very promising due to their absent of toxicity and their excel- lent biocompatibility, as they can react together by coacervation because of their opposite charges (George & Abraham, 2006; Xu, Zhan, Fan, Wang, & Zheng, 2007). 2.3. Optimization of microbeads formation Microbeads were obtained by a vibrating nozzle method using a Buchi B-390 encapsulator (Flawil, Switzerland). An equipment scheme is shown in Fig. 1. Alginate was used as encapsulation agent (1.6% w/v for 150 lm nozzle and 1.8% w/v for 300 lm noz- zle) which were prepared in an ultrasonic bath dissolving the low viscosity sodium alginate in the wine wastes extract. 100 mL of hardening solution was used, which consisted of wine wastes extract in the same concentration as in the alginate solution and g g ) Taking into account the number of variables that might affect the formation of uniform spherical smooth microbeads of calcium-alginate and calcium-alginate-chitosan, the main aim of this work was to optimize the VNM process through an experimen- tal design approach. In this sense, different nozzles and microbead drying methods were assessed to obtain microcapsules of different sizes. Furthermore, in order to assure the mid-term stability of the encapsulated microbeads, the stability of microcapsules formed under optimized conditions was evaluated for 6 months at differ- ent conditions of temperature and light. The effect of chitosan addition and microbead size on long-term stability of encapsulated polyphenols was also studied. Fig. 1. Buchi B-390 encapsulator equipment scheme. (1) Sodium alginate solution along with the extract. (2) Hardening solution: calcium chloride along with the extract at the same concentration. 2.1. Chemicals and materials Encapsulating agents used, low molecular weight sodium alginate from brown algae (viscosity of 1% solution at 25 C, 4–12 cps) and medium molecular weight chitosan (deacetylation degree 75–85%, viscosity of 1% solution with 1% acetic acid, 200–800 cps), as well as the standards (+)catechin (P96%) and ()epicatechin (P90%) were obtained from Sigma–Aldrich (Steinheim, Germany). Analysis grade calcium chloride 2-hydrate used for creating calcium alginate beads and ethanol (96%) used to extract polyphenols were purchased from Panreac (Barcelona, Spain). HPLC grade methanol (MeOH) and analysis grade glacial acetic acid used as mobile phase in chromatographic analysis and tri-sodium citrate used for microbead destabilization were obtained from Merck (Billerica, USA). HPLC grade ethanol (EtOH) used for the extract preparation was purchased from Romil (Cambridge, UK). Ultra-pure water (5 X at 25 C) was generated with a Milli-Q water purification system Element 10 from Millipore (Billerica, USA). 1. Introduction Wine production wastes are an interesting source of many by-products of high added-value, including polyphenols. In fact, the interest of wine wastes comes not only from the high polyphe- nols concentrations (Aizpurua-Olaizola et al., 2015; Makris, Boskou, & Andrikopoulos, 2012) but also from the high waste volumes which are yearly produced (FAO., 2013) and only a small percentage of this it is revalorized in Europe. The production of biofuel and bioenergy, or the preparation of manure or feed for animals are the most used alternatives to avoid costs overrun of waste management. Owing to these drawbacks, grape pomace may become a promising and economically profitable source of polyphenols. The growing rate of industrial food consumption in western societies comes along with the deep changes in nutritional habits and food culture (Jaworowska, Blackham, Davies, & Stevenson, 2013). Surely as a consequence of these changes, the interest of functional and enriched food is growing, either to enhance the health effects or to replace the losses of key compounds during the manufacturing processes (Howlett, 2008). Polyphenols are among these supplements (Shahidi, 2012) since they can neutralize free radicals turning them in less danger- ous and thereby, stopping side reactions (Dugas et al., 2000; Leopoldini, Russo, & Toscano, 2011; Tikhonov, Roginsky, & Pliss, 2009). This feature makes polyphenols an interesting family of compounds with broad applications such as the treatment of can- cer (Cordova & Watson, 2014; Hertog, 1994) or inflammations (Kim, Lee, & Lee, 2014), anti-aging aims in cosmetics (Jean-Gilles et al., 2013; Menaa & Menaa, 2014) or nutraceutical purposes (Gollücke, Peres, Odair, & Ribeiro, 2013). Moreover, polyphenols are known to be nootropics, owing to be helpful in several func- tions of the brain, such as learning, memory, attention or motiva- tion, thereby protecting the brain against neurodegenerative Unfortunately, polyphenols have a poor long-term stability, as they are affected by pH variation, presence of metal ions, light, temperature, oxygen, and enzymatic activities (Bakowska, Kucharska, & Oszmianski, 2003). Moreover, due to low water solubility, they often present a poor bioavailability (Munin & Edwards-Lévy, 2011) and they are unstable in alkaline conditions encountered in biological fluids (Dube, Ng, Nicolazzo, & Larson, 2010). The emergent microencapsulation techniques allow, in this sense, to improve the stability and bioavailability of the products and control the rate of active agent release (Dube et al., 2010; Munin & Edwards-Lévy, 2011; Zvonar, Berginc, Kristl, & O. Aizpurua-Olaizola et al. 2.2. Preparation of wine wastes extract Wine wastes were selected from two most important Basque designations of origin: D.O. Rioja and D.O. Txakoli. Regarding the first one, the wastes were collected from Samaniego, Araba Fig. 1. Buchi B-390 encapsulator equipment scheme. (1) Sodium alginate solution along with the extract. (2) Hardening solution: calcium chloride along with the extract at the same concentration. O. Aizpurua-Olaizola et al. / Food Chemistry 190 (2016) 614–621 616 of calcium chloride at 0.2 M. For complete hardening, microcap- sules were allowed to stir in the hardening solution for 10 min. well-formed microbeads specimens. The precision was estimated from the three replicates of the central point. Among all the instrumental parameters that can affect the appropriate formation of microbeads, in this work the effect of (i) vibration frequency (Hz) used to break up the laminar liquid, (ii) voltage (V) required to create an additional electrostatic field between the nozzle and the hardening solution in order to prevent the coalescence of the microbeads, (iii) air-pressure (bar) to deliver the sodium alginate solution and (iv) the distance between the nozzle and the hardening solution (mm) was assessed for nozzles of 150 lm and 300 lm using an experimental design approach. A central composite design (CCD) was carried out with the The Unscrambler (9.7 Camo Asa, Oslo, Norway) software. The design matrix consisted of 27 randomized experiments allowing the study of the effects of these variables within the following ranges: 100–2000 Hz, 250–2000 V, 140–320 bar and 30–150 mm for nozzle of 150 lm and 100–1500 Hz, 500–2000 V, 56–200 bar and 30–150 mm for nozzle of 300 lm. The amplitude was not opti- mized and it was fixed at 2 in both cases. 2.4. Determination of polyphenols content by means of High Performance Liquid Cromatography with Diode Array Detector (HPLC-DAD) The content of polyphenols entrapped in the microparticles was estimated by dissolving 350 mg of wet capsules or 20 mg of dry capsules in 1.5 mL of 0.2 M sodium citrate sonicating for an hour in an ultrasonic bath. Using sodium citrate, Ca2+ ions of the microbeads were exchanged by Na+ ions of the citrate, destabiliz- ing the calcium alginate gel structure (Deladino, Anbinder, Navarro, & Martino, 2008). Afterwards, alginate was precipitated by adding 3 mL of MeOH and the solution was filtered through a GHP Acrodisc 0.2 lm filter (Pall, NY, USA) prior to the extract anal- ysis by means of HPLC-DAD. 2.2. Preparation of wine wastes extract This last process was carried out to avoid alginate precipitation with the mobile phase. In order to check that no analyte loss happens during the precipitation step, Empty alginate microcapsules were spiked with polyphenol standards and submitted to the whole process. Wine waste extract polyphenol content was analyzed directly by means of HPLC-DAD after filtering through a 0.2 lm filter. These ranges were established considering the equipment restrictions and the characteristics of each nozzle to maintain lam- inar flow breakup. The results were obtained by counting the well formed and not-well formed microbeads in 3 different pictures obtained by optical microscopy (Olympus BH2, Tokyo, Japan) with an adapted digital camera (Nikon DS-Fi2, Tokyo, Japan) operating at 2.5 magnification. Between 100 and 300 microbeads were counted systematically in each sample. l The analysis of polyphenols in microcapsules and wine waste extracts was carried out in a HPLC system consisted of an Agilent 1100 series equipped with a quaternary pump, an autosampler and a DAD. The chromatographic separation was achieved using a Phenomenex Synergy Hydro-RP C18 column (4 lm, 150 mm  4.6 mm i.d.) with a guard column (4 lm Depth Filter  2 mm) (Phenomenex, Torrance (CA), USA) and a binary A/B gradient (Solvent A was Milli Q water with 3% acetic acid and solvent B was 100% MeOH). The gradient program was as follows: initial conditions were 0% B; rise to 80% B over 11 min, decrease to 0% B over the next 0.5 min and hold at 0% B until 18 min for re-equilibration of the system prior to the next injection. A flow Three different responses were considered to get the optimum conditions: (i) the percentage of coalescence formation (clusters of microbeads partially fused), (ii) the percentage of tail formation (microcapsules with an elliptic shape), and (iii) the percentage of well-formed spherical smooth microbeads formation. Some of the microbeads formed in each experiment are illustrated in Fig. 2. The goal of the optimization process was the minimization of the coalescence and tail formation and the maximization of Fig. 2. Optical microscopy photographs of empty alginate microbeads. (a) Well formed microcapsules using optimized conditions with nozzle of 150 lm. (b) Well formed microcapsules using optimized conditions with nozzle of 300 lm. (c) Evidence of coalescence in microbeads obtained with nozzle of 300 lm. (d) Evidence of tails in microbeads obtained with nozzle of 300 lm. Fig. 2. 2.5.3. Long-term stability of microbeads 2.5.3. Long-term stability of microbeads Long-term stability of the microbeads formed with optimized conditions was studied for 6 months at different conditions: (a) 4 C dark (b) room temperature (r.t.) dark, (c) r.t. light. To reinforce the microbeads shell, the addition of 0.05% (w/v) medium molecular weight chitosan to the hardening solution was studied. In order to dissolve the chitosan the hardening solution was prepared at 1% acetic acid concentration. Digital photographs of at least 50 microbeads formed with these optimized conditions were assessed and the average values of diameters were measured. The average size of the obtained wet microcapsules were 278 ± 8 lm and 600 ± 90 lm with the 150 lm and 300 lm nozzles respectively. The different assays were performed using both types of nozzles according to the optimum conditions described in Section 2.3 and freeze-dried afterwards. The (+)catechin and ()epicatechin con- tent of the different assays was analyzed at different days over time (185 days) as described in Section 2.4. In order to investigate the improvement of the encapsulation, free wine wastes extract stability was also studied at the same conditions. 3.2. Assessment of microencapsulation process With the purpose of comparing the degradation of loaded microcapsules and free polyphenol extract, a first-order reaction kinetic model was used. Degradation rate constants (k) and half-lives (t1/2) were calculated according to the Eqs. (1) and (2): 3.1. Optimization of microbeads formation 3.1. Optimization of microbeads formation 3.1. Optimization of microbeads formation In a first attempt, the response surfaces were built by multiple linear regression (MLR) including linear and squared terms and the interactions of any two variables and considering the percentage of well formed microbeads as response. The relative standard devia- tion (RSD%) values were below 10% for all the studied responses with the two nozzle sizes. Model fitness was confirmed by the analysis of variance (ANOVA) of the residuals (lack of fit signifi- cantly lower than pure experimental uncertainty) and the uncer- tainties of each parameter. 2.5.1. Encapsulation efficiency The wine waste extract containing 278 ± 6 lg g1 of (+)catechin and 386 ± 8 lg g1 of ()epicatechin was diluted in 1:1, 3:4, 1:2, 1:4 and 1:8 ratios using Milli-Q water in order to have extracts containing between 35–278 lg g1 and 48–386 lg g1 of (+)cate- chin and ()epicatechin respectively. The encapsulation efficiency was evaluated at these concentration levels. For that purpose, the wine waste extract was diluted in water prior to the addition of low viscosity sodium alginate. The hardening solution was always prepared in the same concentration as in the alginate solution. After incubation in the hardening solution, microcapsules contain- ing different concentrations of polyphenols were rinsed with 100 mL of ultrapure water and analyzed by means of HPLC-DAD as described in Section 2.4. Three microbeads batches were pre- pared for each wine waste dilution and the analyses were carried out in triplicate. Regarding the 300 lm nozzle microbeads formation, similar results were obtained after the analysis of the response surfaces. In fact, MLR data revealed that the significant variables affecting the well formation of microcapsules were also the voltage, the fre- quency and the pressure (p-level <0.05). Again, the distance to the hardening solution was not significant (p-level >0.05) and it was fixed at 60 mm. Similarly to the results found for 150 lm nozzle microbeads, a local maximum was found at high voltage and fre- quency values and at low pressure values (see Fig. 3). Thus, the optimal conditions for 300 lm nozzle were established as follows: 1150 Hz of frequency, 2000 V of voltage and 77 mbar of air-pressure. 2.5. Assessment of microencapsulation approach 2.5. Assessment of microencapsulation approach The microencapsulation approach was assessed taking into account the efficiency of the process as well as the drying method and the stability of the microbeads at different storage conditions. Previously extracted and determinate (with known concentration of (+)catechin and ()epicatechin polyphenols) wine wastes extract was used to evaluate the proposed microbeads formation. MLR data obtained with the experimental design assays of 150 lm nozzle revealed that the significant variables (p-level <0.05) affecting the formation of spherical smooth microbeads were the voltage, the frequency and the pressure. The distance to the hardening solution had no significant effect at this confidence level, but it was fixed at the maximum value (120 mm). As illus- trated in Fig. 3, a relative maximum was found at high voltage and frequency values and at low pressure values with respect to the percentage of well formed microbeads. Hence, high voltage (1565 V) and frequency (1525 Hz) values and low pressure values (185 mbar of air-pressure) were established as optimum condi- tions when nozzle of 150 lm was used. 2.2. Preparation of wine wastes extract Optical microscopy photographs of empty alginate microbeads. (a) Well formed microcapsules using optimized conditions with nozzle of 150 lm. (b) Well formed microcapsules using optimized conditions with nozzle of 300 lm. (c) Evidence of coalescence in microbeads obtained with nozzle of 300 lm. (d) Evidence of tails in microbeads obtained with nozzle of 300 lm. O. Aizpurua-Olaizola et al. / Food Chemistry 190 (2016) 614–621 617 2.5.2. Microbeads drying Once hardened, microcapsules were filtered, rinsed with water and dried with three different techniques: freeze drying (FreeZone Plus Labconco, Kansas, USA) during 48 h, air drying at room tem- perature during 72 h, and drying at constant temperature of 50 C during 24 h. The best drying method condition was estab- lished by the determination of polyphenol content after each dry- ing procedure, which was determined as described in Section 2.4. Once the standard working conditions were established just considering the percentage of well formed microbeads, the coales- cence and tail formation were evaluated. In general, the percentage of tail formation was found to be the highest at low frequencies and at high voltages using both nozzles; and with high working distances in the case of 300 lm nozzle. Regarding the coalescence, maximum values were found at low voltage and distance values; and in the case of 300 lm nozzle also at low frequencies. These results are consistent with the optimal conditions obtained for both microcapsules formation. 3. Results rate of 1 mL min1 was used; the column was set at room temper- ature and the injection volume was 100 lL. Major polyphenols pre- sent in wine wastes, (+)catechin and ()epicatechin were quantified at a detection wavelength of 280 nm. 3.2.1. Encapsulation efficiency The encapsulation efficiency was calculated by comparing the concentration of polyphenols in the loaded capsules with the total amount of polyphenols present in the sodium alginate solution used for the encapsulation. The later was calculated taking into account the flow of alginate used during the encapsulation time. Ln Ct C0   ¼ kt t1=2 ¼ ln2 k Ln Ct C0   ¼ kt ð1Þ t1=2 ¼ ln2 k ð2Þ where C0 is the initial polyphenol content and Ct is the polyphenol Ln Ct C0   ¼ kt ð1Þ t1=2 ¼ ln2 k ð2Þ ð1Þ t1=2 ¼ ln2 k Fig. 4 shows the encapsulation efficiency obtained for each dilu- tion using the two types of studied nozzles. As it can be observed, there were no significant differences (p-level >0.05) in encapsula- tion efficiencies between the different dilutions with any of the ð2Þ when the diameter of the nozzle was increased (Genç & Büyüktiryaki, 2014; Khorram, Samimi, Samimi, & Moghadam, 2014). In addition to the size, another reason to explain the poor encapsulation efficiencies is the used low molecular weight algi- nate. Microcapsules created with medium or high molecular weight alginate have higher encapsulation efficiencies in compar- ison to low molecular weight ones (Bojana, Verica, Viktor, & Branko, 2013). However, the microcapsule size is also bigger and the active ingredient release rate is much slower (Alsberg et al., 2003). Because of this, low molecular weight alginate is more appropriate in applications when the active ingredient degradation must be avoided but fast in vivo release is desired. when the diameter of the nozzle was increased (Genç & Büyüktiryaki, 2014; Khorram, Samimi, Samimi, & Moghadam, 2014). In addition to the size, another reason to explain the poor encapsulation efficiencies is the used low molecular weight algi- nate. Microcapsules created with medium or high molecular weight alginate have higher encapsulation efficiencies in compar- ison to low molecular weight ones (Bojana, Verica, Viktor, & Branko, 2013). However, the microcapsule size is also bigger and the active ingredient release rate is much slower (Alsberg et al., 2003). Because of this, low molecular weight alginate is more appropriate in applications when the active ingredient degradation must be avoided but fast in vivo release is desired. Besides, according to the results shown in Fig. 4, similar encap- sulation efficiencies were obtained for (+)catechin and ()epicate- chin regardless the size of nozzle. However, higher encapsulation efficiencies were obtained using the nozzle of bigger size. In fact, the encapsulation efficiencies for both compounds were around 30% for the 300 lm nozzle microbeads and around 20% for the 150 lm nozzle ones. When bigger nozzle diameters were used bigger microbead sizes were obtained whereas the surface area-to-volume ratio decreased. Consequently, the amount of polyphenols released during the microbeads hardening process was smaller, which favored the encapsulation efficiency. Similar results were previously obtained in other works published in the literature where higher encapsulation efficiencies were obtained ð2Þ ð2Þ where C0 is the initial polyphenol content and Ct is the polyphenol content at time t. where C0 is the initial polyphenol content and Ct is the polyphenol content at time t. O. Aizpurua-Olaizola et al. / Food Chemistry 190 (2016) 614–621 O. Aizpurua-Olaizola et al. / Food Chemistry 190 (2016) 614–621 Fig. 3. Response surfaces of significant variables for the formation of spherical smooth microbeads being the assessed response the percentage of well formed microbeads. For microbeads formed with the 150 lm nozzle: (a) frequency (Hz) and voltage (V), (b) pressure (mbar) and voltage and (c) pressure and frequency. For microbeads formed with the 300 lm nozzle: (d) frequency and voltage, (e) pressure and voltage and (f) pressure and frequency. 618 O. Aizpurua-Olaizola et al. / Food Chemistry 190 (2016) 614–621 618 Fig. 3. Response surfaces of significant variables for the formation of spherical smooth microbeads being the assessed response the percentage of well formed microbeads. For microbeads formed with the 150 lm nozzle: (a) frequency (Hz) and voltage (V), (b) pressure (mbar) and voltage and (c) pressure and frequency. For microbeads formed with the 300 lm nozzle: (d) frequency and voltage, (e) pressure and voltage and (f) pressure and frequency. Fig. 4. (Left) loading efficiencies obtained using different initial wine wastes extract dilutions to prepare the sodium alginate and the hardening solutions. (Right) the effect of different drying procedures on polyphenol loss during drying, compared to non-dried microbeads. n = 3, %95 confidence level. ined using different initial wine wastes extract dilutions to prepare the sodium alginate and the hardening solutions. (Right) the effec henol loss during drying, compared to non-dried microbeads. n = 3, %95 confidence level. Fig. 4. (Left) loading efficiencies obtained using different initial wine wastes extract dilutions to prepare the sodium alginate and th different drying procedures on polyphenol loss during drying, compared to non-dried microbeads. n = 3, %95 confidence level. Fig. 4. (Left) loading efficiencies obtained using different initial wine wastes extract dilutions to prepare the sodium alginate and the hardening solutions. (Right) the effect of different drying procedures on polyphenol loss during drying, compared to non-dried microbeads. n = 3, %95 confidence level. nozzles. Therefore, it can be concluded that the polyphenol con- centration used in the sodium alginate solution and in the harden- ing solution does not affect to the encapsulation efficiency, at least, in the studied range. Table 1 i d Table 1 First order degradation kinetic model parameters and half-lives of free wine wastes extract and the different loaded microbeads (150 lm or 300 lm nozzle, and, with or without chitosan) at different environments (at 4 C in the freezer and at room temperature (r.t.) with light or in darkness). Table 1 First order degradation kinetic model parameters and half-lives of free wine wastes extract and the different loaded microbeads (150 l chitosan) at different environments (at 4 C in the freezer and at room temperature (r.t.) with light or in darkness). First order degradation kinetic model parameters and half-lives of free wine wastes extract and the different loaded microbeads (150 lm or 300 lm nozzle, and, with or without chitosan) at different environments (at 4 C in the freezer and at room temperature (r.t.) with light or in darkness). Environment r2 t(1/2) (days) (+)Catechin ()Epicatechin (+)Catechin ()Epicatechin Free extract 4 C 0.943 0.720 396 408 r.t. Light 0.973 0.984 14 15 r.t. Dark 0.976 0.981 19 20 150 lm, chit. 4 C 0.875 0.788 764 857 r.t. Light 0.980 0.987 89 85 r.t. Dark 0.958 0.966 152 146 150 lm, no chit. 4 C 0.814 0.624 619 680 r.t. Light 0.973 0.980 74 72 r.t. Dark 0.952 0.947 139 139 300 lm, chit. 4 C 0.949 0.929 478 510 r.t. Light 0.971 0.936 462 478 r.t. Dark 0.993 0.992 163 157 300 lm, no chit. 4 C 0.879 0.855 472 465 r.t. Light 0.678 0.647 373 361 r.t. Dark 0.965 0.965 157 159 in presence of light and in darkness) for 6 months. They all presented a first order degradation curve for (+)catechin and ()epicatechin for all storage conditions (Table 1). The changes in (+)catechin and ()epicatechin content of microbeads formed with 2 nozzles in presence or absence of chitosan are shown in Fig. 5. in presence of light and in darkness) for 6 months. They all presented a first order degradation curve for (+)catechin and ()epicatechin for all storage conditions (Table 1). The changes in (+)catechin and ()epicatechin content of microbeads formed with 2 nozzles in presence or absence of chitosan are shown in Fig. 5. According to the results, the best strategy to dry the microbeads was found to be freeze-drying, in which no decrease in polyphenol content was observed for the microcapsules produced with the 300 lm nozzle. Table 1 i d Although a slight decrease of polyphenol content was observed when the nozzle of 150 lm was used, freeze-drying method rendered the lowest losses (30%) in compar- ison to the other two assessed strategies (>70%). In addition, the freeze-dried microbeads were finely granulated whereas microbe- ads dried in air and at constant temperature were bound together. This is important in food applications where mouth-feel is a crucial factor. In the case of microbeads formed with the 150 lm nozzle, at 4 C in darkness half-lives between 619 and 857 days were obtained, whereas at room temperature in light and in darkness were 72–89 days and 139–152 days respectively. When chitosan was added to the gelling bath relatively small differences were found, decreasing the encapsulation efficiency by 3–7% but also decreasing the degradation rate by 3–7%. In contrast, half-lives of the microbeads formed with the 300 lm nozzle at 4 C were between 465 and 510 days, whereas at room temperature in light and in darkness were 361–478 days and 157–163 days respectively. The addition of chitosan improved 3.2.3. Long-term stability of microbeads 3.2.2. Selection of drying method of microbeads The effect of different drying methods on polyphenol encapsu- lation is shown in Fig. 4. The results are expressed as the percent- age of polyphenol loss occurred during the drying techniques. O. Aizpurua-Olaizola et al. / Food Chemistry 190 (2016) 614–621 619 References Aizpurua-Olaizola, O., Ormazabal, M., Vallejo, A., Olivares, M., Navarro, P., Etxebarria, N., et al. (2015). Optimization of supercritical fluid consecutive extractions of fatty acids and polyphenols from Vitis vinifera grape wastes. Journal of Food Science, 80(1), 101–107. Alsberg, E., Kong, H. J., Hirano, Y., Smith, M. K., Albeiruti, A., & Mooney, D. J. (2003). Regulating bone formation via controlled scaffold degradation. Journal of Dental Research, 82(11), 903–908. As it is illustrated in Fig. 5, (+)catechin and ()epicatechin showed close degradation rates. In all cases, much slower degrada- tion rates were obtained at low temperatures. Precisely, the long- est stability in this study was obtained at 4 C in the smallest microcapsules (150 lm nozzle). The addition of chitosan increased slightly the stability of small microcapsules, especially at low temperatures. Bakowska, A. M., Kucharska, A. Z., & Oszmianski, J. (2003). The effects of heating, UV irradiation and storage on stability of anthocyanin–polyphenol copigment complex. Food Chemistry, 81(3), 349–355. Bakowska-Barczak, A. M., & Kolodziejczyk, P. P. (2011). Black currant polyphenols: Their storage stability and microencapsulation. Industrial Crops and Products, 34(2), 1301–1309. ( ) Bojana, I., Verica, D., Viktor, N., Branko, B. (2013). Liposome-in-alginate systems for encapsulation of natural antioxidants. InsideFood Symposium, 9–12 April 2013, Leuven, Belgium. In the same way, 16–22% less degradation was observed when microbeads formed with the 150 lm nozzle were stored in dark- ness. These results were expected as polyphenols are prone to degradate under several environmental conditions, such as light and temperature. Surprisingly, 17–31% lower degradation rates were observed when 300 lm nozzle microbeads were stored in light. It is possible that polyphenols become cross-linked in light, either to the alginate or to each other (McManus, Davis, Lilley, & Haslam, 1981; Munin & Edwards-Lévy, 2011; Nishad Fathima, Ansari, & Raghava rao, 2007), thereby slowering the degradation. Cordova, F. M., & Watson, R. R. (2014). Food and supplement polyphenol action in cancer recurrence. In R. R. Watson, V. R. Preedy, & S. Zibadi (Eds.), Polyphenols in human health and disease (pp. 191–195). Academic Press. ll ( ) l b De Prisco, A., Maresca, D., Ongeng, D., & Mauriello, G. (2015). Microencapsulation by vibrating technology of the probiotic strain Lactobacillus reuteri DSM 17938 to enhance its survival in foods and in gastrointestinal environment. LWT – Food Science and Technology, 61(2), 452–462. Deladino, L., Anbinder, P. S., Navarro, A. S., & Martino, M. N. (2008). References Encapsulation of natural antioxidants extracted from Ilex paraguariensis. Carbohydrate Polymers, 71(1), 126–134. Dorati, R., Genta, I., Modena, T., & Conti, B. (2013). Microencapsulation of a hydrophilic model molecule through vibration nozzle and emulsion phase inversión technologies. Journal of Microencapsulation, 30(6), 559–570. On the other hand, due to lesser surface area-to-volume ratio microbeads formed with the 300 lm nozzle and stored at room temperature were less vulnerable to degradation than the ones formed with the 150 lm nozzle. This pattern was not observed at 4 C. Dube, A., Ng, K., Nicolazzo, J. A., & Larson, I. (2010). Effective use of reducing agents and nanoparticle encapsulation in stabilizing catechins in alkaline solution. Food Chemistry, 122, 662–667. Dugas, A. J., Castañeda-Acosta, J., Bonin, G. C., Price, K. L., Fisher, N. H., & Winston, G. W. (2000). Evaluation of the total peroxyl radical-scavenging capacity of flavonoids: structure–activity relationships. Journal of Natural Products, 63(3), 327–331. Finally, Table 1 shows that the degradation of free wine wastes extract took place much faster than the encapsulated ones, for all environment conditions. This way, due to the protection of polyphenols by the encapsulating agent preventing oxidation, between 5 and 33 times higher half-lives were obtained when microbeads were stored at room temperature. The protection improvement was less when they were stored at 4 C, between 1.2 and 2.1. FAO. (2013). Faostat webpage. http://faostat.fao.org. Genç, L., & Büyüktiryaki, S. (2014). Preparation and characterization of methotrexate-loaded microcapsules. Pharmaceutical Development and Technology, 19(1), 42–47. George, M., & Abraham, E. (2006). Polyionic hydrocolloids for the intestinal delivery of protein drugs: Alginate and chitosan – A review. Journal of Controlled Release, 114(1), 1–14. Gollücke, A. P., Peres, R. C., Odair, A., Jr., & Ribeiro, D. A. (2013). Polyphenols: A nutraceutical approach against diseases. Recent Patents on Food, Nutrition & Agriculture, 5(3), 214–219. Hertog, M. G. L. (1994). Flavonols and flavones in foods and their relation with cancer and coronary heart disease risk. The Netherlands: Agricultural University Wageningen (Ph.D. Thesis). 3.2.3. Long-term stability of microbeads On the other hand, the microcapsules loss some water during this process, lead- ing to a size reduction and therefore to a lower loading efficiency (Deladino et al., 2008). This size reduction effect is much more pro- nunciated in microbeads created with the 150 lm nozzle, as they have higher surface area-to-volume ratio. As a result, their encap- sulation efficiency is lower. Acknowledgements The authors are grateful to Ostatu and Mokoroaga wineries for supplying the required samples, to Mineralogy And Petrology Department of the University of the Basque Country for letting a optical microscopy to perform this study, and to the Basque Government for the SAIOTEK project (SAI12/136). O. Aizpurua is grateful to the Basque Government for the PhD fellowship. 3.2.3. Long-term stability of microbeads Microbeads storage stability was studied along with the non-encapsulated wine wastes extract at different conditions of temperature (i.e., at 4 C and at room temperature) and light (i.e., Fig. 5. Storage stability of two main polyphenol targets (+)catechin and ()epicatechin encapsulated with 150 and 300 lm nozzle and in the presence or absence of chitosan (chit.) at 4 C in the freezer in darkness and at room temperature (r.t.) in darkness and with light (n = 3). RSD values were always below 10%. Fig. 5. Storage stability of two main polyphenol targets (+)catechin and ()epicatechin encapsulated with 150 and 300 lm nozzle and in the presence or absence of chitosan (chit.) at 4 C in the freezer in darkness and at room temperature (r.t.) in darkness and with light (n = 3). RSD values were always below 10%. O. Aizpurua-Olaizola et al. / Food Chemistry 190 (2016) 614–621 620 the encapsulation efficiency by 31–36% and decreased the degra- dation rate by 1–16%. The different effect of chitosan on the encap- sulation efficiency of microbeads for small and large nozzles may lie in the two opposite effects that are happening when chitosan is added to the harderning solution. On the one hand, a membrane is formed through ionic interactions between the carboxylic residues of the alginate and positively charged amino groups of chitosan, reducing the active compounds leaking during encapsulation and decreasing the degradation during storage (Lertsutthiwong, Rojsitthisak, & Nimmannit, 2009). On the other hand, the microcapsules loss some water during this process, lead- ing to a size reduction and therefore to a lower loading efficiency (Deladino et al., 2008). This size reduction effect is much more pro- nunciated in microbeads created with the 150 lm nozzle, as they have higher surface area-to-volume ratio. As a result, their encap- sulation efficiency is lower. the encapsulation efficiency by 31–36% and decreased the degra- dation rate by 1–16%. The different effect of chitosan on the encap- sulation efficiency of microbeads for small and large nozzles may lie in the two opposite effects that are happening when chitosan is added to the harderning solution. On the one hand, a membrane is formed through ionic interactions between the carboxylic residues of the alginate and positively charged amino groups of chitosan, reducing the active compounds leaking during encapsulation and decreasing the degradation during storage (Lertsutthiwong, Rojsitthisak, & Nimmannit, 2009). 4. Conclusions I., Cordopatis, P., Klimis-Zacas, D., & Margarity, M. (2009). Effect of a polyphenol-rich wild blueberry extract on cognitive performance of mice, brain antioxidant markers and acetylcholinesterase activity. Behavioural Brain Research, 198(2), 352–358. cognitive performance of mice, brain antioxidant markers and acetylcholinesterase activity. Behavioural Brain Research, 198(2), 352–358. Lertsutthiwong, P., Rojsitthisak, P., & Nimmannit, U. (2009). Preparation of turmeric oil-loaded chitosan–alginate biopolymeric nanocapsules. Materials Science and Engineering: C, 29, 856–860. Robert, P., Gorena, T., Romero, N., Sepulveda, E., Chavez, J., & Saenz, C. (2010). Encapsulation of polyphenols and anthocyanins from pomegranate (Punica granatum) by spray drying. International Journal of Food Science and Technology, 45(7), 1386–1394. Makris, D. P., Boskou, G., & Andrikopoulos, N. K. (2012). Polyphenolic content and in vitro antioxidant characteristics of wine industry and other agri-food solid waste extracts. Journal of Food Composition and Analysis, 20(2), 125–132. Shahidi, F. (2012). Nutraceuticals, functional foods and dietary supplements in health and disease. Journal of Food and Drug Analysis, 20(1), 226–230. kh k l ( ) h h b k d Mandel, S., & Youdim, M. B. H. (2004). Serial Review: Flavonoids and isoflavones (phytoestrogens): Absorption, metabolism, and bioactivity. Free Radical Biology & Medicine, 37(3), 304–317. Tikhonov, I., Roginsky, V., & Pliss, E. (2009). The chain-breaking antioxidant activity of phenolic compounds with different numbers of OAH groups as determined during the oxidation of styrene. International Journal of Chemical Kinetics, 41(2), 92–100. McManus, J. P., Davis, K. G., Lilley, T. H., & Haslam, E. (1981). The association of proteins with polyphenols. Journal of the Chemical Society, Chemical Communications, 7, 309–311. Vauzour, D., Kerr, J., & Czank, C. (2014). Plant polyphenols as dietary modulators of brain functions. In R. R. Watson, V. R. Preedy, & S. Zibadi (Eds.), Polyphenols in human health and disease (pp. 357–370). Academic Press. Menaa, F., & Menaa, A. (2014). Skin photoprotection by polyphenols in animal models and humans. In R. R. Watson, V. R. Preedy, & S. Zibadi (Eds.), Polyphenols in human health and disease (pp. 831–838). Academic Press. Xu, Y., Zhan, C., Fan, L., Wang, L., & Zheng, H. (2007). Preparation of dual crosslinked alginate–chitosan blend gel beads and in vitro controlled release in oral site- specific drug delivery system. International Journal of Pharmaceutics, 336(2), 329–337. Munin, A., & Edwards-Lévy, F. (2011). Encapsulation of natural polyphenolic compounds: A review. Pharmaceutics, 3(4), 793–829. Zvonar, A., Berginc, K., Kristl, A., & Gasperlin, M. (2010). 4. Conclusions Howlett, J. (2008). Functional Foods: From Science to Health and claims. Brussels, Belgium: ILSI Europe. The encapsulation technique can be a valuable methodology to improve the stability and bioavailability of different type of prod- ucts. In this work, microencapsulation of wine wastes extract rich in polyphenols was successfully optimized and evaluated using two different types of nozzles. Encapsulation efficiencies in cal- cium alginate microbeads were found to be independent to the extract concentration in the studied range but higher efficiencies were rendered by the high size nozzle. Jaworowska, A., Blackham, T., Davies, I. G., & Stevenson, L. (2013). Nutritional challenges and health implications of takeaway and fast food. Nutrition Reviews, 71(5), 310–318. Jean-Gilles, D., Li, I., Vaidyanathan, V. G., King, R., Cho, B., Worthen, D. R., et al. (2013). Inhibitory effects of polyphenol punicalagin on type-II collagen degradation in vitro and inflammation in vivo. Chemico-Biological Interactions, 205(2), 90–99. Khorram, M., Samimi, M., Samimi, A., & Moghadam, H. (2014). Electrospray preparation of propranolol-loaded alginate beads: Effect of matrix reinforcement on loading and release profile. Journal of Applied Polymer Science, 131, 413–434. The stability of microcapsules containing polyphenols was studied at different conditions of light and temperature. The assays showed that all encapsulated systems were much more stable than the free extract at all conditions, with half-life between 72 and 857 days. Lower degradation rates were obtained when chitosan was added to the gelling bath. This study will be helpful for the industrial application of wine wastes polyphenols. Kim, J., Lee, K. W., & Lee, H. J. (2014). Polyphenols suppress and modulate inflammation: Possible roles in health and disease. In R. R. Watson, V. R. Preedy, & S. Zibadi (Eds.), Polyphenols in human health and disease (pp. 393–408). Academic Press. King, A. H. (1983). Brown seaweed extracts (alginates). In M. Glicksman (Ed.). Food hydrocolloids (Vol. 3, pp. 115). Boca Raton, FL: CRC Press. Krasaekoopt, W., Bhandari, B., & Deeth, H. (2003). Evaluation of encapsulation techniques of probiotics for yoghurt. International Dairy Journal, 13, 3–13. O. Aizpurua-Olaizola et al. / Food Chemistry 190 (2016) 614–621 621 Leopoldini, M., Russo, N., & Toscano, M. (2011). The molecular basis of working mechanism of natural polyphenolic antioxidants. Food Chemistry, 125(2), 288–306. Papandreou, M. A., Dimakopoulou, A., Linardaki, Z. I., Cordopatis, P., Klimis-Zacas, D., & Margarity, M. (2009). Effect of a polyphenol-rich wild blueberry extract on Papandreou, M. A., Dimakopoulou, A., Linardaki, Z. 4. Conclusions Microencapsulation of self- microemulsifying system: Improving solubility and permeability of furosemide. International Journal of Pharmaceutics, 388(1–2), 151–158. Nishad Fathima, N., Ansari, T., & Raghava rao, J. (2007). Role of green tea polyphenol crosslinking in alleviating ultraviolet-radiation effects on collagen. Journal of Applied Polymer Science, 106, 3382–3386.
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Trends in healthcare expenditures and resource utilization among a nationally representative population with opioids in the United States: a serial cross-sectional study, 2008 to 2017
Substance abuse treatment, prevention, and policy
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Bounthavong et al. Substance Abuse Treatment, Prevention, and Policy (2021) 16:80 Bounthavong et al. Substance Abuse Treatment, Prevention, and Policy (2021) 16:80 https://doi.org/10.1186/s13011-021-00415-5 RESEARCH Open Access Trends in healthcare expenditures and resource utilization among a nationally representative population with opioids in the United States: a serial cross-sectional study, 2008 to 2017 Trends in healthcare expenditures and resource utilization among a nationally representative population with opioids in the United States: a serial cross-sectional study, 2008 to 2017 Mark Bounthavong1,2* , Kangho Suh3, Meng Li4, Patrick M. Spoutz1, Britney Ann Stottlemyer3 and Aryana Sepassi2 Abstract Background: Previous reports on healthcare costs and expenditures associated with populations prescribed an opioid primarily focused on populations who chronically use opioids or have opioid use disorder. However, studies that characterize the healthcare and expenditures costs among the wider number of people prescribed opioids in a nationally representative population are unavailable. We sought to characterize the healthcare costs and expenditures associated with a population prescribed an opioid in the U.S. from 2008 to 2017. Methods: A serial cross-sectional design was used to compare the economic burden of adult household respondents who were prescribed and not prescribed an opioid using pooled data from the Medical Expenditure Panel Survey (MEPS) between 2008 and 2017. Respondents with an opioid prescription were matched to respondents without an opioid prescription using propensity score match methods with survey weights. Two-part generalized linear models were used to estimate the survey-weighted annual healthcare expenditures and resource utilization adjusting for multiple covariates. Additionally, 10-year trend comparisons between the groups were performed. Costs were adjusted to 2019 US dollars. Introduction on their patient population. Healthcare payers have a fi- nancial incentive to address the opioid epidemic. Under- standing the impact on healthcare expenditures and costs may stimulate policies to improve opioid prescrib- ing for pain management, increase access to harm re- duction treatment (e.g., naloxone), and increase access to medications for opioid use disorder (e.g., buprenorphine). The opioid epidemic in the United States (U.S.) is one of the most devastating public health crises in recent de- cades. In 2018, over 9.9 million Americans 12 years and older misused prescription pain medications, and ap- proximately 2 million Americans were reported to have an opioid use disorder [1]. Opioid use and misuse were responsible for 49,860 drug overdose deaths in 2019 (70.6% of all drug overdose deaths) [2]. In addition to the staggering toll on morbidity and mortality, the opi- oid epidemic has significant impacts on healthcare costs and expenditures. We sought to characterize the healthcare costs and ex- penditures associated with people who were prescribed opioids in the U.S. from 2008 to 2017. Our primary aim was to evaluate whether people who were prescribed opioids have higher healthcare expenditures and re- source utilization compared to people who were not pre- scribed opioids. Secondary aims evaluated whether a higher number of unique opioids prescribed within a given year was associated with healthcare expenditures and resource utilization. The economic burden of the opioid crisis has been re- ported to be approximately $1.02 trillion in 2017, which includes $471 billion for the cost of opioid use disorder and $550 billion for the cost of fatal opioid overdose [3]. The economic burden of the opioid crisis has been re- ported to be approximately $1.02 trillion in 2017, which includes $471 billion for the cost of opioid use disorder and $550 billion for the cost of fatal opioid overdose [3]. Previous reports on healthcare costs and expenditures associated with people who use opioids primarily fo- cused on people who chronically use opioids or people with opioid use disorder or misuse. Chang and col- leagues [4], using prescription administrative claims data between 2012 and 2013, reported that people who chronically use opioids have significantly higher total costs, medical costs, and drug costs compared to non- high-risk people who use opioids. Introduction Kirson and colleagues [5], using administrative claims data between 2011 and 2015, reported that people who abuse opioids had sig- nificantly higher healthcare costs compared to people who use but do not abuse opioids, which were driven by substance use disorder diagnoses, mental health condi- tions, and pain conditions. However, studies that characterize the healthcare and expenditures costs among the wider number of people who use opioids in a nationally representative population could provide payers with insight on the impact opioid prescribing has © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 13 Bounthavong et al. Substance Abuse Treatment, Prevention, and Policy (2021) 16:80 Bounthavong et al. Substance Abuse Treatment, Prevention, and Policy Results: There was a weighted total of 31,696,671 respondents with an opioid and 31,536,639 respondents without an opioid after propensity score matching. The sample had a mean (SD) age of 50.63 years (18.03), 58.9% females, and 81.6% Whites. Total annual economic burden among RPOs was $524 billion. Annual total expenditures per respondent with and without an opioid were $16,542 and $7067, respectively (P < 0.001). Similarly, adjusted prescription, outpatient, emergency department, and inpatient expenditures were significantly higher for respondents with an opioid compared to respondents without an opioid. Average annual increases in expenditures were significantly greater among respondents with an opioid compared to respondents without an opioid for total (+$185; 95% CI: $37–$334) and prescription (+$78; 95% CI: $28–$128) expenditures. There were no differences in the average annual trends for outpatient, emergency department, and inpatient expenditures between respondents with and without an opioid. Conclusions: Respondents with an opioid prescription had higher healthcare expenditures and resource utilization compared to respondents without an opioid prescription from 2008 to 2017. Specifically, significant annual increases were observed for total and prescription expenditures. Additionally, 10-year trends in total and prescription expenditures were higher among respondents with an opioid than respondents without an opioid. Keywords: Health expenditures, Cross-sectional studies, Health resources, Analgesics, opioids, Propensity score Other variables d d Respondent demographics that were collected included age (categorized as 18–24, 25–44, 45–64, and 65 and older), race (White, Black, Native American/Alaskan Na- tive, Asian/Pacific Islander, and Multiple races reported), ethnicity (Hispanic and Non-Hispanic), marital status (Married, Widowed, Divorced, Separated, Never), educa- tion level (No degree, GED/High School, Associated or other degree, Bachelor degree, Master/Doctor degree, Not ascertainable, Don’t know, and Refused to answer), region (Northwest, Midwest, South, and West), poverty status (Poor/Negative, Near Poor, Low Income, Middle Income, and High Income), insurance coverage (Any Private, Public, and Uninsured), and comorbidities. Methods Design A serial cross-sectional design was used to compare the economic burden of adult household respondents with and without an opioid using pooled data from the Med- ical Expenditure Panel Survey (MEPS) between 2008 and 2017. MEPS is a nationally representative sample of the U.S. population and collects data on their use of health services including costs associated with specific services curated by the Agency for Healthcare Research and Quality (AHRQ) [6, 7]. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines for a cross-sectional study design [8]. Page 3 of 13 Page 3 of 13 Bounthavong et al. Substance Abuse Treatment, Prevention, and Policy (2021) 16:80 out-of-pocket payments, and insurance payments (e.g., private, Medicaid, Medicare, and other sources). Pre- scription expenditures include out-of-pocket payments and insurance payers for prescription drugs. MEPS does not report expenditures for over-the-counter medica- tions or inpatient administration of medications. Out- patient expenditures include all provider visits (e.g., physician and non-physician) in the ambulatory setting. Emergency department expenditures included all visits to the emergency department but does not include any visit that resulted in an inpatient admission to avoid double counting. Inpatient expenditures included all ex- penses for direct hospital care (e.g., room, board, diag- nostic and laboratory work, and imaging); MEPS does not record provider services (e.g., anesthesiologists, radi- ologists, and other specialists) as part of the inpatient ex- penditures. All expenditures were adjusted for inflation using the Consumer Price Index to reflect costs in 2019 $US. Missing data for expenditures were imputed using a weighted hot deck procedure where other survey re- sponses were used to input the missing data based on survey-weighted distributions [10]. Respondents with an opioid prescription Respondents with an opioid prescription Respondents with an opioid were defined as household respondents who reported having been prescribed an opioid prescription. Information on opioid use was ac- quired using the Prescription Medicines file, which pro- vides details on the household-reported prescribed medications. Each record describes a unique prescription event (purchased or obtained by the household respond- ent). The therapeutic classes and subclasses of the pre- scription medication were based on Multum Lexicon Variables from Cerner Multum, Inc., which was used to identify household respondents with opioid prescriptions defined as narcotic analgesics. Data from the therapeutic classes and subclasses were cross referenced with the names of the medications and grouped into categories based on the number of unique opioid prescriptions ac- quired during the respective year: household respon- dents that reported having only one unique opioid in a given year, two unique opioids in a given year, and three or more unique opioids in a given year. Unique opioid represents the mutually exclusive generic name of the opioid. Number of prescriptions filled was based on the PC and included the name of the medication, the number of times the medication was acquired, and payments asso- ciated with the medication. Number of outpatient visits was based on MPC and included encounters in office- based settings. Number of emergency department visits was based on the MPC and included the count of emer- gency department visits reported. Number of inpatient night stays were based on the MPC and included the total number of nights associated with a discharge event. Sample p Data from adult MEPS household respondents (18 years old or older) between 2008 and 2017 were pooled. The pooled population was based on the subsample of the National Health Interview Survey households, which is a national representative sample of the non- institutionalized U.S. population. We used the consoli- dated MEPS Household Component, Prescription Medi- cines and Medical Conditions files to identify and characterize respondents with and without an opioid on their prescription profile. The Household Component file contains information on responder demographics, socioeconomic information, insurance information, em- ployment information, and health status. The Prescrip- tion Medicines file contains information of respondents’ self-reported prescription drug fills. Medical condition file contains information about the respondents’ self- reported diagnoses. Statistical analysis Descriptive analysis on demographics were compared between respondents with and without an opioid using independent t tests and chi square tests for continuous and discrete data, respectively, and applying the appro- priate survey weights. We used Stata’s set of svy com- mands to properly survey weight the pooled matched data to reflect a national representative noninstitutional- ized U.S. population. Means and standard deviations were presented for continuous data and frequency and proportions were presented for discrete data. We applied a survey-weighted two-part generalized linear model to compare the annual health expenditures and resource utilization between respondents with and without an opioid prescription adjusting for their char- acteristics [18, 19]. In the first part, we used a logistic re- gression model to assess the likelihood of having nonzero healthcare expenditures. In the second part, we used a generalized linear model with gamma distribution to evaluate the association between healthcare expendi- tures with treatment assignment conditions on whether the respondents had nonzero healthcare expenditures adjusting for respondent characterisitcs [20]. Results were reported as annual mean expenditures and re- sources utilized with corresponding 95% confidence in- tervals (CIs). Goodness of fit tests included the Pearson correlation of the predicted values and residuals, Pregi- bon’s link test, and modified Hosmer-Lemeshow test [21]. p p p A propensity score matching method for complex sur- vey data was used to balance the measurable covariates between the two groups [11]. This allowed us to create weighted matched cohorts that would be generalizable to the original survey population. We applied this method to generate a 1:1 propensity match between re- spondents with and without an opioid. Propensity scores were generated using a logistic regression by regressing the covariates to the treatment assignment variable (re- spondents with and without an opioid). We included variables into the propensity score matching based on the Anderson-Newman Behavioral Health Model [12– 14], which provides a framework for the social and indi- vidual determinants of health care utilization. These in- clude age, gender, race, ethnicity, marital status, education level, region, poverty status, and insurance coverage, and comorbidities. Moreover, we selected these comorbidities based on their impact on health care utilization and availability from the MEPS data [15, 16]. Matches were made using the nearest neighbor ap- proach with a caliper distance of 0.01 without replace- ments. Statistical analysis Balance between the groups was assessed using standardized differences; a value of 0.1 or less was con- sidered balanced [17]. Comparison of trends between respondents with and without an opioid prescription were evaluated using lin- ear regression models adjusting for covariates. An inter- action term between the respondents with an opioid prescription variable and time was used to estimate the average annual differences in expenditures (differences in trends) between respondents with and without an opi- oid prescription across 2008 to 2017. These findings were presented as mean annual differences with corre- sponding 95% CIs. Statistical significance was defined as a two-tailed alpha < 0.05. Propensity score matching was performed using the MatchIt [22] package for R software version 4.0.3 (The R Foundation for Statistical Computing; http://www.r-project.org) [23]. All other analyses were performed using Stata SE version 15 (Stata Corp, Inc., College Station, TX). For the primary aim, we evaluated whether respon- dents with an opioid prescription have higher healthcare expenditures and resource utilization compared to matched respondents without an opioid. Healthcare ex- penditures included total, prescription, outpatient, emer- gency department, and inpatient expenditures. Healthcare resource utilization included number of pre- scriptions filled, number of office-based visits, number Healthcare expenditures and resources The outcomes of interest included total healthcare ex- penditures, prescription expenditures, outpatient expen- ditures, emergency department expenditures, inpatient expenditures, number of prescriptions filled, number of office-based visits, number of emergency department visits, and number of inpatient night stays. Expenditure estimates in MEPS are based on the Medical Provider Component (MPC) and Pharmacy Component (PC) of the survey, which include payments and not charges [9]. Total healthcare expenditures captured all payments re- lated to healthcare services including direct payments, Federal poverty status was categorized based on the federal poverty level (FPL) defined by the Current Popu- lation Survey for the respective years: Poor/Negative (less than 100% of FPL), Near Poor (100% to less than 120% of FPL), Low Income (125% to less than 200% of FPL), Middle Income (200% to less than 400% of FPL), Bounthavong et al. Substance Abuse Treatment, Prevention, and Policy Page 4 of 13 Page 4 of 13 (2021) 16:80 and High Income (greater than or equal to 400% of FPL). of emergency department visits, and number of inpatient night stays. Additionally, we compared the trends in healthcare expenditures and resource utilization between respondents with and without an opioid across 2008 to 2017. In the secondary aim, we performed a subgroup analysis to evaluate whether a higher number of unique opioids prescribed within a given year was associated with greater healthcare expenditures and resource utilization. Respondents were grouped into three cat- egories based on the number of unique opioid prescrip- tions they received during the given year: one opioid prescription, two unique opioid prescriptions, and three or more unique opioid prescriptions. Comorbidities included high blood pressure, coronary heart disease, angina, myocardial infarction, other heart diseases, stroke, high cholesterol, cancer, diabetes, and arthritis. Comorbidities were identified using MEPS pri- ority conditions definitions that ask respondents if they were ever diagnosed with these conditions. Priority con- ditions were selected due to their high prevalence and established standards for clinical care. Results Among the 350,831 respondents who were pooled be- tween 2008 and 2017, a total of 32,779 (9.3%) had ac- quired or purchased an opioid prescription (see Supplement Table A). After propensity score matching, a total of 30,703 respondents with an opioid prescription were matched to an equal number of respondents with- out an opioid prescription. Visual inspection of the stan- dardized mean difference plot indicated that appropriate balance was achieved between the two groups (see Sup- plement Figure A). The matched cohorts represented a survey weighted population of 63.2 million total respon- dents with 31.7 million people with and without an opi- oid prescription in each group. Characteristics between the two matched cohorts were balanced with no mean- ingful differences (Table 1). p p p p In the subgroup analysis, respondents with 3 or more unique opioid prescriptions had significantly higher average annual total, prescription, and inpatient expendi- tures compared to respondents with 2 unique opioid prescriptions and respondents with 1 unique opioid pre- scription (Table 3). However, respondents with 2 unique opioid prescriptions had significantly higher average an- nual outpatient and emergency department expenditures compared to respondents with 3 or more unique opioids prescriptions and respondents with 1 unique opioid pre- scription. Regarding healthcare resource utilization, re- spondents with 3 or more unique opioid prescriptions had higher number of prescriptions filled, number of office-based visits, number of emergency department visits, and number of inpatient night stays compared to respondents with 2 unique opioid prescriptions and re- spondents with 1 unique opioid prescription. (The 10- year trends for healthcare expenditures and resource utilization for the subgroup analyses are illustrated in see Supplement Figure B). The total annual economic burden of respondents with an opioid prescription as reflected by healthcare ex- penditures was estimated as $524 billion (31.7 million weighted number of people with an opioid X $16,542). Between 2008 and 2017, respondents with an opioid pre- scription had significantly higher average annual total ($16,542 versus $7067; P < 0.001), prescription ($3067 versus $2293; P < 0.001), outpatient ($1804 versus $650; P < 0.001), emergency department ($703 versus $249; P < 0.001), and inpatient ($5610 versus $1640; P < 0.001) expenditures compared to respondents without an opi- oid prescription after adjusting for characteristic vari- ables (Table 2). The 10-year trends for healthcare expenditures for re- spondents with and without an opioid prescription are illustrated in Fig. 1. Results In the trend analysis, the average an- nual increase in expenditure was significantly greater among respondents with an opioid prescription com- pared to respondents without an opioid prescription for total (difference in trends: +$185; 95% CI: $37, $334) and prescription (difference in trends: +$78; 95% CI: $28, $128) expenditures (see Supplement Table B). There were no differences in the average annual trends for outpatient, emergency department, and inpatient ex- penditures between respondents with and without an opioid prescription. Patient and public involvement Since this study used household respondent data from MEPS, patients were not involved in the design nor the development of the research questions. Results of our Page 5 of 13 Bounthavong et al. Substance Abuse Treatment, Prevention, and Policy Page 5 of 13 (2021) 16:80 finding will be disseminated through the peer-review form in addition to presentations at scientific meetings. prescription adjusting for characteristic variables (Table 2). In the trend analysis, the average annual in- crease in healthcare resources used was significantly greater among respondents with an opioid prescription compared to respondents without an opioid prescription for number of prescriptions filled (difference in trends: + 0.27; 95% CI: 0.10, 0.45) and number of office-based visits (difference in trends: + 0.15; 95% CI: 0.05, 0.25; see Supplement Table B). There were no differences in the average annual trends for number of emergency depart- ment visits and number of inpatient night stays between respondents with and without an opioid prescription. Discussion The findings from our study highlights the differences in healthcare expenditures and resource utilization between respondents with and without an opioid prescription in a nationally representative population. Over a period of 10 years, the trends for total and prescription expendi- tures grew at a faster rate among respondents with opi- oids versus those without suggesting underlying issues that continue to exacerbate the opioid crisis. These in- creases in expenditures are likely driven by increases in the number of prescriptions filled and number of office- based visits, which are significantly higher among re- spondents with an opioid than respondents without an opioid prescription. However, it remains unclear whether the increases in healthcare expenditures are due to the use of opioid or other factors that predispose the patients to receiving opioids. The healthcare resources used for respondents with and without an opioid prescription are illustrated in Fig. 2. Respondents with an opioid prescription had sig- nificantly higher average annual number of prescriptions filled (29.5 versus 19.0; P < 0.001), number of office- based visits (12.4 versus 8.1; P < 0.001), number of emer- gency department visits (0.59 versus 0.25; P < 0.001), and number of inpatient night stays (1.61 versus 0.58; p < 0.001) compared to respondents without an opioid Previous studies have investigated factors associated with elevated costs among respondents with an opioid prescription. Kirson and colleagues [5] reported that opi- oid drug dependence, poisoning, drug-induced mental Bounthavong et al. Substance Abuse Treatment, Prevention, and Policy (2021) 16:80 Page 6 of 13 Bounthavong et al. Substance Abuse Treatment, Prevention, and Policy (2021) 16:80 Pag Bounthavong et al. Substance Abuse Treatment, Prevention, and Policy (2021) 16:80 Bounthavong et al. Discussion Substance Abuse Treatment, Prevention, and Policy (2021) 16:80 Bounthavong et al. Discussion Substance Abuse Treatment, Prevention, and Policy Page 6 of 13 Table 1 Demographic characteristics of matched adult (> = 18 Characteristics Total Respondents with an prescription Number of adults 61,406 30,703 Weighted sample 63,233,310 31,696,671 Age (years), mean (SD) 50.63 (18.03) 50.77 (17.42) Age category, n (%) 18 to 24 years 5,799,596 (9.2%) 2,453,290 (7.7%) 25 to 44 years 18,010,168 (28.5%) 9,182,160 (29.0%) 45 to 64 years 23,967,616 (28.5%) 12,572,602 (39.7%) 65 + years 15,455,930 (24.4%) 7,488,618 (23.6%) Gender, n (%) Male 25,984,623 (41.4%) 13,032,419 (41.1%) Female 37,248,687 (58.9%) 18,664,252 (58.9%) Race, n (%) White 52,228,967 (82.6%) 26,128,470 (82.4%) Black 7,473,006 (11.8%) 3,851,219 (12.2%) Native American / Alaskan Native 550,687 (0.9%) 337,100 (1.1%) Asian / Pacific Islander 1,737,580 (2.8%) 649,421 (2.1%) Multiple races reported 1,243,070 (2.0%) 730,460 (2.3%) Ethnicity, n (%) Hispanic 6,108,537 (9.7%) 3,116,758 (9.8%) Not Hispanic 57,124,773 (90.3%) 28,579,913 (90.2%) Marital status, n (%) Married 32,985,524 (52.2%) 16,515,381 (52.1%) Widowed 5,605,103 (8.9%) 2,744,706 (8.9%) Divorced 9,232,188 (14.6%) 5,082,374 (16.0%) Separated 1,661,458 (2.6%) 948,179 (3.0%) Never 13,749,037 (21.7%) 6,406,031 (20.2%) Education, n (%) No degree 9,345,503 (14.8%) 4,620,405 (14.6%) GED / High School 25,689,371 (40.6%) 12,824,112 (40.5%) Associates or Other degree 12,778,135 (20 2%) 6,926,184 (21.9%) Table 1 Demographic characteristics of matched adult (> = 18 years) responders from the MEPS, 2008 to 2017 Characteristics Total Respondents with an opioid prescription Respondents without an opioid prescription Standardized difference Number of adults 61,406 30,703 30,703 Weighted sample 63,233,310 31,696,671 31,536,639 Age (years), mean (SD) 50.63 (18.03) 50.77 (17.42) 50.44 (18.62) 0.018 Age category, n (%) 18 to 24 years 5,799,596 (9.2%) 2,453,290 (7.7%) 3,346,306 (10.6%) −0.100 25 to 44 years 18,010,168 (28.5%) 9,182,160 (29.0%) 8,828,008 (28.0%) 0.022 45 to 64 years 23,967,616 (28.5%) 12,572,602 (39.7%) 11,395,014 (36.1%) 0.073 65 + years 15,455,930 (24.4%) 7,488,618 (23.6%) 7,967,312 (25.3%) −0.038 Gender, n (%) Male 25,984,623 (41.4%) 13,032,419 (41.1%) 12,952,203 (41.1%) −0.001 Female 37,248,687 (58.9%) 18,664,252 (58.9%) 18,584,436 (58.9%) 0.001 Race, n (%) White 52,228,967 (82.6%) 26,128,470 (82.4%) 26,100,497 (82.8%) −0.009 Black 7,473,006 (11.8%) 3,851,219 (12.2%) 3,621,787 (11.5%) 0.021 Native American / Alaskan Native 550,687 (0.9%) 337,100 (1.1%) 213,587 (0.7%) 0.042 Asian / Pacific Islander 1,737,580 (2.8%) 649,421 (2.1%) 1,088,159 (3.5%) −0.086 Multiple races reported 1,243,070 (2.0%) 730,460 (2.3%) 512,609 (1.6%) 0.049 Ethnicity, n (%) Hispanic 6,108,537 (9.7%) 3,116,758 (9.8%) 2,991,778 (9.5%) 0.012 Not Hispanic 57,124,773 (90.3%) 28,579,913 (90.2%) 28,544,861 (90.5%) −0.012 Marital status, n (%) Married 32,985,524 (52.2%) 16,515,381 (52.1%) 16,470,143 (52.2%) −0.002 Widowed 5,605,103 (8.9%) 2,744,706 (8.9%) 2,860,397 (9.1%) −0.014 Divorced 9,232,188 (14.6%) 5,082,374 (16.0%) 4,149,814 (13.2%) 0.082 Separated 1,661,458 (2.6%) 948,179 (3.0%) 713,279 (2.3%) 0.046 Never 13,749,037 (21.7%) 6,406,031 (20.2%) 7,343,006 (23.3%) −0.075 Education, n (%) No degree 9,345,503 (14.8%) 4,620,405 (14.6%) 4,725,097 (15.0%) −0.011 GED / High School 25,689,371 (40.6%) 12,824,112 (40.5%) 12,865,260 (40.8%) −0.007 A i t Oth d 12 778 135 6 926 184 (21 9%) 5 851 951 (18 6%) 0 082 Respondents without an opioid prescription Standardiz difference 30,703 31,536,639 50.44 (18.62) 0.018 3,346,306 (10.6%) −0.100 8,828,008 (28.0%) 0.022 11,395,014 (36.1%) 0.073 7,967,312 (25.3%) −0.038 12,952,203 (41.1%) −0.001 18,584,436 (58.9%) 0.001 26,100,497 (82.8%) −0.009 3,621,787 (11.5%) 0.021 213,587 (0.7%) 0.042 1,088,159 (3.5%) −0.086 512,609 (1.6%) 0.049 2,991,778 (9.5%) 0.012 28,544,861 (90.5%) −0.012 16,470,143 (52.2%) −0.002 2,860,397 (9.1%) −0.014 4,149,814 (13.2%) 0.082 713,279 (2.3%) 0.046 7,343,006 (23.3%) −0.075 4,725,097 (15.0%) −0.011 12,865,260 (40.8%) −0.007 5,851,951 (18.6%) 0.082 Bounthavong et al. Discussion Substance Abuse Treatment, Prevention, and Policy (2021) 16:80 Bounthavong et al. Substance Abuse Treatment, Prevention, and Policy Page 8 of 13 Table 1 Demographic characteristics of matched adult (> = 18 years) responders from the MEPS, 2008 to 2017 (Continued) Characteristics Total Respondents with an opioid prescription Respondents without an opioid prescription Standardized difference Diabetes 9,237,334 (14.6%) 4,792,081 (14.6%) 4,445,253 (14.1%) 0.029 Arthritis 31,506,810 (49.8%) 15,846,310 (50.0%) 15,660,500 (49.7%) 0.007 the loss of life due to a fatal overdose, which are associ- ated with a large proportion of the societal costs of the opioid epidemic. According to Florence and colleagues, fatal overdoses and reduced quality of life costs made up 53.9 and 38.2% of the total societal costs for the opioid crisis, respectively [3]. However, these findings were fo- cused on the population with opioid use disorder. Our study looked at the entire population with an opioid pre- scription, which may result in a larger economic burden when other factors (e.g., fatal overdoses, loss productiv- ity, criminal justice costs) are incorporated into our esti- mates. Future investigations will need to incorporate these additional costs to determine the potential overall economic burden among all people with an opioid prescription. health disorders, and alcohol and non-opioid drug de- pendence and abuse were major cost drivers among opi- oid abusers. Leider and colleagues [24] reported that people who chronically use opioids had significantly more ambulatory, emergency department, and hospital visits; and higher total annual healthcare costs compared to people who do not use opioids. Moreover, they identi- fied nonadherence to opioid regimen as a major driver for healthcare costs. Nonadherent patients filled more prescriptions and had more unique opioid types, dis- pensing, and fills than adherent patients thereby driving up healthcare costs and resource utilization. We estimated the average annual economic burden of respondents with an opioid prescription to be approxi- mately $524 billion based on health care costs from MEPS data. Discussion Substance Abuse Treatment, Prevention, and Policy Page 7 of 13 Table 1 Demographic characteristics of matched adult (> = 18 years) res Characteristics Total Respondents with an opioid prescription Bachelor 9,752,704 (15.4%) 4,631,816 (14.6%) Master / Doctor 5,433,179 (8.6%) 2,564,625 (8.1%) Not Ascertainable 16,402 (0.03%) 9050 (0.03%) Don’t know 179,207 (0.3%) 104,307 (0.3%) Refused to answer 38,808 (0.06%) 16,172 (0.05%) Region, n (%) Northwest 9,961,981 (15.8%) 4,674,845 (14.7%) Midwest 14,577,600 (23.1%) 7,582,641 (23.9%) South 24,726,287 (39.1%) 12,468,361 (39.3%) West 13,967,442 (22.1%) 6,970,825 (22.0%) Poverty status, n (%) Poor / Negative 10,313,443 (16.3%) 5,104,930 (16.1%) Near Poor 3,413,335 (5.4%) 1,697,182 (5.4%) Low Income 9,119,538 (14.4%) 4,758,503 (15.0%) Middle Income 17,785,938 (28.1%) 8,869,788 (28.0%) High Income 22,601,056 (35.7%) 11,266,268 (35.5%) Insurance coverage, n (%) Any Private 41,247,710 (65.2%) 20,323,558 (64.1%) Public 17,036,658 (26.9%) 9,043,432 (28.5%) Uninsured 4,948,942 (7.8%) 2,329,680 (7.4%) Comorbidities, n (%) High blood pressure 29,518,235 (46.7%) 15,038,099 (47.4%) Coronary heart disease 5,893,689 (9.3%) 2,904,943 (9.2%) Angina 3,365,407 (5.3%) 1,708,626 (5.4%) Myocardial infarction 4,485,031 (7.1%) 2,225,777 (7.0%) Other heart disease 11,633,422 (18.4%) 5,750,760 (18.1%) Stroke 4,660,588 (7.4%) 2,372,209 (7.5%) High cholesterol 26,295,752 (41.6%) 13,374,570 (42.2%) Cancer 11,018,769 (17.4%) 5,539,973 (17.5%) Table 1 Demographic characteristics of matched adult (> = 18 years) responders from the MEPS, 2008 to 2017 (Continued) Characteristics Total Respondents with an opioid prescription Respondents without an opioid prescription Standardized difference Bachelor 9,752,704 (15.4%) 4,631,816 (14.6%) 5,120,887 (16.2%) −0.045 Master / Doctor 5,433,179 (8.6%) 2,564,625 (8.1%) 2,868,554 (9.1%) −0.036 Not Ascertainable 16,402 (0.03%) 9050 (0.03%) 7352 (0.02%) 0.003 Don’t know 179,207 (0.3%) 104,307 (0.3%) 74,901 (0.2%) 0.017 Refused to answer 38,808 (0.06%) 16,172 (0.05%) 22,636 (0.07%) −0.008 Region, n (%) Northwest 9,961,981 (15.8%) 4,674,845 (14.7%) 5,287,137 (16.8%) −0.055 Midwest 14,577,600 (23.1%) 7,582,641 (23.9%) 6,994,959 (22.2%) 0.041 South 24,726,287 (39.1%) 12,468,361 (39.3%) 12,257,926 (38.9%) 0.010 West 13,967,442 (22.1%) 6,970,825 (22.0%) 6,996,617 (22.2%) −0.005 Poverty status, n (%) Poor / Negative 10,313,443 (16.3%) 5,104,930 (16.1%) 5,208,513 (16.5%) −0.001 Near Poor 3,413,335 (5.4%) 1,697,182 (5.4%) 1,716,153 (5.4%) −0.004 Low Income 9,119,538 (14.4%) 4,758,503 (15.0%) 4,361,035 (13.%) 0.034 Middle Income 17,785,938 (28.1%) 8,869,788 (28.0%) 8,916,150 (28.3%) −0.006 High Income 22,601,056 (35.7%) 11,266,268 (35.5%) 11,334,788 (35.9%) −0.008 Insurance coverage, n (%) Any Private 41,247,710 (65.2%) 20,323,558 (64.1%) 20,924,152 (66.3%) −0.047 Public 17,036,658 (26.9%) 9,043,432 (28.5%) 7,993,226 (25.3%) 0.072 Uninsured 4,948,942 (7.8%) 2,329,680 (7.4%) 2,619,262 (8.3%) −0.036 Comorbidities, n (%) High blood pressure 29,518,235 (46.7%) 15,038,099 (47.4%) 14,480,136 (45.9%) 0.031 Coronary heart disease 5,893,689 (9.3%) 2,904,943 (9.2%) 2,988,746 (9.5%) 0.011 Angina 3,365,407 (5.3%) 1,708,626 (5.4%) 1,656,781 (5.3%) 0.006 Myocardial infarction 4,485,031 (7.1%) 2,225,777 (7.0%) 2,259,254 (7.2%) 0.006 Other heart disease 11,633,422 (18.4%) 5,750,760 (18.1%) 5,882,663 (18.7%) 0.013 Stroke 4,660,588 (7.4%) 2,372,209 (7.5%) 2,288,378 (7.3%) 0.009 High cholesterol 26,295,752 (41.6%) 13,374,570 (42.2%) 12,921,182 (41.0%) 0.025 Cancer 11,018,769 5,539,973 (17.5%) 5,478,796 (17.4%) 0.003 haracteristics of matched adult (> = 18 years) responders from the MEPS, 2008 to 2017 (Continued) 20,924,152 (66.3%) 14,480,136 (45.9%) Bounthavong et al. *Adjusted using a two-part model controlling for the following covariates: age, sex, race, ethnicity, marital status, year, region, poverty status, insurance status, education, high blood pressure, coronary heart disease, angina, myocardial infarction, orthostatic hypertension, stroke, high cholesterol, cancer, diabetes, d th iti *Adjusted using a two-part model controlling for the following covariates: age, sex, race, ethnicity, marital status, year, region, povert education, high blood pressure, coronary heart disease, angina, myocardial infarction, orthostatic hypertension, stroke, high cholester and arthritis Discussion This does not account for criminal justice costs, worker productive loss, reduced quality of life, and Our findings are different from those of previous lit- erature among people who use and abuse opioids, which Table 2 Adjusted healthcare expenditures and resource utilization between matched adult (> = 18 years) respondents with and without an opioid prescription from the MEPS, 2008 to 2017 Outcome Total (weighted n = 63,233,310)* Respondents with an opioid prescription (weighted n = 31,696,671)* Respondents without an opioid prescription (weighted n = 31,536,639)* P- value** Expenditures Total expenditures ($), mean (SD) $11,817 (9592) $16,542 (10,805) $7067 (4683) < 0.001 Prescription expenditures ($), mean (SD) $2681 (2481) $3067 (2767) $2293 (2084) < 0.001 Outpatient expenditures ($), mean (SD) $1228 (995) $1804 (1052) $650 (449) < 0.001 Emergency department expenditures ($), mean (SD) $476 (300) $703 (253) $249 (112) < 0.001 Inpatient expenditures ($), mean (SD) $3630 (3374) $5610 (3595) $1640 (1384) < 0.001 Resources Number of prescriptions filled, mean (SD) 24.3 (19.1) 29.5 (21.7) 19.0 (14.2) < 0.001 Number of office-based visits, mean (SD) 10.2 (5.1) 12.4 (5.4) 8.1 (3.7) < 0.001 Number of emergency department visits, mean (SD) 0.42 (0.29) 0.59 (0.29) 0.25 (0.15) < 0.001 Number of inpatient night stays, mean (SD) 1.10 (1.20) 1.61 (1.39) 0.58 (0.65) < 0.001 *Adjusted using a two-part model controlling for the following covariates: age, sex, race, ethnicity, marital status, year, region, poverty status, insurance status, education, high blood pressure, coronary heart disease, angina, myocardial infarction, orthostatic hypertension, stroke, high cholesterol, cancer, diabetes, and arthritis **Margins command althcare expenditures and resource utilization between matched adult (> = 18 years) respondents with and escription from the MEPS 2008 to 2017 Bounthavong et al. Substance Abuse Treatment, Prevention, and Policy (2021) 16:80 Bounthavong et al. Substance Abuse Treatment, Prevention, and Policy (2021) 16:80 Page 9 of 13 Bounthavong et al. Substance Abuse Treatment, Prevention, and Policy Page 9 of 13 varied widely Scarpati and colleagues [25] using med- 2008 USD) were reported by Leider and colleagues [24] Fig. 1 The 10-year trends for healthcare expenditures for respondents with and without an opioid prescription. Total expenditures for respondents with and without an opioid prescription (A). Prescription expenditures for respondents with and without an opioid prescription (B). Outpatient expenditures for respondents with and without an opioid prescription (C). Emergency department expenditures for respondents with and without an opioid prescription (D). Discussion Inpatient expenditures for respondents with and without an opioid prescription (E). Respondents with an opioid were defined as a household respondent who reporting having been prescribed an opioid prescription on the MEPS Prescription Medicines file Fig. 1 The 10-year trends for healthcare expenditures for respondents with and without an opioid prescription. Total expenditures for respondents with and without an opioid prescription (A). Prescription expenditures for respondents with and without an opioid prescription (B). Outpatient expenditures for respondents with and without an opioid prescription (C). Emergency department expenditures for respondents with and without an opioid prescription (D). Inpatient expenditures for respondents with and without an opioid prescription (E). Respondents with an opioid were defined as a household respondent who reporting having been prescribed an opioid prescription on the MEPS Prescription Medicines file Fig. 1 The 10-year trends for healthcare expenditures for respondents with and without an opioid prescription. Total expenditures for respondents with and without an opioid prescription (A). Prescription expenditures for respondents with and without an opioid prescription (B). Outpatient expenditures for respondents with and without an opioid prescription (C). Emergency department expenditures for respondents with and without an opioid prescription (D). Inpatient expenditures for respondents with and without an opioid prescription (E). Respondents with an opioid were defined as a household respondent who reporting having been prescribed an opioid prescription on the MEPS Prescription Medicines file varied widely. Scarpati and colleagues [25], using med- ical and prescription claims data from a commercially insured population, reported that people who abuse opi- oids had $7346 (2015 USD) excess costs compared to non-abusers, which is somewhat close to our average an- nual difference of $9475 between respondents with and without opioids. However, large differences ($18,074, 2008 USD) were reported by Leider and colleagues [24] between people who chronically use opioids and people who do not use opioids among geographically diverse populations from U.S. commercial, Medicare Advantage, and Medicaid health plans. Reasons for the differences may be due to the study cohort which was mostly older compared to our sample. Baser and colleagues [26] Bounthavong et al. Substance Abuse Treatment, Prevention, and Policy (2021) 16:80 Bounthavong et al. Substance Abuse Treatment, Prevention, and Policy (2021) 16:80 Page 10 of 13 Bounthavong et al. Substance Abuse Treatment, Prevention, and Policy Page 10 of 13 Fig. 2 The 10-year trends for healthcare resources used for respondents with and without an opioid prescription. Discussion Average number of office- based visits for respondents with and without an opioid prescription (A). Average number of emergency department visits for respondents with and without an opioid prescription (B). Average number of inpatient nights for respondents with and without an opioid prescription (C). Average number of prescription fills for respondents with and without an opioid prescription (D). Respondents with an opioid were defined as a household respondent who reporting having been prescribed an opioid prescription on the MEPS Prescription Medicines file Fig. 2 The 10-year trends for healthcare resources used for respondents with and without an opioid prescription. Average number of office- based visits for respondents with and without an opioid prescription (A). Average number of emergency department visits for respondents with and without an opioid prescription (B). Average number of inpatient nights for respondents with and without an opioid prescription (C). Average number of prescription fills for respondents with and without an opioid prescription (D). Respondents with an opioid were defined as a household respondent who reporting having been prescribed an opioid prescription on the MEPS Prescription Medicines file weights from MEPS in our propensity score matching to make population-level inferences. Currently, there are no gold standard recommendations for applying survey weights from complex survey designs to propensity score matching; however, there is consensus that using these weights are necessary for generalizability to the survey target population [27, 30]. Future research will need to validate these methods in applying survey weights to complex survey designs. examined the healthcare expenditures between veterans with and without an opioid prescription at the U.S. De- partment of Veterans Affairs (VA) and reported that 12- month follow-up healthcare costs were higher among veterans with an opioid prescription than veterans with- out an opioid prescription by $18,847 (2010 USD). Des- pite these differences, people who use opioids that develop dependence or misuse are associated with dra- matic increases in healthcare costs. Healthcare payers have an important public policy role in addressing the opioid epidemic. Given the high costs associated with opioid use, healthcare payers are finan- cially incentivized to reduce opioid prescribing, mitigate opioid overdoses, and provide care to those with opioid addiction and misuse. For example, removal of formu- lary restrictions for buprenorphine-naloxone, which is used for treatment of opioid use disorder, was associated with an increase of 17.9 prescriptions per plan per year among Medicare beneficiaries [34]. Discussion Substance Abuse Treatment, Prevention, and Policy (2021) 16:80 Page 11 of 13 Table 3 Adjusted healthcare expenditures and resource utilizations among adults (> = 18 years) with different unique opioid fills from the MEPS, 2008 to 2017 Outcome 1 unique opioid fill (weighted n = 15,943,576) 2 unique opioid fills (weighted n = 4,669,310) 3 or more unique opioid fills (weighted n = 11,083,785) P- value* P- value** Expenditures Total expenditures ($), mean (SD) $11,633 (7349) $18,131 (11,001) $23,471 (13,786) < 0.001 < 0.001 Prescription expenditures ($), mean (SD) $1425 (1280) $2468 (2006) $6355 (4623) < 0.001 < 0.001 Outpatient expenditures ($), mean (SD) $1576 (933) $2212 (1197) $1949 (1139) < 0.001 < 0.001 Emergency department expenditures ($), mean (SD) $641 (203) $842 (268) $731 (302) < 0.001 < 0.001 Inpatient expenditures ($), mean (SD) $4073 (2436) $6573 (3829) $7364 (4481) < 0.001 < 0.001 Resources Number of prescriptions filled, mean (SD) 15.7 (10.5) 22.9 (14.6) 52.0 (29.3) < 0.001 < 0.001 Number of office-based visits, mean (SD) 9.6 (4.2) 12.9 (5.2) 16.3 (5.8) < 0.001 < 0.001 Number of emergency department visits, mean (SD) 0.49 (0.23) 0.67 (0.29) 0.69 (0.33) < 0.001 < 0.001 Number of inpatient night stays, mean (SD) 1.0 (0.8) 1.6 (1.2) 2.4 (1.8) < 0.001 < 0.001 *Comparison between 2 unique narcotic fills and 1 unique narcotic fill **Comparison between 3 unique narcotic fills and 1 unique narcotic fill Table 3 Adjusted healthcare expenditures and resource utilizations among adults (> = 18 years) with different unique opioid fills althcare expenditures and resource utilizations among adults (> = 18 years) with different unique opioid fills to 2017 *Comparison between 2 unique narcotic fills and 1 unique narcotic fill **Comparison between 3 unique narcotic fills and 1 unique narcotic fill *Comparison between 2 unique narcotic fills and 1 unique narcotic fill **Comparison between 3 unique narcotic fills and 1 unique narcotic fill associated with increased healthcare expenditures and resource utilization. Diagnostic codes in MEPS only in- clude the first three digits of the International Classifica- tion of Diseases, Tenth Edition codes due to patient confidentiality protection, which limited our ability to identify respondents with opioid use disorder or depend- ency. Additionally, we were unable to determine whether responders were using opioids for the first time or chronically using opioids. Limitations There are several limitations with our study. Although we based our findings on a nationally representative sample of the non-institutionalized U.S. population, re- spondents are subject to recall bias, in particular when reporting on their healthcare expenditures and resource utilization. MEPS mitigates this problem by cross- referencing self-reports with the Medical Provider Com- ponent follow-back surveys collected from medical pro- viders and pharmacies; however, we cannot rule out the possibility of error. Furthermore, we do not have data on illicit opioid use or opioid misuse which have been Discussion Moreover, increased expendi- tures may be driven by increases in the number of pre- scriptions filled and number of office-based visits; however, we were unable to determine the reasons for these behaviors. Furthermore, propensity score method requires inclusion of relevant potential confounders to balance the groups; however, unobserved confounders or omitted variables can compromise the internal valid- ity of the method [36, 37]. Finally, the results from our findings were based on a nationally representative sam- ple of the civilian, non-institutionalized U.S. population, which may limit the generalizability to other countries. However, other nations have reported similar experi- ences with the opioid crises and may find these findings useful [38–41]. substance use-related inpatient admissions (2.0 admis- sions per plan per year) and emergency department visits (1.4 visits per plan per year) [34]. The U.S. Depart- ment of Veterans Affairs, the largest integrated health- care system in the United States implemented the Opioid Safety Initiative in 2013 to reduce opioid pre- scribing and reported a 56% reduction in opioid pre- scribing, an 83% reduction in opioid and benzodiazepine co-prescribing, and a 77% reduction in high-dose opioid prescribing from 2012 to 2019 [35]. These policy deci- sions by healthcare payers have a meaningful impact on the opioid crisis, which not only improve the quality of life for their patients, but they may potentially impact the increased expenditures associated with opioid use in their system. Discussion Moreover, removal of formulary restriction resulted in a reduction in Interest in using complex survey weights in propensity score matching methods has been an important area for methodologists [11, 27–30]. Conventional propensity score matching is commonly used to balance the charac- teristics of cohorts and to generate unbiased estimates [31–33]. With complex survey designs, the use of survey weights are necessary for generalizing the findings to the original survey population. DuGoff and colleagues devel- oped a method to apply the survey weights from com- plex surveys to generate propensity score [11]. We used this method because it allowed us to apply the survey Bounthavong et al. Substance Abuse Treatment, Prevention, and Policy (2021) 16:80 Page 11 of 13 Bounthavong et al. Acknowledgements N/A 8. von Elm E, Altman DG, Egger M, Pocock SJ, Gøtzsche PC, Vandenbroucke JP. The strengthening the reporting of observational studies in epidemiology (STROBE) statement: guidelines for reporting observational studies. Ann Intern Med. 2007;147(8):573–7. https://doi.org/10.7326/0003-4 819-147-8-200710160-00010. Availability of data and materials Data are available in a public, open access repository. Data used in our study are from publicly available source from the U.S. AHRQ. The MEPS data is located at the following URL: https://www.meps.ahrq.gov/mepsweb/data_ stats/download_data_files.jsp 12. Andersen R, Newman JF. Societal and individual determinants of medical care utilization in the United States. Milbank Q. 2005;83(4). https://doi.org/1 0.1111/j.1468-0009.2005.00428.x. 12. Andersen R, Newman JF. Societal and individual determinants of medical care utilization in the United States. Milbank Q. 2005;83(4). https://doi.org/1 0.1111/j.1468-0009.2005.00428.x. 13. Andersen RM. Revisiting the behavioral model and access to medical care: does it matter? J Health Soc Behav. 1995;36(1):1–10. https://doi.org/10.23 07/2137284. 13. Andersen RM. Revisiting the behavioral model and access to medical care: does it matter? J Health Soc Behav. 1995;36(1):1–10. https://doi.org/10.23 07/2137284. Funding Thi This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. 11. Dugoff EH, Schuler M, Stuart EA. Generalizing observational study results: applying propensity score methods to complex surveys. Health Serv Res. 2014;49(1):284–303. https://doi.org/10.1111/1475-6773.12090. Conclusion Our findings indicate that respondents with an opioid prescription have higher healthcare expenditures and re- source utilization than respondents without an opioid Page 12 of 13 Page 12 of 13 Bounthavong et al. Substance Abuse Treatment, Prevention, and Policy (2021) 16:80 (2021) 16:80 Accepted: 6 October 2021 prescription. Furthermore, those with greater numbers of unique opioids had higher average annual total, pre- scription, and inpatient expenditures compared to re- spondents with one or two unique opioid prescriptions. Findings from this study will inform stakeholders of the economic burden among people with an opioid prescrip- tion that could influence policy, guidelines, and strat- egies to address the opioid crisis. Abbreviations AHRQ: Agency for Healthcare Research and Quality; CIs: Confidence intervals; FPL : Federal poverty level; MEPS : Medical Expenditure Panel Survey; MPC: Medical Provider Component; PC : Pharmacy Component; STROBE: Strengthening the Reporting of Observational Studies in Epidemiology; U.S: United States AHRQ: Agency for Healthcare Research and Quality; CIs: Confidence intervals; FPL : Federal poverty level; MEPS : Medical Expenditure Panel Survey; MPC: Medical Provider Component; PC : Pharmacy Component; STROBE: Strengthening the Reporting of Observational Studies in Epidemiology; U.S: United States 2. National Institute on Drug Abuse. Overdose death rates: trends and statistics. National Institute on Drug Abuse. Published January 29, 2021. https://www.drugabuse.gov/drug-topics/trends-statistics/overdose-death-ra tes. Accessed 8 Feb 2021. 2. National Institute on Drug Abuse. Overdose death rates: trends and statistics. National Institute on Drug Abuse. Published January 29, 2021. https://www.drugabuse.gov/drug-topics/trends-statistics/overdose-death-ra tes. Accessed 8 Feb 2021. 3. Florence C, Luo F, Rice K. The economic burden of opioid use disorder and fatal opioid overdose in the United States, 2017. Drug Alcohol Depend. 2021;218:108350. https://doi.org/10.1016/j.drugalcdep.2020.108350. 3. Florence C, Luo F, Rice K. The economic burden of opioid use disorder and fatal opioid overdose in the United States, 2017. Drug Alcohol Depend. 2021;218:108350. https://doi.org/10.1016/j.drugalcdep.2020.108350. References S b 1. Substance Abuse and Mental Health Services Administration. Key substance use and mental health indicators in the United States: results from the 2018 National Survey on Drug Use and Health. (HHS Publication No. PEP19–5068, NSDUH Series H-54): Center for Behavioral Health Statistics and Quality, Substance Abuse and Mental Health Services Administration; 2019. https:// www.samhsa.gov/data/sites/default/files/cbhsqreports/NSDUHNationa lFindingsReport2018/NSDUHNationalFindingsReport2018.pdf. Accessed 8 Feb 2021. Authors’ contributions Conception or design of the work: MB, KS, ML, PS; Data acquisition: MB, AS; Data analysis: MB, AS, BS; Interpretation of data for the work: All authors; Drafting of the work: All authors; Revising work critically for important intellectual content: All authors; Final approval of the version to be published: All authors; Agreement to be accountable for all aspects of the work: All authors. 9. Agency for Healthcare Research and Quality. Medical expenditure panel survey topics: health care costs/expenditures. Published April 3, 2019. https://meps.ahrq.gov/mepsweb/data_stats/MEPS_topics.jsp?topicid=5Z-1. Accessed 2 Mar 2021. 10. Machlink SR, Dougherty DD. Overview of methodology for imputing missing expenditure data in the medical expenditure data survey: Agency for Healthcare Research and Quality; 2007. http://www.meps.ahrq.gov/ mepsweb/data_files/publications/mr19/mr19.pdf. Accessed 8 Feb 2021. Supplementary Information h l l 4. Chang H-Y, Kharrazi H, Bodycombe D, Weiner JP, Alexander GC. Healthcare costs and utilization associated with high-risk prescription opioid use: a retrospective cohort study. BMC Med. 2018;16(1):69. https://doi.org/10.1186/ s12916-018-1058-y. 4. Chang H-Y, Kharrazi H, Bodycombe D, Weiner JP, Alexander GC. Healthcare costs and utilization associated with high-risk prescription opioid use: a retrospective cohort study. BMC Med. 2018;16(1):69. https://doi.org/10.1186/ s12916-018-1058-y. The online version contains supplementary material available at https://doi. org/10.1186/s13011-021-00415-5. The online version contains supplementary material available at https://doi. org/10.1186/s13011-021-00415-5. The online version contains supplementary material available at https://doi. org/10.1186/s13011-021-00415-5. Additional file 1: Table A. Demographic characteristics of unmatched adult (> = 18 years) responders from the MEPS, 2008 to 2017. Table B. Average annual trends estimations for respondents with and without an opioid from MEPS, 2008 to 2017. Figure A. Visual inspection of the standardized mean difference plot after propensity score matching. Figure B. Trends for unique opioid groups (1 opioid, 2 opioids, 3 or more opioids). Additional file 1: Table A. Demographic characteristics of unmatched adult (> = 18 years) responders from the MEPS, 2008 to 2017. Table B. Average annual trends estimations for respondents with and without an opioid from MEPS, 2008 to 2017. Figure A. Visual inspection of the standardized mean difference plot after propensity score matching. Figure B. Trends for unique opioid groups (1 opioid, 2 opioids, 3 or more opioids). 5. Kirson NY, Scarpati LM, Enloe CJ, Dincer AP, Birnbaum HG, Mayne TJ. The economic burden of opioid abuse: updated findings. J Manag Care Spec Pharm. 2016;23(4):427–45. https://doi.org/10.18553/jmcp.2017.16265. 5. Kirson NY, Scarpati LM, Enloe CJ, Dincer AP, Birnbaum HG, Mayne TJ. The economic burden of opioid abuse: updated findings. J Manag Care Spec Pharm. 2016;23(4):427–45. https://doi.org/10.18553/jmcp.2017.16265. 6. Agency for Healthcare Research and Quality. Medical Expenditure Panel Survey (MEPS). Published August 2018. https://www.ahrq.gov/data/meps. html. Accessed 2 Mar 2021. 6. Agency for Healthcare Research and Quality. Medical Expenditure Panel Survey (MEPS). Published August 2018. https://www.ahrq.gov/data/meps. html. Accessed 2 Mar 2021. 7. Cohen JW, Cohen SB, Banthin JS. The medical expenditure panel survey: a national information resource to support healthcare cost research and inform policy and practice. Med Care. 2009;47(7 Suppl 1). https://doi.org/1 0.1097/MLR.0b013e3181a23e3a. 7. Cohen JW, Cohen SB, Banthin JS. The medical expenditure panel survey: a national information resource to support healthcare cost research and inform policy and practice. Med Care. 2009;47(7 Suppl 1). https://doi.org/1 0.1097/MLR.0b013e3181a23e3a. Ethics approval and consent to participate 14. Andersen RM. National health surveys and the behavioral model of health services use. Med Care. 2008;46(7):647–53. https://doi.org/10.1097/MLR. 0b013e31817a835d. 14. Andersen RM. National health surveys and the behavioral model of health services use. Med Care. 2008;46(7):647–53. https://doi.org/10.1097/MLR. 0b013e31817a835d. The Institutional Review Board of the Veterans Affairs San Diego Healthcare System approved this as a non-human subject research project (N210071). 15. Zhang JX, Rathouz PJ, Chin MH. Comorbidity and the concentration of healthcare expenditures in older patients with heart failure. J Am Geriatr Soc. 2003;51(4):476–82. https://doi.org/10.1046/j.1532-5415.2003.51155.x. 15. Zhang JX, Rathouz PJ, Chin MH. Comorbidity and the concentration of healthcare expenditures in older patients with heart failure. J Am Geriatr Soc. 2003;51(4):476–82. https://doi.org/10.1046/j.1532-5415.2003.51155.x. Consent for publication N/A 16. Dieleman JL, Cao J, Chapin A, Chen C, Li Z, Liu A, et al. US health care spending by payer and health condition, 1996-2016. JAMA. 2020;323(9):863– 84. https://doi.org/10.1001/jama.2020.0734. 16. Dieleman JL, Cao J, Chapin A, Chen C, Li Z, Liu A, et al. US health care spending by payer and health condition, 1996-2016. JAMA. 2020;323(9):863– 84. https://doi.org/10.1001/jama.2020.0734. Competing interests 17. Zhang Z, Kim HJ, Lonjon G, Zhu Y. Balance diagnostics after propensity score matching. Ann Transl Med. 2019;7(1). https://doi.org/10.21037/atm.201 8.12.10. 17. Zhang Z, Kim HJ, Lonjon G, Zhu Y. Balance diagnostics after propensity score matching. Ann Transl Med. 2019;7(1). https://doi.org/10.21037/atm.201 8.12.10. Author details 1 18. Manning WG. A two-part model of the demand for medical care : preliminary results from the Health Insurance Study. Health Econ Health Econ Proc World Congr Health Econ Leiden Neth Sept 1980. Published online 1981. 18. Manning WG. A two-part model of the demand for medical care : preliminary results from the Health Insurance Study. Health Econ Health Econ Proc World Congr Health Econ Leiden Neth Sept 1980. Published online 1981. Page 13 of 13 Page 13 of 13 Page 13 of 13 Bounthavong et al. Substance Abuse Treatment, Prevention, and Policy (2021) 16:80 (2021) 16:80 19. Belotti F, Deb P, Manning WG, Norton EC. twopm: Two-part models. Stata J. 2015;15(1):3–20. https://doi.org/10.1177/1536867X1501500102. 41. Organisation for Economic Co-operation and Development. Addressing problematic opioid use in OECD countries: OECD Publishing. https://doi. org/10.1787/a18286f0-en 41. Organisation for Economic Co-operation and Development. Addressing problematic opioid use in OECD countries: OECD Publishing. https://doi. org/10.1787/a18286f0-en 20. Glick HA, Doshi JA, Sonnad SS, Polsky D. Economic evaluation in clinical trials (handbooks in health economic evaluation). 2nd ed: Oxford University Press; 2015. https://doi.org/10.1093/med/9780199685028.001.0001. Publisher’s Note 21. Manning WG, Basu A, Mullahy J. Generalized modeling approaches to risk adjustment of skewed outcomes data. J Health Econ. 2005;24(3):465–88. https://doi.org/10.1016/j.jhealeco.2004.09.011. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 22. Ho D, Imai K, King G, Stuart EA. MatchIt: nonparametric preprocessing for parametric causal inference. J Stat Softw. 2011;42(1):1–28. https://doi.org/1 0.18637/jss.v042.i08. 23. R Core Team. R: a language and environment for statistical computing: R Foundation for Statistical Computing; 2020. https://www.R-project.org. Accessed 8 Feb 2021. 24. Leider HL, Dhaliwal J, Davis EJ, Kulakodlu M, Buikema AR. Healthcare costs and nonadherence among chronic opioid users. Am J Manag Care. 2011; 17(1):32–40. 25. Scarpati LM, Kirson NY, Jia ZB, Wen J, Howard J. Opioid abuse: a detailed examination of cost drivers over a 24-month follow-up period. J Manag Care Spec Pharm. 2017;23(11):1110–5. https://doi.org/10.18553/jmcp.2017.1 7019. 26. Baser O, Xie L, Mardekian J, Schaaf D, Wang L, Joshi AV. Prevalence of diagnosed opioid abuse and its economic burden in the veterans health administration. Pain Pract Off J World Inst Pain. 2014;14(5):437–45. https:// doi.org/10.1111/papr.12097. 27. Austin PC, Jembere N, Chiu M. Propensity score matching and complex surveys. Stat Methods Med Res. 2018;27(4):1240–57. https://doi.org/10.1177/ 0962280216658920. 28. Ridgeway G, Kovalchik SA, Griffin BA, Kabeto MU. Propensity score analysis with survey weighted data. J Causal Inference. 2015;3(2):237–49. https://doi. org/10.1515/jci-2014-0039. 29. Zanutto EL. A comparison of propensity score and linear regression analysis of complex survey data. J Data Sci. 2006;4(1):67–91. https://doi.org/10.6339/ JDS.2006.04(1).233. 30. Dong N, Stuart EA, Lenis D, Quynh NT. Using propensity score analysis of survey data to estimate population average treatment effects: a case study comparing different methods. Eval Rev. 2020;44(1):84–108. https://doi.org/1 0.1177/0193841X20938497. 30. Dong N, Stuart EA, Lenis D, Quynh NT. Using propensity score analysis of survey data to estimate population average treatment effects: a case study comparing different methods. Eval Rev. 2020;44(1):84–108. https://doi.org/1 0.1177/0193841X20938497. 31. Austin PC. An introduction to propensity score methods for reducing the effects of confounding in observational studies. Multivar Behav Res. 2011; 46(3):399–424. https://doi.org/10.1080/00273171.2011.568786. 32. D’Agostino RB. Propensity score methods for bias reduction in the comparison of a treatment to a non-randomized control group. Stat Med. 1998;17(19):2265–81. https://doi.org/10.1002/(SICI)1097-0258(19981015)17:1 9<2265::AID-SIM918>3.0.CO;2-B. 33. Rosenbaum PR, Rubin DB. The central role of the propensity score in observational studies for causal effects. Biometrika. 1983;70(1):41–55. https:// doi.org/10.1093/biomet/70.1.41. 33. Rosenbaum PR, Rubin DB. The central role of the propensity score in observational studies for causal effects. Biometrika. 1983;70(1):41–55. https:// doi.org/10.1093/biomet/70.1.41. 34. Publisher’s Note Mark TL, Parish WJ, Zarkin GA. Association of formulary prior authorization policies with buprenorphine-naloxone prescriptions and hospital and emergency department use among Medicare beneficiaries. JAMA Netw Open. 2020;3(4):e203132. https://doi.org/10.1001/jamanetworkopen.202 0.3132. 35. Sandbrink F, Oliva EM, McMullen TL, et al. Opioid prescribing and opioid risk mitigation strategies in the veterans health administration. J Gen Intern Med. 2020;35(3):927–34. https://doi.org/10.1007/s11606-020-06258-3. 36. Okoli GN, Sanders RD, Myles P. Demystifying propensity scores. BJA Br J Anaesth. 2014;112(1):13–5. https://doi.org/10.1093/bja/aet290. 37. Winkelmayer WC, Kurth T. Propensity scores: help or hype? Nephrol Dial Transplant. 2004;19(7):1671–3. https://doi.org/10.1093/ndt/gfh104. 38. Jakovljevic M, Lazarevic M, Jurisevic M, Jovanovic M. When cure becomes an illness—abuse of addictive prescription medicines. Front Pharmacol. 2015;6:193. https://doi.org/10.3389/fphar.2015.00193. 39. Amsterdam J van, Brink W van den, Pierce M. Explaining the differences in opioid overdose deaths between Scotland and England/Wales: implications for European opioid policies. Eur Addict Res. Published online May 7, 2021: 1–14. https://doi.org/10.1159/000516165. 39. Amsterdam J van, Brink W van den, Pierce M. Explaining the differences in opioid overdose deaths between Scotland and England/Wales: implications for European opioid policies. Eur Addict Res. Published online May 7, 2021: 1–14. https://doi.org/10.1159/000516165. 40. Organisation for Economic Co-operation and Development. Addressing problematic opioid use in OECD countries: OECD Publishing. https://doi. org/10.1787/a18286f0-en. Accessed 8 Feb 2021. 40. Organisation for Economic Co-operation and Development. Addressing problematic opioid use in OECD countries: OECD Publishing. https://doi. org/10.1787/a18286f0-en. Accessed 8 Feb 2021.
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Sistem Manajemen Potensi Anak Sejak Dini (SIMPONI) Berdasarkan Teori Kecerdasan Majemuk Menggunakan Metode Simple Additive Weighting (SAW)
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Infotek : Jurnal Informatika dan Teknologi Vol. 3 No. 2, Juli 2020, hal. 194-203 Infotek : Jurnal Informatika dan Teknologi Vol. 3 No. 2, Juli 2020, hal. 194-203 Sistem Manajemen Potensi Anak Sejak Dini (SIMPONI) Berdasarkan Teori Kecerdasan Majemuk Menggunakan Metode Simple Additive Weighting (SAW) Mustafa1 *, Mustafid2, R. Rizal Isnanto3 1Program Studi Teknik Informatika, Universitas Islam Sultan Agung Semarang 2,3Program Studi Magister Sistem Informasi, Universitas Diponegoro Semarang *cakmustafa@unissula.ac.id Abstract Learning program for elementary school age children will be more easily presented when using learning strategies according to the child’s learning style or multiple intelligences profile. Identification of multiple intelligences profile child can be done through a process of observation of parents and teachers on children's daily activities. Children multiple intelligences profile is dynamic so it’s necessary to identify multiple intelligence profiles on a regular basis for at least one year. This research aims to implement Simple Additive Weight (SAW) methode in Management Information Systems to Identification of Potential Early Childhood - SIMPONI based on the Theory of Multiple Intelligences. Simple Additive weighting methode (SAW) is used for calculation of the ranking of Multiple Intelligences profile child. The Results of this research is management information systems to identificaton Multiple Intelligences Profile with the data processing using Simple Additive weighting methode (SAW). The product advantages of this research is parents can identify multiple intelligence profile of children more easily than previous models that use a method that requires face-to-face interviews. Keywords: Identification, Learning Style, Management Information Systems, Multiple Intelligences, Potential, SAW Teori Kecerdasan Majemuk mengambarkan kecerdasan dengan perspektif yang lebih luas sehingga kecerdasan tidak hanya dipandang dari konsep abstrak namun lebih pada fungsional yang dapat terlihat dalam perilaku sehari-hari dengan beragam cara yang lebih komprehensif Abstrak Program pembelajaran untuk anak Sekolah Dasar (SD) lebih mudah disampaikan apabila menggunakan strategi pembelajaran yang sesuai dengan gaya belajar atau profil kecerdasan majemuk anak. Identifikasi profil kecerdasan majemuk anak dapat dilakukan melalui proses observasi orang tua dan guru sekolah terhadap kegiatan sehari-hari anak. Profil kecerdasan majemuk anak bersifat dinamis sehingga perlu dilakukan identifikasi profil kecerdasan majemuk secara berkala minimal satu tahun sekali. Penelitian ini bertujuan untuk mengimplementasikan metode Simple Additive Weight (SAW) pada Sistem Manajemen Potensi Anak Sejak Dini (SIMPONI) berdasarkan Teori Kecerdasan Majemuk. Metode Simple Additive Weighting (SAW) digunakan untuk perhitungan penentuan rangking profil Kecerdasan Majemuk anak. Hasil penelitian ini adalah sistem informasi manajemen identifikasi Profil Kecerdasan Majemuk dengan pengolahan data menggunakan metode Simple Additive Weighting (SAW). Keunggulan produk yang dihasilkan penelitian ini adalah orang tua dapat melakukan identifikasi profil kecerdasan majemuk anak secara lebih mudah dibandingkan model sebelumnya yang menggunakan metode wawancara yang mengharuskan tatap muka. Kata kunci: Gaya Belajar, Identifikasi, Kecerdasan Majemuk, Metode SAW, Potensi, Sistem Informasi Manajemen 1. Pendahuluan Setiap orang tua pasti ingin memberikan program pendidikan yang terbaik untuk anak tercinta. Seharusnya program pendidikan yang tidak sekedar mengikuti keinginan orang tua namun sesuai dengan bakat atau potensi anak. e-ISSN 2614-8773 194 Infotek : Jurnal Informatika dan Teknologi Vol. 3 No. 2, Juli 2020, hal. 194-203 Infotek : Jurnal Informatika dan Teknologi Vol. 3 No. 2, Juli 2020, hal. 194-203 mencakup delapan kecerdasan, yakni linguistik, logis-matematis, spasial, kinestetis, musikal, interpersonal, intrapersonal, naturalis [1]. dan guru tentang strategi pembelajaran yang paling sesuai digunakan karena profil kecerdasan majemuk sekaligus sebagai gambaran gaya belajar anak/siswa. Profil kecerdasan majemuk selain memberikan gambaran potensi kecerdasan anak juga dapat menginformasikan gaya belajar anak. Dengan gaya belajar yang sudah teridentifikasi tersebut maka orang tua ataupun guru dapat menyusun strategi pembelajaran terbaik untuk anak. Dengan strategi pembelajaran yang tepat maka anak akan dengan mudah menyerap materi pembelajaran yang disampaikan [1]. 1. Peran Teori Kecedasan Majemuk Dalam Pendidikan Dalam penelitian identifikasi profil kecerdasan majemuk untuk siswa sekolah dasar, selain metode observasi dapat pula dilakukan melalui identifikasi portofoliso karya siswa dan wawancara dengan pihak yang sering berhubungan dengan siswa. Melalui proses analisa hasil identifikasi dari tiga metode tersebut maka profil kecerdasan majemuk siswa dapat teridentifikasi dengan baik [7]. Identifikasi profil kecerdasan majemuk melalui kegiatan obervasi dapat dilakukan dengan mengembangkan metode summated rating dari Likert [8]. Pemahaman terhadap kecerdasan mulai mengalami perubahan tidak hanya dilihat dari IQ sejak munculnya teori Multiple Inteligences (kecerdasan majemuk) dari seorang ahli Psikologi Kognitif Harvard University, Dr Howard Garnerd pada tahun 1983. Individu yang selama ini sering dikatakan kurang atau bahkan tidak cerdas hanya karena nilai pelajaran Matematika, Bahasa, Sains di bawah rata-rata meskipun mereka memiliki nilai rata-rata atau bahkan diatas rata-rata untuk mata pelajaran olahraga ataupun seni [1]. 2.1. Penelitian Sebelumnya Teori kecerdasan majemuk dengan metode perhitungan AHP dapat digunakan dalam sistem pendukung keputusan untuk memberikan rekomendasi pilihan peminatan mahasiswa sebagai bahan pertimbangan. Dalam implementasinya didapatkan sebanyak 49% pemilihan peminatan sesuai antara rekomendasi berdasarkan sistem yang peminatan yang dipilih oleh mahasiswa [2]. Oleh Karena itu dalam penelitian ini akan dirancang bangun Sistem Manajemen Potensi Anak Sejak Dini menggunakan metode SAW berdasarkan teori Kecerdasan Majemuk melalui proses observasi. Dalam pembelajaran, guru perlu memahami profil kecerdasan majemuk siswa sehingga dapat menerapkan strategi yang paling sesuai [3]. Profil kecerdasan majemuk siswa dapat menjadi pintu gerbang guru dalam penyampaian materi sehingga materi yang disampaikan dapat lebih mudah dipahami oleh siswa [4]. Identifikasi profil kecerdasan majemuk dapat menggunakan kuesioner menggunakan skala likert [5]. Tujuan penelitian ini adalah mengimplementasikan metode rangking Simple Additive Weight (SAW) untuk Sistem Manajemen Potensi Anak Sejak Dini (SIMPONI) berdasarkan Teori Kecerdasan Majemuk. penelitian ini Dengan adanya penelitian ini diharapkan dapat memberikan manfaat sebagai berikut; Dengan adanya penelitian ini diharapkan dapat memberikan manfaat sebagai berikut; 1. Mengoptimalkan fungsi observasi kegiatan harian untuk melakukan identifikasi profil kecerdasan majemuk anak/siswa. Identifikasi profil kecerdasan majemuk dapat dilakukan melalui sistem komputer berbasis web menggunkaan metode certainty factor. Profil kecerdasan majemuk yang dapat di identifiaksi tersebut dapat menjadi referensi potensi dan bakat siswa [6]. 2. Sistem Manajemen Potensi Anak Sejak Dini (SIMPONI) membantu orang tua dan guru untuk identifikasi potensi kecerdasan anak berdasarkan teori kecerdasan majemuk lebih cepat dan tepat. Hasil identifikasi profil kecerdasan anak melalui SIMPONI memberikan informasi kepada orang tua 195 e-ISSN 2614-8773 Infotek : Jurnal Informatika dan Teknologi Vol. 3 No. 2, Juli 2020, hal. 194-203 Infotek : Jurnal Informatika dan Teknologi Vol. 3 No. 2, Juli 2020, hal. 194-203 komprehensif mengenai kecerdasan manjemuk siswa selain melalui Observasi [1]. Dalam penelitian identifikasi profil kecerdasan majemuk untuk siswa sekolah dasar, selain metode observasi dapat pula dilakukan melalui identifikasi portofoliso karya siswa dan wawancara dengan pihak yang sering berhubungan dengan siswa. Melalui proses analisa hasil identifikasi dari tiga metode tersebut maka profil kecerdasan majemuk siswa dapat teridentifikasi dengan baik [7]. Identifikasi profil kecerdasan majemuk melalui kegiatan obervasi dapat dilakukan dengan mengembangkan metode summated rating dari Likert [8]. komprehensif mengenai kecerdasan manjemuk siswa selain melalui Observasi [1]. 3.1. Tahap Perencanaan Penelitian Pada tahapan ini akan dilakukan perencanaan untuk melakukan penelitian meliputi tahapan untuk studi pustaka, desain kuesioner, pengembangan SIMPONI. Dimana 𝑟 adalah rating kinerja ternormalisasi dari alternatif pada atribut ; i=1,2,…,m dan j=1,2,…,n. 2. Simple Additive Weight (SAW) Melalui konsep kecerdasan majemuk (Multiple Intelligences), kecerdasan tidak sekedar dilihat dari kemampuan logika-matematika dan bahasa tetapi juga harus dilihat dari aspek lain diantarnya kinestetis, musical, visual-spasial, interpersonal, intrapersonal, dan naturalis. Simple Additive Weighting (SAW) merupakan metode penentuan rangking melalui perhitungan metode penjumlahan terbobot dari beberapa alternative pilihan. Perhitungan menggunakan metode SAW dapat menghasilkan nilai terbesar yang akan dipilih sebagai alternatif yang terbaik. Metode ini banyak digunakan untuk berbagai sistem pendukung keputusan karena metode ini konsepnya simpel, mudah digunakan dan dipahami [9]. Simple Additive Weighting (SAW) merupakan metode penentuan rangking melalui perhitungan metode penjumlahan terbobot dari beberapa alternative pilihan. Perhitungan menggunakan metode SAW dapat menghasilkan nilai terbesar yang akan dipilih sebagai alternatif yang terbaik. Profil kecerdasan majemuk siswa dalam Pendidikan menjadi pintu gerbang bagi guru dalam Menyusun Rencan Pelaksanaan Pembelajaran (RPP). Penggunaan strategi pembelajaran yang disesuaikan dengan profil kecerdasan majemuk siswa akan membuat materi lebih mudah disampaikan kepada siswa. Profil kecerdasan majemuk siswa dapat diidentifikasi melalui proses observasi kegaitan harian siswa baik dirumah maupun disekolah [4]. Hal ini sesuai dengan pemahaman bahwa tidak ada satupun “test tercanggih” di masyarakat yang dapat menghasilkan survey yang Metode ini banyak digunakan untuk berbagai sistem pendukung keputusan karena metode ini konsepnya simpel, mudah digunakan dan dipahami [9]. Tujuan utama dalam perhitunggan menggunakan metode SAW pada dasarnya untuk mengevaluiasi m alternatif terhadap sekumpulan atribut atau kriteria , dimana setiap atribut saling tidak bergantung satu dengan yang lainnya. Matriks keputusan setiap alternatif 196 e-ISSN 2614-8773 Infotek : Jurnal Informatika dan Teknologi Vol. 3 No. 2, Juli 2020, hal. 194-203 Infotek : Jurnal Informatika dan Teknologi Vol. 3 No. 2, Juli 2020, hal. 194-203 Infotek : Jurnal Informatika dan Teknologi Vol. 3 No. 2, Juli 2020, hal. 194-203 Infotek : Jurnal Informatika dan Teknologi Vol. 3 No. 2, Juli 2020, hal. 194-203 terhadap setiap atribut X diberikan sebagai berikut: [10] mendapatkan nilai Vi dapat dilihat pada rumus berikut: 𝑉 ∑ 5 Keterangan : [ ] (1) [ ] (1) 5 (1) Keterangan : Keterangan : Vi = Nilai untuk rangking setiap alternatif wj = Nilai bobot dari setiap kriteria rij = Nilai rating kinerja ternormalisasi Dimana Xij merupakan rating kinerja alternatif ke-i terhadap atribut ke-j. Nilai bobot yang menunjukkan tingkat kepentingan relatif setiap atribut, diberikan sebagai, W: 3. Metode Penelitian { } (2) (2) Tahapan penelitian pengembangan Sistem Manajemen Potensi Anak Sejak Dini (SIMPONI) Berdasarkan Teori Kecerdasan Majemuk Menggunakan Metode Simple Additive Weighting (SAW) meliputi tahapan-tahapan sebagai berikut Dalam perhitungan untuk menghasilkan rangking menggunakan metode SAW dikenal adanya 2 atribut yaitu kriteria keuntungan (benefit) dan kriteria biaya (Cost) serta membutuhkan proses normalisasi matriks keputusan (X) ke suatu skala yang dapat diperbandingkan dengan semua rating alternatif yang ada. Formula untuk melakukan proses normalisasi berdasarkan kriteria keuntungan ataupun kriteria biaya dapat dilihat pada rumus berikut [11]: a. Proses Aplikasi SIMPONI a. Proses Aplikasi SIMPONI Proses aplikasi Sistem Manajemen Potensi Anak Sejak Dini (SIMPONI) dari input-proses dan keluaran dapat dilihat pada Gambar 1.; Gambar 1 Proses aplikasi SIMPONI dari Input- Proses-Keluaran 1) Diagram Use Case Tahap implementasi adalah tahapan untuk pembuatan aplikasi sesuai dengan perancangan sistem yang telah disusun. Pada tahapan ini dilakukan juga ujicoba sistem secara bertahap sehingga jika ada kesalahan dapat diperbaiki sejak awal. Diagram use case secara keseluruhan dapat dilihat pada Gambar 2. Diagram use case secara keseluruhan dapat dilihat pada Gambar 2. Gambar 2 Diagaram Use Case SIMPONI b. Perancangan Sistem b. Perancangan Sistem 3.2. Tahap Penyusunan kuesioner dan Pembobotan observer Dimana 𝑟 adalah rating kinerja ternormalisasi dari alternatif pada atribut ; i=1,2,…,m dan j=1,2,…,n. Dalam proses penyusunan kuesioner ini akan dilakukan diskusi mendalam dengan psikolog. Pembobotan pada tiap kategori observer yang melakukan input kuesioner terdiri dari; Selanjutnya untuk menentukan pilihan alternatif terbaik dapat dilihat dari perhitungan nilai preferensi. Nilai prefesensi untuk setiap alternatif (𝑉 ) untuk menentukan alternatif mana yang akan menjadi yang terbaik. Dalam hal ini nilai 𝑉 yang lebih besar mengindikasikan bahwa alternatif lebih terpilih. Rumus untuk Tabel 1. Pembobotan dalam perhitungan metode SAW sesuai dengan hubungan observer dengan siswa yang diobservasi Bobot Keterangan Nilai Bobot Sangat Tinggi - Keluarga inti meliputi orang tua, kakek/nenek, kakak/adik yang tinggal satu rumah 5 e-ISSN 2614-8773 197 Infotek : Jurnal Informatika dan Teknologi Vol. 3 No. 2, Juli 2020, hal. 194-203 Infotek : Jurnal Informatika dan Teknologi Vol. 3 No. 2, Juli 2020, hal. 194-203 Tinggi - Keluarga support yang konsisten memiliki interaksi dengan anak namun tidak tinggal serumah. - Guru di sekolah yang memiliki interaksi konsisten setiap hari dengan anak 3 Cukup - Guru di sekolah yang memiliki interaksi setidaknya sehari dalam 1 minggu - Guru kursus/les yang memiliki interaksi setidaknya sehari dalam 1 minggu 2 Pada tahap ujicoba validasi peneliti melakukan pengambilan data input kuesioner observasi kegiatan anak secara manual kepada orang tua siswa, guru harian dan guru pendamping di sekolah. 3.3. Tahap Perancangan sistem Pada tahap ini melakukan penyusunan Use Case Diagram dan diagram aktivitas untuk input dan keluaran. Selanjutnya akan dilakukan pembuatan kode program sesuai dengan perancangan sistem. Gambar 1 Proses aplikasi SIMPONI dari Input- Proses-Keluaran 3.5. Tahap Pengujian Sistem Tahap pengujian sistem meliputi proses kegiatan verifikasi dan validasi. Untuk kegiatan verifikasi akan dilakukan pengujian menggunakan data dummy yang selanjutnya dilakukan perbandingan dengan requirement sistem yang telah ditentukan. Gambar 2 Diagaram Use Case SIMPONI 198 e-ISSN 2614-8773 Infotek : Jurnal Informatika dan Teknologi Vol. 3 No. 2, Juli 2020, hal. 194-203 kualitas interaksi observer dengan kegiatan harian anak. 2) Diagram Aktivitas akun orang tua Proses aktivitas yang dapat dilakukan oleh orang tua dapat dilihat pada gambar 3.3 berikut ini: Proses pengolahan data dari masukan kuesioner hasil observasi menggunakan metode SAW untuk mendapatkan hasil rangking potensi kecerdasan anak. Selanjutnya hasil rangking potensi kecerdasan tersebut ditampilkan pada luaran SIMPONI dalam bentuk grafik dan khusus 4 (empat) nilai tertinggi hasil identifikasi potensi kecerdasan tersebut akan dideskripsikan sebagai profil kecerdasan majemuk. Gambar 3 : Diagram aktivitas akun orang tua Hasil identifikasi potensi kecerdasan anak berdasarkan teori kecerdasan majemuk sekaligus dapat memberikan informasi gaya belajar paling tepat yang dimiliki oleh anak sehingga berdasarkan hasil identifikasi ini para orang tua dan guru dapat menyusun strategi pembelajarn atau program pendidikan yang paling tepat kepada anak/siswa. Gambar 3 : Diagram aktivitas akun orang tua c. Pembahasan c. Pembahasan Hasil penelitian ini adalah Sistem Manajemen Potensi Anak Sejak Dini (SIMPONI) dengan menerapkan metode SAW untuk menentukan rangking yang paling dominan dari 8 (delapan) jenis potensi kecerdasan anak berdasarkan teori kecerdasan majemuk. Masukan untuk aplikasi SIMPONI didapatkan melalui pengisian kuesioner observasi kegiatan harian anak. Masukan data observasi kegiatan harian anak disediakan dalam aplikasi SIMPONI dalam bentuk list pernyataan yang selanjutnya observer melakukan checklist berdasarkan kesesuaian antara pernyataan dengan kegiatan harian anak. Untuk mengoptimalkan hasil identifikasi potensi kecerdasan anak, SIMPONI menyediakan formulir pengisian kuesioner dari observer selain dari orang tua dengan pembobotan sesuai Berdasarkan hasil diskusi dengan konsultan pendidikan kecerdasan majemuk, didapatkan bahwa potensi kecerdasan atau gaya belajar anak tidak bisa dilakukan dengan satu kali proses karena pada dasarnya potensi kecerdasan atau gaya belajar anak sangat dipengaruhi oleh lingkungan sekitar. Proses identifikasi bertahap ini dapat dilakukan dengan rentang waktu paling tidak 3 bulan sehingga hasilnya akan lebih valid. Hasil identifikasi tahap sebelumnya juga menjadi referensi perhitungan menentukan potensi kecerdasan anak. Oleh karena itu pengembangan sistem informasi dalam penelitian ini akan memberi manfaat yang besar untuk kecepatan dan ketepatan dalam e-ISSN 2614-8773 199 Infotek : Jurnal Informatika dan Teknologi Vol. 3 No. 2, Juli 2020, hal. 194-203 Gambar 5. Formulir pendaftaran untuk menggunakan aplikasi SIMPONI Gambar 5. Formulir pendaftaran untuk menggunakan aplikasi SIMPONI melakukan perhitungan hasil observasi kegiatan keseharian anak. 1) Sistem Manajemen Potensi Anak Sejak Dini (SIMPONI) Penelitian ini menghasilkan aplikasi Sistem Manajemen Potensi Anak Sejak Dini (SIMPONI) yang memiliki fungsi utama yaitu untuk menentukan rangking potensi anak melalui proses observasi berdasarkan teori Kecerdasan majemuk menggunakan metode perhitungan Simple Additive Weighting (SAW) atau disebut sebagai profil kecerdasan majemuk. Penelitian ini menghasilkan aplikasi Sistem Manajemen Potensi Anak Sejak Dini (SIMPONI) yang memiliki fungsi utama yaitu untuk menentukan rangking potensi anak melalui proses observasi berdasarkan teori Kecerdasan majemuk menggunakan metode perhitungan Simple Additive Weighting (SAW) atau disebut sebagai profil kecerdasan majemuk. Gambar 5. Formulir pendaftaran untuk menggunakan aplikasi SIMPONI Setelah melakukan pendaftaran maka sistem akan mengirimkan email pemberitahuan untuk melakukan aktivasi. Setelah melakukan aktivasi maka user dapat menggunakan aplikasi SIMPONI. Setelah melakukan pendaftaran maka sistem akan mengirimkan email pemberitahuan untuk melakukan aktivasi. Setelah melakukan aktivasi maka user dapat menggunakan aplikasi SIMPONI. Pengembangan aplikasi SIMPONI dapat optimalkan oleh orang tua maupun sekolah. Tampilan utama aplikasi SIMPONI ditunjukan pada Gambar 4.1. Proses pendaftaran untuk mendapatkan akun dilakukan melalui admin sistem. d. Pengujian Sistem Pembahasan hasil penelitian ini akan dibagi menjadi dua yaitu pengujian sistem SIMPONI mulai dari proses input data dalam sistem hingga tampilan keluaran dan pengujian validitas analisa data perhitungan menggunakan metode Simple Addtive Weighting (SAW). Dalam pengujian ini peneliti menggunakan metode back box yaitu pengujian yang berfokus pada fungsional aplikasi SIMPONI berdasarkan perancangan desain yang sudah disusun. Selain itu, dalam pengujian akan dilakukan pula validitas pengeolahan data dengan membandingkan hasil keluaran pengolahan melalui sistem dengan hasil pengolahan data secara manual. Gambar 4 Tampilan utama aplikasi SIMPONI Gambar 4 Tampilan utama aplikasi SIMPONI Gambar 4 Tampilan utama aplikasi SIMPONI Berikut penjelasan penggunaan aplikasi SIMPONI oleh orang tua: a) Pendaftaran aplikasi SIMPONI Pendaftaran akun pengguna orang tua oleh admin dapat dilakukan melalui menu kelola user dengan tampilan aplikasi seperti pada Gambar 5 Selain pengelolaan user, admin dapat melakukan update kuesioner pada menu master data dengan pilihan data survey. Berdasarkan metode pengujian sistem blackbox test didapatkan hasil pengujian untuk akun orang tua sebagai berikut: 200 e-ISSN 2614-8773 Infotek : Jurnal Informatika dan Teknologi Vol. 3 No. 2, Juli 2020, hal. 194-203 Tabel 2. Hasil pengujian SIMPONI menggunakan metode Blackbox Test No Butir Pengujian Hasil yang diharapkan Hasil 1 Member area dan pendaftaran Tampil kepada halaman formulir pendaftaran Sesuai 2 Pengelolaan data pengguna Akun orang tua berhasil dibuat Sesuai 3 Pengelolaan kuesioner Tampil halaman pengelolaan data kuesioner Sesuai 4 Login pengguna User dapat login dan halaman dashboard aplikasi SIMPONI Sesuai 5 Update profil orang tua Halaman profil orang tua terupdate Sesuai 6 Update profil anak Data anak bisa update Sesuai 7 Permintaan dukungan observasi Data dukungan untuk observasi terupdate dan dapat melakukan input data kuesioner observasi. Sesuai 8 Keluaran observasi Tampilan halaman pilihan untuk melihat hasil dukungan observasi untuk setiap anak yang sudah di input oleh orang tua Sesuai Infotek : Jurnal Informatika dan Teknologi Vol. 3 No. 2, Juli 2020, hal. 194-203 kepada pihak lain. Data yang digunakan berdasarkan hasil observasi dari orang tua untuk Ahmad Naufal Aziz. Berdasarkan input hasil observasi tersebut, output dari SIMPONI ditunjukan pada Gambar 6. abel 2. Hasil pengujian SIMPONI menggunakan Tabel 2. Hasil pengujian SIMPONI menggunakan metode Blackbox Test Gambar 6 Keluaran SIMPONI dari observasi orang tua Gambar 6 Keluaran SIMPONI dari observasi orang tua Gambar 6 Keluaran SIMPONI dari observasi orang tua 3) Pengujian Validitas Pengolahan data dengan dukungan observasi bobot hubungan tinggi dan cukup 3) Pengujian Validitas Pengolahan data dengan dukungan observasi bobot hubungan tinggi dan cukup Selanjutnya pada kondisi kedua yaitu orang tua melakukan permintaan dukungan observasi kepada pihak lain.sehingga SIMPONI menampilkan output yang ditunjukan pada Gambar 7. 1) Pengujian Validitas Pengolahan Data akun orang tua Pada akun orang tua kami melakukan pengujian validitas pengolahan data melalui dua kondisi yaitu pengolahan data jika orang tua tidak melakukan permintaan dukungan observasi kepada pihak manapun dan pengujian pengolahan data ketika orang tua melakukan permintaan dukungan observasi kepada pihak lain. Gambar 7. Keluaran SIMPONI observasi orang tua dan dukungan observasi hubungan tinggi dan cukup Gambar 7. Keluaran SIMPONI observasi orang tua dan dukungan observasi hubungan tinggi dan cukup Gambar 7. a) Pendaftaran aplikasi SIMPONI Keluaran SIMPONI observasi orang tua dan dukungan observasi hubungan tinggi dan cukup 2) Pengujian Validitas Pengolahan Data tanpa dukungan observasi Pada kondsi pertama yaitu orang tua tidak melakukan permintaan dukungan observasi 201 e-ISSN 2614-8773 e-ISSN 2614-8773 Infotek : Jurnal Informatika dan Teknologi Vol. 3 No. 2, Juli 2020, hal. 194-203 Infotek : Jurnal Informatika dan Teknologi Vol. 3 No. 2, Juli 2020, hal. 194-203 6. Daftar Pustaka [1] T. Amstrong, Sekolah Para Juara. Kaifa Bandung, 2002. Metode Simple Additive Weighting (SAW) dapat diterapkan untuk menentukan rangking potensi kecerdasan anak yang didapatkan dari hasil input data pernyataan kuesioner berdasarkan observasi kegiatan harian anak untuk menghasilkan profil kecerdasan majemuk. [1] T. Amstrong, Sekolah Para Juara. Kaifa Bandung, 2002. [2] F. Purnomo, B. Sarikho, A. Sutanto, R. Peminatan, and A. Database, “Analisis Dan Perancangan Decision Support System Untuk Rekomendasi Peminatan Berdasarkan 9 Kecerdasan Manusia Dengan Metode Constrained Fuzzy Ahp,” vol. 2010, no. Snati, pp. 7–12, 2010. [2] F. Purnomo, B. Sarikho, A. Sutanto, R. Peminatan, and A. Database, “Analisis Dan Perancangan Decision Support System Untuk Rekomendasi Peminatan Berdasarkan 9 Kecerdasan Manusia Dengan Metode Constrained Fuzzy Ahp,” vol. 2010, no. Snati, pp. 7–12, 2010. Penelitian ini menggunakan alternatif yang dijadikan dasar pengambilan keputusan diantaranya: Linguistik, Matematis logis, Visual Spasial, Kinestetik, Musikal, Interpersonal, Intrapersonal, Naturalis. Selanjutnya kriteria yang dijadikan parameter dalam perhitungan metode SAW berdasarkan kualitas hubungan antara observer dengan anak yang akan di observasi. Kualitas hubungan ini terdiri dari tiga kriteris yaitu sangat tinggi, tinggi dan cukup. [3] I. M. González-Treviño, G. M. Núñez- Rocha, J. M. Valencia-Hernández, and A. Arrona-Palacios, “Assessment of multiple intelligences in elementary school students in Mexico: An exploratory study,” Heliyon, vol. 6, no. 4, p. e03777, 2020, doi: https://doi.org/10.1016/j.heliyon.2020.e0377 7 Nilai alternatif Ai pada setiap kriteria Cj didapatkan dari hasil input data observasi kegiatan keseharian anak dengan menggunakan skala pengukuran tipe skala Likert. Nilai bobot (W) untuk tiap kriteria adalah 5 untuk kualitas hubungan sangat tinggi, 3 untuk kualitas hubungan tinggi dan 2 untuk kualitas hubungan cukup. [4] M. Qomarudin, M. Mustafa, and M. A. Basir, “Pengembangan Model Pembelajaran Adaptive Berdasarkan Teori Kecerdasan Majemuk,” JSHP ( J. Sos. Hum. dan Pendidikan), vol. 2, no. 1, p. 69, 2018, doi: 10.32487/jshp.v2i1.294. [5] Farida Agus Setiawati, “Penskalaan Tipe Likert Dan Thurstone Dengan Teori Klasik Dan Modern : Studi Pada Instrumen Multiple Intelligences,” 2013. Aplikasi SIMPONI memudahkan orang tua dan guru untuk manajemen profil potensi Kecerdasan Majemuk anak/siswa karena semua tersimpan dalam basis data yang dapat diakses setiap saat. Hasil pengolahan data dengan metode SAW baik secara manual menggunakan excel dan pengolahan data melalui sistem menghasilkan keluaran yang sama [6] S. A. Seputro and F. Masya, “Assessment of Student’S Interests and Talents Using the Web-Based Certainty Factor Method,” J. Ris. Inform., vol. 2, no. 3, pp. 131–136, 2020, doi: 10.34288/jri.v2i3.139. [7] E. Ladyawati and O. T. S. Infotek : Jurnal Informatika dan Teknologi Vol. 3 No. 2, Juli 2020, hal. 194-203 Kecerdasan Majemuk (Multiple Intellegences) pada Siswa Sekolah Dasar,” Wahana, vol. 62, no. 1. pp. 15–23, 2014. 664, 2019, doi: 10.25126/jtiik.201961636. 6. Daftar Pustaka Dewi, “Identifikasi [7] E. Ladyawati and O. T. S. Dewi, “Identifikasi e-ISSN 2614-8773 202 (Multiple [10] V. C. Hardita, E. Utami, and E. T. Luthfi, “Penerapan Simple Additive Weighting pada Pemilihan Canvasser Terbaik PT.Eratel Prima,” J. Teknol. Inf. dan Ilmu Komput., vol. 6, no. 5, p. 567, 2019, doi: 10.25126/jtiik.2019651218. [8] F. A. Setiawati, “Penskalaan multiple intelligences sebagai salah satu alternatif instrumen untuk mengukur karakter bangsa yang cerdas,” Konf. HEPI di Unesa., 2012. [11] J. Hutahaean and M. Badaruddin, “Sistem Pendukung Keputusan Pemilihan Sekolah SMK Swasta Penerima Dana Bantuan Menerapkan Metode Simple Additive Weighting ( SAW ),” vol. 4, no. April, pp. 466–471, 2020, doi: 10.30865/mib.v4i2.2109. [9] S. Rinaldi, T. Informatika, and K. Dosen, “Implementasi Metode Analytical Hierarchy Proces S Dan Simple Additive Weighting Untuk Pemilihan Dosen Terbaik Studi Kasus Decision Support System To Choose the Best Lecturer Performance With Analytical Hierarchy Proces S and Simple,” J. Teknol. Inf. dan Ilmu Komput., vol. 6, no. 6, pp. 655– 203 e-ISSN 2614-8773
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Strong generalized synchronization with a particular relationship<i>R</i>between the coupled systems
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Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under licence by IOP Publishing Ltd 1 Strong generalized synchronization with a particular relationship R between the coupled systems Clara Gr´acio1, Sara Fernandes2 and Lu´ıs M´ario Lopes3 1Department of Mathematics, ECT - Universidade de ´Evora, CIMA-UE Rua Rom˜ao Ramalho, 59, 7000-585 ´Evora, Portugal 2Department of Mathematics, ECT - Universidade de ´Evora, CIMA-UE Rua Rom˜ao Ramalho, 59, 7000-585 ´Evora, Portugal 3Department of Mathematics, ISEL Rua Conselheiro Em´ıdio Navarro, 1, 1959-007 Lisboa, Portugal E-mail: 1mgracio@uevora.pt, 2saf@uevora.pt, 3llopes@adm.isel.pt Abstract. The question of the chaotic synchronization of two coupled dynamical systems is an issue that interests researchers in many fields, from biology to psychology, through economics, chemistry, physics, and many others. The different forms of couplings and the different types of synchronization, give rise to many problems, most of them little studied. In this paper we deal with general couplings of two dynamical systems and we study strong generalized synchronization with a particular relationship R between them. Our results include the definition of a window in the domain of the coupling strength, where there is an exponentially stable solution, and the explicit determination of this window. In the case of unidirectional or symmetric couplings, this window is presented in terms of the maximum Lyapunov exponent of the systems. Examples of applications to chaotic systems of dimension one and two are presented. This content was downloaded from IP address 194.210.196.15 on 10/12/2018 at 11:33 European Conference - Workshop "Nonlinear Maps and Applications" IOP Conf. Series: Journal of Physics: Conf. Series 990 (2018) 012005 European Conference - Workshop "Nonlinear Maps and Applications" IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 990 (2018) 012005 doi:10.1088/1742-6596/990/1/012005 IOP Publishing doi:10.1088/1742-6596/990/1/012005 doi:10.1088/1742-6596/990/1/012005 2. A c-family of couplings that admit generalized synchronization with a particular relationship R p p We consider the general coupling of two discrete n-dimensional chaotic dynamical systems xt and yt that is widely used in this context [28, 29, 30, 26], given by  xt+1 = f (xt) + c · [F1(xt) + F2(yt)] yt+1 = g (yt) + c · [G1(xt) + G2(yt)] , (1) (1) where c is the coupling strength, with values in [0, 1] and f, g, F1, F2, G1 and G2 are appropriate f ti where c is the coupling strength, with values in [0, 1] and f, g, F1, F2, G1 and G2 are appropriate functions. The ability of the systems to synchronize depends, not only on the functions F1, F2, G1 and G2, but also on the coupling strength c. In order to consider couplings where the values of the coupling strength c for which the coupling achieves generalized synchronization with a particular relationship R is not a discrete set, we want to consider couplings that admit this synchronization for all values of c (even if it is not an exponentially stable one). That will allow us to define “synchronization windows” in a similar way that was often done for complete synchronization (either in couplings or in networks [1]). So, we consider the following c-family of couplings that provide a similar frame of analysis for the generalized synchronization with a particular relationship R:  xt+1 = f (xt) + c · [−F2(R(xt) + F2(yt)] yt+1 = g (yt) + c · [−G2(R(xt)) + G2(yt)] , (2) (2) with R ◦f = g ◦R. In fact, (xt, yt) = (st, R (st)) with st+1 = f(st) is a solution of (2) for all values of c, since (2) reduces to with R ◦f = g ◦R. In fact, (xt, yt) = (st, R (st)) with st+1 = f(st) is a solution of (2) for all values of c, since (2) reduces to  st+1 = f (st) R (st+1) = g (R (st))  st+1 = f (st) R (st+1) = g (R (st)) and both equations are verified because R ◦f = g ◦R and st+1 = f(st). We note that the complete synchronization corresponds to R(u) = u and the lag synchronization corresponds to R(u) = f(∆t)(u) [17, 19, 31]. Further, if R is a diffeomorphism, then f and g are topologically conjugate maps by the topological conjugacy R. European Conference - Workshop "Nonlinear Maps and Applications" IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 990 (2018) 012005 doi:10.1088/1742-6596/990/1/012005 European Conference - Workshop "Nonlinear Maps and Applications" IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 990 (2018) 012005 doi:10.1088/1742-6596/990/1/012005 [22, 23, 24, 25, 21] and even fewer to bidirectional couplings [20, 26, 21] since generalized synchronization is traditionally introduced for two unidirectionally coupled systems [11, 17, 27]. [ ] In this paper we discuss the design of a c-family of couplings in order to achieve generalized synchronization with a desired relationship R between the coupled systems. Similar discussions have already been done for unidirectional couplings or for couplings of continuous-time systems [28, 29, 30], but now we are considering those less studied situations: bidirectional couplings of discrete-time systems. We consider conditions for which a c-family of couplings achieves the referred synchronization and for which we will be able to calculate the R-synchronization window. In fact, only some values of the coupling strength will provide exponentially stable synchronized solutions, defining the referred window. 2. A c-family of couplings that admit generalized synchronization with a particular relationship R So, a coupling of type (2) of two dynamical systems described by maps that are topologically conjugate by a topological conjugacy R, admits generalized synchronization with a relationship R between the coupled systems. 1. Introduction Chaotic synchronization is a very important phenomenon in many fields involving mathematical, physical, sociological, physiological, biological or other systems [1, 2, 3, 4]. Several different types of synchronization have been studied: complete synchronization [5, 6], phase synchronization [1, 7], lag synchronization [8, 9, 10], generalized synchronization [11, 12], projective synchronization [13, 14], etc. The complete synchronization is the simplest type of synchronization and a lot of work have been produced about it. Much less study have been devoted to one of the most interesting types of synchronous chaotic behavior, the generalized synchronization. When there is generalized synchronization in a coupling of two dynamical systems xt and yt, it is established a relationship yt = R (xt) between the system states after the transient is finished. Depending on the properties of the relationship R, the synchronization is said to be either strong or weak. Namely, strong synchronization corresponds to a smooth relationship R, while weak synchronization corresponds to a fractal one [17]. Several methods have been suggested to detect generalized synchronization: the method of the mutual false nearest neighbors [11, 15], the auxiliary system method [16], using the conditional Lyapunov exponents [17], the modified system approach [18, 19], the phase tube approach [20, 21]. Nevertheless, most of the studies done focuses on continuous-time systems. Few of them are related to discrete-time systems 1 3. R-synchronization window Even if a coupling admits generalized synchronization with a particular relationship R, only some values of the coupling strength (or even none) correspond to a coupling that admits a function st such that (xt, yt) = (st, R (st)) is an exponentially stable solution. So, we consider the following definition. 2 European Conference - Workshop "Nonlinear Maps and Applications" IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 990 (2018) 012005 doi:10.1088/1742-6596/990/1/012005 Definition 1. For a c-family of couplings that admit generalized synchronization with a particular relationship R, we call R-synchronization window, RSW, the set of values of the coupling strength c for which there is a function st such that (xt, yt) = (st, R (st)) is an exponentially stable solution of (1). The following proposition establishes conditions for a value of c to belong to the RSW. P iti 1 Th R h i ti i d RSW f th li (2) ti fi The following proposition establishes conditions for a value of c to belong to the RSW Proposition 1. The R-synchronization window, RSW, of the coupling (2) satisfies {c ∈[0, 1] : µrs < 0} ⊂RSW ⊂{c ∈[0, 1] : µrs ≤0} Proposition 1. The R-synchronization window, RSW, of the coupling (2) satisfies {c ∈[0, 1] : µrs < 0} ⊂RSW ⊂{c ∈[0, 1] : µrs ≤0} {c ∈[0, 1] : µrs < 0} ⊂RSW ⊂{c ∈[0, 1] : µrs ≤0} where where µrs = max u0 lim T→+∞ 1 T ln DMT (s0) · u0 ∥u0∥ , M = g ◦R + c · (G2 ◦R −R ◦f · F2 ◦R) , DMT (s0) = DM(sT−1) · DM(sT−2) · ... · DM(s0) and st+1 = f(st). and st+1 = f(st). Proof. 3. R-synchronization window Series: Journal of Physics: Conf. Series 990 (2018) 012005 doi:10.1088/1742-6596/990/1/012005 European Conference - Workshop "Nonlinear Maps and Applications" IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 990 (2018) 012005 doi:10.1088/1742-6596/990/1/012005 We note that this proposition is valid for dynamical systems of any dimension. In the next section, we use it to confirm numerical examples of dimensions one and two. For some couplings it is possible to explicit the RSW as a function of the maximal Lyapunov exponent of the coupled dynamical systems. In fact, if R is a diffeomorphism and G2 = −g we have for unidirectional couplings (i.e. couplings with F2 = 0) or for the ones that F2 = R−1 ◦g (we will call them ”symmetric couplings”) the following propositions. Remark 1. If G2 = −g and F2 = R−1 ◦g, the coupling is symmetric since  xt+1 = (1 −c) · f (xt) + c · f ◦R−1 (yt) yt+1 = c · (R ◦f) (xt) + (1 −c) · R ◦f ◦R−1 (yt) , . If G2 = −g and F2 = R−1 ◦g, the coupling is symmetric since (2) reduces to Remark 1. If G2 = −g and F2 = R−1 ◦g, the coupling is symmetric since (2) reduces to  xt+1 = (1 −c) · f (xt) + c · f ◦R−1 (yt) yt+1 = c · (R ◦f) (xt) + (1 −c) · R ◦f ◦R−1 (yt) ,  xt+1 = (1 −c) · f (xt) + c · f ◦R−1 (yt) yt+1 = c · (R ◦f) (xt) + (1 −c) · R ◦f ◦R−1 (yt) , Proposition 2. For an unidirectional coupling (2) with G2 = −g, Proposition 2. For an unidirectional coupling (2) with G2 = −g,  1 −e−µ0, 1  ⊂RSW ⊂  1 −e−µ0, 1  , where µ0 is the maximal Lyapunov exponent of st+1 = f (st). where µ0 is the maximal Lyapunov exponent of st+1 = f (st). For such a coupling, since G2 = −g and F2 = 0, (4) reduces to DM (st) = Dg (R(st)) −c · Dg(R(st)) = (1 −c) · Dg(est), with est+1 = R(st) satisfying est+1 = g(est). In fact, when st+1 = f(st), we have est+1 = R (st+1) = R (f(st)) = g (R(st)) = g(est). with est+1 = R(st) satisfying est+1 = g(est). 3. R-synchronization window Considering ut = yt −R(xt), or equivalently, yt = R(xt) + ut, ut+1 = yt+1 −R (xt+1) = = g (R(xt) + ut) + c · [−G2(R(xt)) + G2(R(xt) + ut)] − −R (f (xt) + c · [−F2(R(xt)) + F2(R(xt) + ut)]) and, near the synchronized solution, that corresponds to ut = 0 and xt+1 = f(xt), we have and, near the synchronized solution, that corresponds to ut = 0 and xt+1 = f(xt), we have ut+1 ≃g (R (xt)) + Dg (R(xt)) · ut + c · DG2(R(xt)) · ut − −R (f(xt)) −c · DR (f (xt)) · DF2(R(xt)) · ut Since R ◦f = g ◦R, we obtain Since R ◦f = g ◦R, we obtain ut+1 ≃Dg (R(xt)) · ut + c · DG2(R(xt)) · ut −c · DR (f (xt)) · DF2(R(xt)) · ut Then, the linearization of the ut evolution is given by Then, the linearization of the ut evolution is given by ut+1 = DM(st) · ut (3) ut+1 = DM(st) · ut (3) with DM (st) = Dg (R(st)) + c · [DG2(R(st)) −DR (f (st)) · DF2(R(st))] (4) (4) and st+1 = f(st), and if, for a particular value of c, this system is exponentially stable, then the synchronized state (xt, yt) = (st, R (st)) of the coupling (2) is also exponentially stable, i.e. c belongs to its RSW. Further and st+1 = f(st), and if, for a particular value of c, this system is exponentially stable, then the synchronized state (xt, yt) = (st, R (st)) of the coupling (2) is also exponentially stable, i.e. c belongs to its RSW. h Further, uT = DM(sT−1) · DM(sT−2) · ... · DM(s0) · u0 = DMT (s0) · u0, and if lim T→+∞ 1 T ln DMT (s0) · u0 is negative for all u0 then (3) is exponentially stable. So we conclude that {c ∈[0, 1] : µrs < 0} ⊂RSW. and if lim T→+∞ 1 T ln DMT (s0) · u0 is negative for all u0 then (3) is exponentially stable. So we conclude that {c ∈[0, 1] : µrs < 0} ⊂RSW. { [ , ] µ } On the contrary, if µrs is positive, then (3) is unstable and we conclude that RSW ⊂ {c ∈[0, 1] : µrs ≤0}. 3 European Conference - Workshop "Nonlinear Maps and Applications" IOP Publishing IOP Conf. 3. R-synchronization window In fact, when st+1 = f(st), we have est+1 = R (st+1) = R (f(st)) = g (R(st)) = g(est). ( ( )) ( ( )) ( ) Using DgT (es0) to stand for Dg(esT−1) · Dg(esT−2) · ... · Dg(es0), we obtain µrs = max u0 lim T→+∞ 1 T ln (1 −c)T · DgT (es0) · u0 ∥u0∥ = ln |1 −c| + eµ0 ( ( )) ( ( )) ( ) Using DgT (es0) to stand for Dg(esT−1) · Dg(esT−2) · ... · Dg(es0), we obtain µrs = max u0 lim T→+∞ 1 T ln (1 −c)T · DgT (es0) · u0 ∥u0∥ = ln |1 −c| + eµ0, where eµ0 is the maximal Lyapunov exponent of est+1 = g(est). Since f and g are diffeomorphically conjugate, eµ0 = µ0 [32] and we obtain µrs < 0 ⇔1 −e−µ0 < c < 1 + e−µ0 So, we conclude that ]1 −e−µ0, 1] ⊂RSW ⊂[1 −e−µ0, 1]. µrs < 0 ⇔1 −e−µ0 < c < 1 + e−µ0 So, we conclude that ]1 −e−µ0, 1] ⊂RSW ⊂[1 −e−µ0, 1]. Proposition 3. For a symmetric coupling (2) with G2 = −g, 1 −e−µ0 2 , 1 + e−µ0 2  ⊂RSW ⊂ 1 −e−µ0 2 , 1 + e−µ0 2  , 1 −e−µ0 2 , 1 + e−µ0 2  ⊂RSW ⊂ 1 −e−µ0 2 , 1 + e−µ0 2  , where µ0 is the maximal Lyapunov exponent of st+1 = f (st). Proof. For such a coupling, since G2 = −g and F2 = R−1 ◦g, (4) reduces to where µ0 is the maximal Lyapunov exponent of st+1 = f (st). Proof. For such a coupling, since G2 = −g and F2 = R−1 ◦g, (4) reduces to DM (st) = Dg (R(st)) + c ·  −Dg(R(st)) −DR (f (st)) · D R−1 ◦g  (R(st))  = Dg (R(st)) + c · h −Dg(R(st)) −DR (f (st)) · (DR)−1 R−1 (g(R(st)) where µ0 is the maximal Lyapunov exponent of st+1 = f (st). Proof. 4. Examples of the R-synchronization windows p y In this section, in order to consider the general scope of the results obtained in the previous one, we illustrate them in situations that do not reduce neither to the complete synchronization nor to the lag synchronization, since those particularizations of the general problem have already been considered before [5, 8, 6, 9, 10], i.e. we consider R(u) different from u and from f(∆t)(u). We consider couplings of one dimensional and two dimensional dynamical systems, using emblematic maps for both situations: for the couplings of one dimensional dynamical systems we consider that f is the logistic map and R(u) = u2 and for the couplings of two dimensional dynamical systems we consider that f is the H´enon map and R(u1, u2) = (u2, u1). We begin by the examples of unidirectional couplings, i.e. couplings with G2 = −g and F2 = 0. In figure 1 we show the computed results for both the one dimensional and two dimensional situations. In order to the RSW appear clearly, we show the graphs of the difference of the iterates yt and R (xt) (for the two dimensional situation we show the first component of this difference), namely yt −x2 t for the one dimensional situation and (yt)1 −(xt)2 for the two dimensional situation. The RSW corresponds to the values of c for which the ordinates of all the iterates are zero (after the transients died out). The RSW shown in the figures are confirmed by proposition 2, since the maximal Lyapunov exponents for the logistic and H´enon maps (ln 2 and 0.419, respectively) provide ]1 −e−µ0, 1] equal to  1 2, 1  and ]0.342, 1], respectively. In the same Figure 1. Graphs of the post-transient iterates yt−R (xt) as a function of c for an unidirectional coupling with G2 = −g. At the left, we consider that f is the logistic map and R (u) = u2. At the right, we consider that f is the H´enon map and R (u1, u2) = (u2, u1). way, we show in figure 2 the computed results for both the one dimensional and two dimensional situations corresponding to symmetric couplings, i.e. couplings with G2 = −g and F2 = R−1 ◦g. Figure 1. Graphs of the post-transient iterates yt−R (xt) as a function of c for an unidirectional coupling with G2 = −g. European Conference - Workshop "Nonlinear Maps and Applications" IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 990 (2018) 012005 doi:10.1088/1742-6596/990/1/012005 European Conference - Workshop "Nonlinear Maps and Applications" IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 990 (2018) 012005 doi:10.1088/1742-6596/990/1/012005 European Conference - Workshop "Nonlinear Maps and Applications" IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 990 (2018) 012005 doi:10.1088/1742-6596/990/1/012005 We note that in both of the previous propositions RSW only depends on f, which means that using an unidirectional or a symmetric coupling in order to obtain an exponentially stable R-synchronization, the values of the coupling strength c that must be used are independent of R. In both cases the width of the RSW is e−µ0 but for a symmetric coupling an exponentially stable R-synchronization is obtained for smaller values of the coupling strength. 3. R-synchronization window For such a coupling, since G2 = −g and F2 = R−1 ◦g, (4) reduces to DM (st) = Dg (R(st)) + c ·  −Dg(R(st)) −DR (f (st)) · D R−1 ◦g  (R(st))  = Dg (R(st)) + c · h −Dg(R(st)) −DR (f (st)) · (DR)−1 R−1 (g(R(st)))  · Dg(R(st)) i DM (st) = Dg (R(st)) + c ·  −Dg(R(st)) −DR (f (st)) · D R−1 ◦g  (R(st))  = Dg (R(st)) + c · h −Dg(R(st)) −DR (f (st)) · (DR)−1 R−1 (g(R(st)))  = Dg (R(st)) + c · h −Dg(R(st)) −DR (f (st)) · (DR)−1 R−1 (g(R(st)))  · Dg(R(st)) i and, since R ◦f = g ◦R, we obtain, g ( ( t)) + h g( ( t)) (f ( t)) ( ) (g( ( t)))  g( ( t)) i and, since R ◦f = g ◦R, we obtain, DM (st) = Dg (R(st)) + c · [−Dg(R(st)) −Dg(R(st))] = (1 −2c) · Dg (est) with est+1 = R(st), as in the previous proposition. with est+1 = R(st), as in the previous proposition. ( ) Also, in a similar way to the proof of the previous propos Also, in a similar way to the proof of the previous proposition, that leads to µrs < 0 ⇔1 −e−µ0 2 < c < 1 + e−µ0 2 at i 1−e−µ0 2 , 1+e−µ0 2 h ⊂RSW ⊂ h 1−e−µ0 2 , 1+e−µ0 2 i . µrs < 0 ⇔1 −e−µ0 2 < c < 1 + e−µ0 2 So, we conclude that i 1−e−µ0 2 , 1+e−µ0 2 h ⊂RSW ⊂ h 1−e−µ0 2 , 1+e−µ0 2 i . µrs < 0 ⇔1 −e µ0 2 < c < 1 + e µ0 2 hat i 1−e−µ0 2 , 1+e−µ0 2 h ⊂RSW ⊂ h 1−e−µ0 2 , 1+e−µ0 2 i . 4 4. Examples of the R-synchronization windows At the left, we consider that f is the logistic map and R (u) = u2. At the right, we consider that f is the H´enon map and R (u1, u2) = (u2, u1). way, we show in figure 2 the computed results for both the one dimensional and two dimensional situations corresponding to symmetric couplings, i.e. couplings with G2 = −g and F2 = R−1 ◦g. way, we show in figure 2 the computed results for both the one dimensional and two dimensional situations corresponding to symmetric couplings, i.e. couplings with G2 = −g and F2 = R−1 ◦g. 5 European Conference - Workshop "Nonlinear Maps and Applications" IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 990 (2018) 012005 doi:10.1088/1742-6596/990/1/012005 IOP Publishing Again, the RSW shown in the graphs are confirmed by the proposition 3, since i 1−e−µ0 2 , 1+e−µ0 2 h for the one and two dimensional situations considered are  1 4, 3 4  and ]0.171, 0.829[, respectively. Figure 2. Graphs of the post-transient iterates yt −R (xt) as a function of c for a symmetric coupling. At the left, we consider that f is the logistic map and R (u) = u2. At the right, we consider that f is the H´enon map and R (u1, u2) = (u2, u1). Figure 2. Graphs of the post-transient iterates yt −R (xt) as a function of c for a symmetric coupling. At the left, we consider that f is the logistic map and R (u) = u2. At the right, we consider that f is the H´enon map and R (u1, u2) = (u2, u1). We consider also two examples of couplings for which the RSW can not be calculated neither by proposition 2 nor by proposition 3. Namely, we consider examples of unidirectional couplings (i.e. couplings with F2 = 0) with G2(u) = −g2 (u) for the one dimensional situation and G2(u1, u2) = −g2 1 (u1,u2) , −g2 2 (u1,u2)  for the two dimensional situation. We show in figure 3 the computed results and we verify that a non-empty RSW exists. In order to show that this window is confirmed by proposition 1 we show in figure 4 the value of µrs. 4. Examples of the R-synchronization windows In fact the values for which µrs are negative in the figure 4 are the ones for which the ordinates of all the post-transient iterates of yt −R (xt) are zero. 5. Conclusions If some conditions are satisfied, when we couple two discrete chaotic dynamical systems, they achieve generalized synchronization with a particular relationship R between the coupled systems. In fact, if the two systems are topologically conjugate by a topological conjugacy R, a coupling of the type (2) admits that synchronization and there is a synchronized exponentially stable solution for the values of the coupling strength c that belong to the R-synchronization window RSW. So, if we are dealing with two topologically conjugate dynamical systems, we are able to synchronize them in an exponentially stable way. We just have to couple them with a coupling of type (2) and use a value of c in RSW. Or, the other way round, if we want that a dynamical system R-synchronizes with another, we choose that other system topologically conjugate to the first one by a topological conjugacy R and then we couple them in the referred way. For couplings of type (2) an analytical expression of the RSW can be calculated. In some situations, as in the unidirectional and symmetric cases considered in propositions 2 and 3, that 6 6 European Conference - Workshop "Nonlinear Maps and Applications" IOP Conf. Series: Journal of Physics: Conf. Series 990 (2018) 012005 European Conference - Workshop "Nonlinear Maps and Applications" IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 990 (2018) 012005 doi:10.1088/1742-6596/990/1/012005 doi:10.1088/1742-6596/990/1/012005 Figure 3. Graphs of the post-transient iterates yt −R (xt) as a function of c for an unidirectional coupling. At the left, we consider that f is the logistic map, R (u) = u2 and G2(u) = −g2 (u). At the right, we consider that f is the H´enon map, R (u1, u2) = (u2, u1) and G2(u1, u2) = −g2 1 (u1,u2) , −g2 2 (u1,u2)  . Figure 3. Graphs of the post-transient iterates yt −R (xt) as a function of c for an unidirectional coupling. At the left, we consider that f is the logistic map, R (u) = u2 and G2(u) = −g2 (u). At the right, we consider that f is the H´enon map, R (u1, u2) = (u2, u1) and G2(u1, u2) = −g2 1 (u1,u2) , −g2 2 (u1,u2)  . Figure 4. Graphs of µrs as a function of c for an unidirectional coupling. European Conference - Workshop "Nonlinear Maps and Applications" IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 990 (2018) 012005 doi:10.1088/1742-6596/990/1/012005 European Conference - Workshop "Nonlinear Maps and Applications" IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 990 (2018) 012005 doi:10.1088/1742-6596/990/1/012005 note that those windows are independent of R. p For all the examples that we considered, even the ones that RSW is not expressed as a function of the Lyapunov exponent of the free dynamical systems, the numerical approach used provides the RSW analytically calculated, revealing that the basin of attraction of the exponentially stable synchronized solution is sufficiently large in order to avoid that the RSW is masked by trajectories corresponding to random initial values not belonging to the basin of attraction. Acknowledgments Research partially sponsored by national funds through the Funda¸c˜ao Nacional para a Ciˆencia e Tecnologia, Portugal-FCT, under the project UID/MAT/04674/2013 (CIMA) References References [1] Pikovsky A, Rosenblum M and Kurths J 2001 Synchronization, a universal concept in nonlinear sciences (England: Cambridge University Press) [1] Pikovsky A, Rosenblum M and Kurths J 2001 Synchronization, a universal concept in nonlinear sciences (England: Cambridge University Press) ( [2] Glass L 2001 Nature 410 277–84 [2] Glass L 2001 Nature 410 277 84 [3] Boccaletti S, Kurths J, Osipov G V, Valladares D L and Zhou C S 2002 Phys. Reports 366 1–10 [3] Boccaletti S, Kurths J, Osipov G V, Valladares D L and Zhou C S 2002 Phys. Reports [3] Boccaletti S, Kurths J, Osipov G V, Valladares D L and Zhou C S 2002 Phys. Reports 366 [4] K kii A A M k l k O I d H A E 2009 Ph i U khi 52 1213 38 [ ] , , p , y p [4] Koronovskii A A, Moskalenko O I and Hramov A E 2009 Physics-Uspekhi 52 1213–38 ] , , p , y p 4] Koronovskii A A, Moskalenko O I and Hramov A E 2009 Physics-Uspekhi 52 1213–38 [ ] , y [5] Pecora L M and Carroll T L 1990 Phys. Rev. Lett. 64 821–4 [5] Pecora L M and Carroll T L 1990 Phys. Rev. Lett. 64 821–4 [6] Luo C and Wang X 2013 Nonlinear Dyn. 71 241–257 ] g y 7] Mahmoud G M and Mahmoud E E 2010 Nonlinear Dyn. 61 141–152. [ ] [7] Mahmoud G M and Mahmoud E E 2010 Nonlinear Dyn. 61 141–152. osenblum M G, Pikovsky A S and Kurths J 1997 Ph [8] Rosenblum M G, Pikovsky A S and Kurths J 1997 Phys. Rev. Lett. 78 4193–6 [8] Rosenblum M G, Pikovsky A S and Kurths J 1997 Phys. Rev. Lett. 78 419 [9] Lopes L, Fernandes S and Gr´acio C 2014 Nonlinear Dyn. 79 1615–24 9] Lopes L, Fernandes S and Gr´acio C 2014 Nonlinear Dyn. 79 1615–24 [10] Zhang F 2015 Entropy 17 4974–85 [ ] g py [11] Rulkov N F, Sushchik M M, Tsimring L S and Abarbanel H D I 1995 Phys. Rev. E 51 980–94 [11] Rulkov N F, Sushchik M M, Tsimring L S and Abarbanel H D I 1995 Phys. Rev. 5. Conclusions At the left, we consider that f is the logistic map, R (u) = u2 and G2(u) = −g2 (u). At the right, we consider that f is the H´enon map, R (u1, u2) = (u2, u1) and G2(u1, u2) = −g2 1 (u1,u2) , −g2 2 (u1,u2)  . Figure 4. Graphs of µrs as a function of c for an unidirectional coupling. At the left, we consider that f is the logistic map, R (u) = u2 and G2(u) = −g2 (u). At the right, we consider that f is the H´enon map, R (u1, u2) = (u2, u1) and G2(u1, u2) = −g2 1 (u1,u2) , −g2 2 (u1,u2)  . expression can be expressed in an easy way as a function of the Lyapunov exponent of the free dynamical systems. The width of the RSW of that unidirectional coupling is the same as the one of the symmetric coupling, but if we choose a symmetric coupling instead of an unidirectional one, the systems synchronize for smaller values of the coupling strength. It is also relevant to 7 References E 51 980–94 [12] Zhang G, Liu Z R and Ma Z J 2007 Chaos, Solitons & Fractals 32 773–779 [ ] [13] Grassi G and Miller D A 2007 Int. J. Bifurcation Chaos 17 1337-42 d Miller D A 2007 Int. J. Bifurcation Chaos 17 1337 [14] Nian F, Wan X and Zheng P 2013 Int. J. Mod. Phys. B 27 2252–79 5] Pecora L M, Carroll T L and Heagy J F 1995 Phys. Rev. E 52 3420–39 [15] Pecora L M, Carroll T L and Heagy J F 1995 Phys. Rev. E 52 3420–39 [16] Abarbanel H D I, Rulkov N F and Sushchik M M 1996 Phys. Rev. E 53 4528–35. [17] Pyragas K 1996 Phys. Rev. E 54 R4508–11 [ ] [18] Hramov A E and Koronovskii A A 2005 Phys. Rev. E 71 067201 [18] Hramov A E and Koronovskii A A 2005 Phys [19] Koronovskii A A, Moskalenko O I and Hramov A E 2006 Technical Physics 51 143–150 Koronovskii A A, Moskalenko O I and Hramov A E 2011 Phys. Rev. E 84 037201 [20] Koronovskii A A, Moskalenko O I and Hramov A E 2011 Phys. Rev. E 84 037201 [21] Koronovskii A A, Moskalenko O I, Shurygina S A and Hramov A E 2013 Chaos, Solitons & Fractals 46 12-18 [22] Rulkov N F, Afraimovich V S, Lewis C T, Chazottes J R and Cordonet A 2001 Phys. Rev. E 64 0 [23] Afraimovich V S, Chazottes J R and Cordonet A 2001 Discrete Contin. Dyn. Syst. B 1 21–442 [24] So P, Barreto E, Josic K, Sander E and SchiffS J 2002 Phys. Rev. E 65 046225 [25] Grassi G 2012 Chin. Phys. B 21 050505 [25] Grassi G 2012 Chin. Phys. B 21 050505 [ ] y [26] Yuan Z, Xu Z and Guo L 2012 Commun. Nonlinear Sci. Numer. Simulat. 17 992-1002 [26] Yuan Z, Xu Z and Guo L 2012 Commun. [27] Hramov A E, Koronovskii A A and Moskalenko O I 2005 Europhys. Lett. 72 901–7. References [28] Yang X S and Chen G 2002 Chaos, Solitons & Fractals 131303–8 28] Yang X S and Chen G 2002 Chaos, Solitons & F [29] Lu J and Xi Y 2003 Chaos, Solitons & Fractals 17 825–31 [29] Lu J and Xi Y 2003 Chaos, Solitons & Fractals 17 825–31 [30] Jie S Bollt E M and Nishikawa T 2009 SIAM Journal on Applied Dynamical Systems 8 202 221 [ ] , [30] Jie S, Bollt E M and Nishikawa T 2009 SIAM Journal on Applied Dynamical Systems 8 202–221 e S, Bollt E M and Nishikawa T 2009 SIAM Journal [31] Lopes L, Fernandes S and Gr´acio C 2016 Journal of Physics: Conference Series 692 012008 [ ] [32] Bollt E M and Santitissadeekorn N 2013 Applied and computational measurable dynamics (Society for Industrial and Applied Mathematics) 8
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La promesa de lo bello: consideraciones acerca de la estética filosófica hacia finales del siglo XVIII
Anales del Seminario de Historia de la Filosofía
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María Verónica GALFIONE Universidad Nacional de Córdoba - CONICET (Argentina) Universidad Nacional de Córdoba - CONICET (Argentina) Recibido: 21-05-2013 Aceptado: 21-11-2013 Resumen El presente trabajo analiza algunas de las estrategias por medio de las cuales la refle- xión estética de finales del siglo XVIII intentó dar respuesta al problema de la legitimación de la representación artística. Entre ellas es considerada con particular atención la redefini- ción del concepto de belleza que propone Immanuel Kant en la Crítica de la facultad de juzgar. A partir de la revisión de la postura kantiana, se pone en evidencia la conexión exis- tente entre la crisis de fundamentación de la belleza artística y la debacle de la historia natu- ral clásica, por una parte. Por la otra, se procura dar cuenta de la modificación del estatuto y de las funciones de la esfera estética que introduce el vuelco kantiano hacia el ámbito de la subjetividad. Palabras clave: belleza, naturaleza, representación artística, teleología. La promesa de lo bello: consideraciones acerca de la estética filosófica hacia finales del siglo XVIII La promesa de lo bello: consideraciones acerca de la estética filosófica hacia finales del siglo XVIII Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 131 ISSN: 0211-2337 http://dx.doi.org/10.5209/rev_ASHF.2014.v31.n1.45615 Abstract This paper analyzes the strategies that the aesthetic reflection in the late eighteenth cen- tury set out to address the problem of artistic representation legitimacy. Among them, we closely consider the redefinition of “beauty” proposed by Kant in the Kritik der Urteilskraft. By reviewing Kant’s position, we intend to show, on the one hand, the connection between the justification of artistic beauty crisis and the debacle of classical natural history. On the other hand, we seek to highlight the aesthetic sphere’s change of status and function intro- duced by the Kantian turn towards subjectivity. Keywords: Beauty, Nature, Artistic representation, Teleology. ISSN: 0211-2337 http://dx.doi.org/10.5209/rev_ASHF.2014.v31.n1.45615 ISSN: 0211-2337 http://dx.doi.org/10.5209/rev_ASHF.2014.v31.n1.45615 ISSN: 0211-2337 http://dx.doi.org/10.5209/rev_ASHF.2014.v31.n1.45615 The promise of beauty: considerations about philosophical aesthetics in the late eighteenth century María Verónica GALFIONE Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 131 Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 La promesa de lo bello: consideraciones acerca de la... María Verónica Galfione Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 1 Cf. Wolf Lepenies. Das Ende der Naturgeschichte. Frankfurt: Suhrkamp, 1978, pp. 16-8; Michel Foucault. Las palabras y las cosas. Trad. Elsa Cecilia Frost. Barcelona: Planeta, 1984, p. 137. 2 El preformismo biológico negaba la posibilidad de que en el lapso temporal que mediaba entre la fecundación y la formación definitiva del organismo se produjesen transformaciones morfológicas. El desarrollo de los seres naturales consistía así en un proceso mecánico a lo largo del cual maduraban una serie de disposiciones que se hallaban depositadas antes de la fecundación o bien en el óvulo o bien en el espermatozoide. En cualquier caso, el desarrollo evolutivo no suponía la creación de partes nuevas sino el desenvolvimiento de aquellos órganos que se hallaban replegados desde un comienzo. En este sentido, el preformismo le atribuía al concepto de “evolución” un significado contrario al que esta palabra adquiriría a partir del siglo XIX. Cf. Elías Palti. Aporías. Tiempo, modernidad, sujeto, his- toria, nación. Buenos Aires: Alianza, 2001, pp. 35-6; Gottfried W. Leibniz. Los principios de la natu- raleza y de la gracia. Trad. García Morente. México: Porrúa, 1977, p. 64. En el plano filogenético el preformismo tenía como correlato la teoría del encapsulamiento múltiple. Pues si cada ser vivo esta- ba prefigurado en las semillas depositadas por su progenitor o su progenitora, en su propio germen debían hallarse replegados también, en un tamaño aún más reducido, los organismos completos de su entera descendencia. En última instancia, debía existir un óvulo o un espermatozoide primero en el cual se encontrasen encapsuladas, unas dentro de otras, cada una de las generaciones posteriores. Sobre este tema, se pueden consultar los trabajos de: Igor Polianski. “Natursystem, Systemästhetik und das Überleben der Physikotheologie. Eine Jenaer Botanikgeschichte um 1800“. Reinhard Wegner (Ed.) Kunst–Die andere Natur. Göttingen: Vandenhoeck y Ruprecht, 2004, pp. 125-134; Gian France Frigo. „Die stete und feste Gang der Natur zur Organisation“. Olaf Breidbach y Paul Ziche (Eds.) Naturwissenschaften um 1800. Weimar: Hermann Böhlaus Nachfolger, 2001, pp. 27-46; Johannes Bierbrodt. Naturwissenschaft und Ästhetik, 1750-1810. Würzburg: Königshausen und Neumann, 2000, p. 187; Stefan Metzger. Die Konjektur des Organismus Wahrscheinlichkeitsdenken und Performanz im späten 18. Jahrhundert. München: Wilhelm Fink, 2002, pp. 29-40. 3 En este sentido señalaba Leibniz: “si Dios hace milagros, esto sucede, según creo, no porque la natu- raleza se lo exija, sino su piedad: juzgar esto de otra forma supondría tener una representación real- mente baja de la fuerza y la sabiduría de Dios” Gottfried W. Leibniz. Der Briefwechsel mit G.W. 1. Introducción El presente trabajo se inscribe en una investigación de mayor alcance que indaga los vínculos existentes entre el problema de la legitimación de la representación artística, hacia finales del siglo XVIII, y la crisis de las categorías histórico-naturales clásicas. Con esto último hacemos referencia al proceso de temporalización de los fenómenos naturales que puso en cuestión aquella articulación entre el abordaje mecanicista y la explicación teleoló- gica que habían hecho posible las teorías biológicas del siglo XVII1. Estas corrientes, cuyas primeras formulaciones remitían a Marcello Malpighia o Jan Jakob Swammerdam, y cuya fundamentación filosófica puede encontrarse en Gottfried Wilhelm Leibniz, sostenían que los diferentes momentos del desarrollo de un ser vivo se hallaban prefigurados en el germen desde el momento de la fecundación.2 De esta manera, las concepciones preformistas podí- an dar cuenta del carácter teleológico de las configuraciones orgánicas sin renunciar a la posibilidad de explicar el desarrollo de las mismas en términos estrictamente mecánico-cau- sales. En este sentido, la teoría de los gérmenes prefigurados permitía establecer un puente entre la ciencia y la religión. Pues, la configuración de los seres orgánicos era remitida a la voluntad divina, pero el alcance de esta última quedaba restringido al momento inicial de la creación.3 Sin embargo, este procedimiento introducía un drástico recorte de las facultades 1 Cf. Wolf Lepenies. Das Ende der Naturgeschichte. Frankfurt: Suhrkamp, 1978, pp. 16-8; Michel Foucault. Las palabras y las cosas. Trad. Elsa Cecilia Frost. Barcelona: Planeta, 1984, p. 137. 2 El preformismo biológico negaba la posibilidad de que en el lapso temporal que mediaba entre la fecundación y la formación definitiva del organismo se produjesen transformaciones morfológicas. El desarrollo de los seres naturales consistía así en un proceso mecánico a lo largo del cual maduraban una serie de disposiciones que se hallaban depositadas antes de la fecundación o bien en el óvulo o bien en el espermatozoide. En cualquier caso, el desarrollo evolutivo no suponía la creación de partes nuevas sino el desenvolvimiento de aquellos órganos que se hallaban replegados desde un comienzo. En este sentido, el preformismo le atribuía al concepto de “evolución” un significado contrario al que esta palabra adquiriría a partir del siglo XIX. Cf. Elías Palti. Aporías. Tiempo, modernidad, sujeto, his- toria, nación. Buenos Aires: Alianza, 2001, pp. 35-6; Gottfried W. Leibniz. Los principios de la natu- raleza y de la gracia. Trad. García Morente. México: Porrúa, 1977, p. 64. 1. Introducción En el plano filogenético el preformismo tenía como correlato la teoría del encapsulamiento múltiple. Pues si cada ser vivo esta- ba prefigurado en las semillas depositadas por su progenitor o su progenitora, en su propio germen debían hallarse replegados también, en un tamaño aún más reducido, los organismos completos de su entera descendencia. En última instancia, debía existir un óvulo o un espermatozoide primero en el cual se encontrasen encapsuladas, unas dentro de otras, cada una de las generaciones posteriores. Sobre este tema, se pueden consultar los trabajos de: Igor Polianski. “Natursystem, Systemästhetik und das Überleben der Physikotheologie. Eine Jenaer Botanikgeschichte um 1800“. Reinhard Wegner (Ed.) Kunst–Die andere Natur. Göttingen: Vandenhoeck y Ruprecht, 2004, pp. 125-134; Gian France Frigo. „Die stete und feste Gang der Natur zur Organisation“. Olaf Breidbach y Paul Ziche (Eds.) Naturwissenschaften um 1800. Weimar: Hermann Böhlaus Nachfolger, 2001, pp. 27-46; Johannes Bierbrodt. Naturwissenschaft und Ästhetik, 1750-1810. Würzburg: Königshausen und Neumann, 2000, p. 187; Stefan Metzger. Die Konjektur des Organismus Wahrscheinlichkeitsdenken und Performanz im späten 18. Jahrhundert. München: Wilhelm Fink, 2002, pp. 29-40. 3 En este sentido señalaba Leibniz: “si Dios hace milagros, esto sucede, según creo, no porque la natu- raleza se lo exija, sino su piedad: juzgar esto de otra forma supondría tener una representación real- mente baja de la fuerza y la sabiduría de Dios” Gottfried W. Leibniz. Der Briefwechsel mit G.W. 132 María Verónica Galfione La promesa de lo bello: consideraciones acerca de la... sobrenaturales. Por este motivo, Pierre-Simon Laplace llegaría a caracterizar a Dios como una hipótesis de carácter innecesario4. Ya que, aun cuando la voluntad divina dispusiese las formas originarias, el desenvolvimiento de las mismas solo podía ser explicado a partir de la acción mecánica de los diversos factores naturales. Este acuerdo entre ciencia y religión resultaba dependiente de una particular concepción de la temporalidad que excluía por principio toda posible asimetría entre el pasado y el futu- ro. Esta concepción “reversible” de la temporalidad permitía garantizar la conservación de las intenciones divinas a lo largo del desarrollo evolutivo y aseguraba, de esta forma, la coincidencia entre las disposiciones finales y las cadenas mecánico-causales. No obstante, las nuevas investigaciones científicas del siglo XVIII tendieron a poner en duda este presu- puesto y a socavar, en tal sentido, la confianza preformista en la finalidad de las formas orgánicas naturales. 5 Cf. Georges-Louis Leclerc Buffon. Histoire Naturelle, Générale et Particulière, avec la description du Cabinet du Roy. Tome Sixième. Paris: de l’Imprimerie Royale, 1756, pp. 299-301 (entre otros pasa- jes). historia natural de Buffon”. História, Ciências, Saúde – Manguinhos. Vol.20/n.°3, pp. 813-829. 7 En un principio se afirmó que los fósiles correspondían a especies que habían debido emigrar a zonas inexploradas del globo a raíz de grandes transformaciones climáticas. Sin embargo, la progresiva exploración del planeta tornó cada vez más improbable el hallazgo de rastros de las especies extintas en rincones alejados del mismo. Estas circunstancias llevaron a que, hacia mediados del siglo XVIII, no quedase prácticamente otra opción más que admitir el hecho de que los fósiles correspondían a res- tos de especies extintas. Cf. Nicholas Kupke. “Caves, fossils and the history of the earth”. A. Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 6 Cf. María Verónica Galfione. “Historia Natural y temporalización: Algunas consideraciones sobre la historia natural de Buffon”. História, Ciências, Saúde – Manguinhos. Vol.20/n.°3, pp. 813-829. Leibniz von 1715/1716. Hamburg: Felix Meiner, 1990, pp. 10-1. Aquí y en todos los casos en los cua- les no se consigne lo contrario, la traducción ha sido realizada por la autora de este artículo. 4 Cf. Elías Palti. Op. cit. p. 33. Leibniz von 1715/1716. Hamburg: Felix Meiner, 1990, pp. 10-1. Aquí y en todos los casos en los cua- les no se consigne lo contrario, la traducción ha sido realizada por la autora de este artículo. 4 Cf. Elías Palti. Op. cit. p. 33. 5 Cf. Georges-Louis Leclerc Buffon. Histoire Naturelle, Générale et Particulière, avec la description du Cabinet du Roy. Tome Sixième. Paris: de l’Imprimerie Royale, 1756, pp. 299-301 (entre otros pasa- jes). 6 Cf. María Verónica Galfione. “Historia Natural y temporalización: Algunas consideraciones sobre la historia natural de Buffon”. História, Ciências, Saúde – Manguinhos. Vol.20/n.°3, pp. 813-829. 7 En un principio se afirmó que los fósiles correspondían a especies que habían debido emigrar a zonas inexploradas del globo a raíz de grandes transformaciones climáticas. Sin embargo, la progresiva exploración del planeta tornó cada vez más improbable el hallazgo de rastros de las especies extintas en rincones alejados del mismo. Estas circunstancias llevaron a que, hacia mediados del siglo XVIII, no quedase prácticamente otra opción más que admitir el hecho de que los fósiles correspondían a res- tos de especies extintas. Cf. Nicholas Kupke. “Caves, fossils and the history of the earth”. A. 1. Introducción No obstante, para que esta interpretación resultase inteligible era necesario dar cuenta de la existencia de una causa que se encon- trase en condiciones de producir los fenómenos observados. En este contexto, el embriólogo alemán apelaba a una misteriosa vis essentialis que debía garantizar la unicidad y articulación interna del pro- ceso generativo. Con esto, Wolff no lograba resolver el problema de la generación, pero ponía en evi- dencia al menos la necesidad de concebir principios explicativos que, a diferencia de las fuerzas mecá- nicas, fuesen capaces de dar cuenta de la producción de una cadena de estructuras diferenciadas y coordinadas entre sí. 9 Las investigaciones de Wolff ponían en evidencia la existencia de transformaciones morfológicas en el lapso que mediaba entre la fecundación y la formación definitiva del embrión. Wolff remitía estas modificaciones a la diferenciación y sedimentación gradual de un material originariamente indiferen- ciado Cf. Caspar Friedrich Wolff. Theorie von der Generation in zwei Abhandlungen erklärt und bewiesen. Hildesheim: Robert Herrlinger, 1966, p. 163. De esta forma, las diferentes partes de los cuerpos organizados se presentaban como momentos sucesivos y progresivos del proceso de estructu- ración de la materia inorgánica que se hallaba disponible en el embrión. No obstante, para que esta interpretación resultase inteligible era necesario dar cuenta de la existencia de una causa que se encon- trase en condiciones de producir los fenómenos observados. En este contexto, el embriólogo alemán apelaba a una misteriosa vis essentialis que debía garantizar la unicidad y articulación interna del pro- ceso generativo. Con esto, Wolff no lograba resolver el problema de la generación, pero ponía en evi- dencia al menos la necesidad de concebir principios explicativos que, a diferencia de las fuerzas mecá- nicas, fuesen capaces de dar cuenta de la producción de una cadena de estructuras diferenciadas y coordinadas entre sí. 10 En este sentido resulta paradigmático el trabajo de Johann Christoph Gottsched. Gottsched le atri- buía a dios la creación de todo “según número, masa y medida” y, asumiendo el carácter bello de la naturaleza, infería de allí la necesidad de una imitación artística de la misma. “Las cosas naturales son bellas en sí mismas y si el arte quiere producir también algo bello, debe imitar el modelo de la natu- raleza. La relación exacta, el orden y la armonía correcta de todas las partes, en la que consiste una cosa, es la fuente de toda belleza. 1. Introducción La posibilidad de incorporar el principio de la temporalidad en los aná- lisis naturales fue considerada por primera vez en el ámbito de las ciencias taxonómicas clá- sicas. Esto último puede apreciarse de manera paradigmática en el caso de Georges-Louis Leclerc Buffon, quien sostuvo la existencia de una “degeneración” de las formas vivas e incorporó la hipótesis de una “historia de la tierra”. Sin embargo, tampoco Buffon llegó a concebir la posibilidad de que el tiempo desempeñara un papel determinante en la constitu- ción de los seres naturales. Pues, desde su punto de vista, las transformaciones orgánicas que se registraban a lo largo de la historia respondían al efecto limitante que ejercían los dife- rentes factores climáticos sobre el poder de estructuración de un principio de carácter atem- poral: la moule intérieur5. Según hemos mostrado en otro lugar6, la resistencia del naturalista francés a admitir una concepción irreversible de la temporalidad se desprendía de las consecuencias epistémicas de la misma. Puesto que, si se asumía la existencia de transformaciones orgánicas no-rever- sibles se volvía necesario postular la existencia de una guía de carácter providencial a los fines de explicar el surgimiento de las formas naturales organizadas. Sin embargo, la idea de una temporalidad de naturaleza irreversible recibiría un notable empuje durante las últi- mas décadas del siglo XVIII. Entre los elementos que es necesario mencionar a la hora de dar cuenta del progresivo abandono de la noción clásica de la temporalidad merecen una atención especial sucesos tan dispares como el descubrimiento de nuevos yacimientos fósi- les,7 el terremoto de Lisboa8 o la publicación de las investigaciones embriológicas de Caspar Leibniz von 1715/1716. Hamburg: Felix Meiner, 1990, pp. 10-1. Aquí y en todos los casos en los cua- les no se consigne lo contrario, la traducción ha sido realizada por la autora de este artículo. 4 Cf. Elías Palti. Op. cit. p. 33. 6 Cf. María Verónica Galfione. “Historia Natural y temporalización: Algunas consideraciones sobre la historia natural de Buffon”. História, Ciências, Saúde – Manguinhos. Vol.20/n.°3, pp. 813-829. 133 Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 María Verónica Galfione La promesa de lo bello: consideraciones acerca de la... Friedrich Wolff.9 Pues, todos estos acontecimientos contribuyeron a poner en cuestión la existencia de procesos teleológicos naturales. Pero la progresiva temporalización de la experiencia natural no sólo tornó problemáti- ca la explicación de las formas orgánicas. Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 Cunningham et al. (Eds.) Romanticism and the sciences. New York: Cambridge Univ. Press, 1990, pp. 241-259). Así lo haría Blumenbach, quien señalaba: “cada roca de Göttingen da fe de que especies –incluso géneros enteros– de animales deben haber perecido… las estructuras de un enorme número de fósiles en nuestra vecindad son tan divergentes de las formas presentes que nadie tomaría seria- mente ninguna comparación con las formas de vida existente.” Johann Friedrich Blumenbarch. Beiträge zur Naturgeschichte. Göttingen: Johann Christian Dieterich, 1790, pp. 6-8. 8 El terremoto de Lisboa de 1755 fue asumido como la refutación definitiva de la tendencia de la teo- logía natural a concebir a la naturaleza como una entidad armónica en la cual podían combinarse la belleza, el orden y la utilidad. Este acontecimiento tornó evidente el hecho de que la naturaleza no podía ser dominada por medio de un orden racional preconcebido y subrayó, además, la finitud de los hombres y su insuperable estado de orfandad. Cf. Odo Marquard. „Die Krise des Optimismus und die Geburt der Geschichtsphilosophie“. G. Lauer et al. (Eds.) Das Erdbeben von Lissabon und der Katastrophendiskurs im 18. Jahrhundert. Göttingen: Wallstein, 2008, pp. 205–215. 1. Introducción Más allá de este hecho, que no podemos analizar aquí en toda su extensión, la misma socavó aquellos presupuestos sobre la base de los cua- les la estética racionalista de comienzos del siglo XVIII había logrado sostener una concep- ción secular, pero normativa, de lo bello.10 Pues el proceso de temporalización obligaba a limitar las pretensiones nomológicas al ámbito de los fenómenos inorgánicos y tornaba inconcebible, de este modo, aquellas fundamentaciones de la belleza artística que tomaban como punto de referencia la racionalidad intrínseca del orden natural. De hecho, hacia fina- les del siglo XVIII, la reflexión estética se hallaba obligada a enfrentar una situación de Cunningham et al. (Eds.) Romanticism and the sciences. New York: Cambridge Univ. Press, 1990, pp. 241-259). Así lo haría Blumenbach, quien señalaba: “cada roca de Göttingen da fe de que especies –incluso géneros enteros– de animales deben haber perecido… las estructuras de un enorme número de fósiles en nuestra vecindad son tan divergentes de las formas presentes que nadie tomaría seria- mente ninguna comparación con las formas de vida existente.” Johann Friedrich Blumenbarch. Beiträge zur Naturgeschichte. Göttingen: Johann Christian Dieterich, 1790, pp. 6-8. 8 El terremoto de Lisboa de 1755 fue asumido como la refutación definitiva de la tendencia de la teo- logía natural a concebir a la naturaleza como una entidad armónica en la cual podían combinarse la belleza, el orden y la utilidad. Este acontecimiento tornó evidente el hecho de que la naturaleza no podía ser dominada por medio de un orden racional preconcebido y subrayó, además, la finitud de los hombres y su insuperable estado de orfandad. Cf. Odo Marquard. „Die Krise des Optimismus und die Geburt der Geschichtsphilosophie“. G. Lauer et al. (Eds.) Das Erdbeben von Lissabon und der Katastrophendiskurs im 18. Jahrhundert. Göttingen: Wallstein, 2008, pp. 205–215. 9 Las investigaciones de Wolff ponían en evidencia la existencia de transformaciones morfológicas en el lapso que mediaba entre la fecundación y la formación definitiva del embrión. Wolff remitía estas modificaciones a la diferenciación y sedimentación gradual de un material originariamente indiferen- ciado Cf. Caspar Friedrich Wolff. Theorie von der Generation in zwei Abhandlungen erklärt und bewiesen. Hildesheim: Robert Herrlinger, 1966, p. 163. De esta forma, las diferentes partes de los cuerpos organizados se presentaban como momentos sucesivos y progresivos del proceso de estructu- ración de la materia inorgánica que se hallaba disponible en el embrión. 1. Introducción Puesto que, con este desplazamiento, la filosofía kantiana sentaba las bases sobre las cuales las estéticas idealistas de finales del siglo XVIII y comienzos del XIX articularían la pro- blemática filosófica de la autofundamentación de la subjetividad con el problema de la legi- timación estética de la representación artística.12 Ya que, aun cuando Kant no llegara a con- cebir a la belleza artística como auto-objetivación de una subjetividad unificada, convertía a la experiencia estética en un espacio adecuado para apuntalar una posible aplicación extra- estética exitosa de las diferentes capacidades subjetivas.13 Hasta qué punto se atentaba de 11 Asumimos aquí la existencia de una continuidad entre el juicio teleológico y el juicio estético no solo en tanto ambos intentan dar respuesta a un mismo problema – que tiene su origen en el proceso de temporalización- sino también porque lo hacen por medio de un desplazamiento análogo más allá del ámbito de la representación. Una lectura de la obra kantiana que enfatiza, en cambio, las diferen- cias existentes entre los dos momentos de la Crítica de la facultad de juzgar puede encontrarse en Lewis White Beck y Paul Guyer. Cf. Lewis White Beck. Early German Philosophy. Cambridge: Belknap Press, 1969, pp. 496-498; Paul Guyer. Kant and the Claims of Taste. Cambridge: Harvard Univ. Press, 1979, pp. 213-18. No obstante, estas posturas han sido particularmente discutidas en los últimos años. En este sentido pueden ser consultadas las interpretaciones de Rachel Zuckert y de Hannah Ginsborg. Cf. Rachel Zuckert. Kant on Beauty and Biology. Cambridge: Cambridge Univ. Press, 2007 y Hannah Ginsborg. “Kant on Aesthetic and Biological Purposiveness”. Andrews Reath et al. (Eds.) Reclaiming the History of Ethics: Essays for John Rawls. Cambridge: Cambridge Univ. Press, 1997, pp. 329-360. 12 Una lectura opuesta a la que proponemos aquí ha sido desarrollada durantes los años 70 por Rüdiger Bubner. Este autor establece un hiato entre la estética kantiana y las filosofías idealistas de arte. Desde su perspectiva, la estética kantiana se concentraría en la experiencia estética, y no en la obra de arte, y resignaría el contenido de verdad de esta última. Cf. Rüdiger Bubner. “Über einige Bedingungen gegenwärtiger Ästhetik“. Neue hefte für Philosophie. Göttingen, Vandenhoeck & Ruprecht, V. 5, 1973, p. 65ss. 13 En este punto, nuestra interpretación se orienta en un sentido similar al análisis que propone Zuckert. 1. Introducción La imitación de la naturaleza perfecta puede dar entonces a una obra artística la perfección por medio de la cual aquella guste y resulte agradable para el entendimiento. La desviación con respecto a este modelo, producirá siempre algo informe y contrario al gusto.” Johann Christoph Gottsched. Versuch einer Critischen Dichtkunst. Leipzig: Breitkopf, 1751, p. 132. 134 María Verónica Galfione La promesa de lo bello: consideraciones acerca de la... carácter paradójico. Pues, de admitir la existencia de leyes naturales para la creación artís- tica, debía renunciar a la posibilidad de concebirlas en términos estéticos, ya que, en un sen- tido estricto, las formas naturales no se hallaban dotadas de belleza ni de fealdad. En las páginas que siguen intentaremos revisar algunas de las estrategias por medio de las cuales la reflexión estética de las últimas décadas del siglo XVIII intentó superar la pér- dida de legitimidad ontológica de la representación artística a la que dieron lugar los proce- sos mencionados. Antes que nada, consideraremos los orígenes de la estética ilustrada de los efectos, en tanto primera respuesta a la crisis de la estética racionalista clásica. En segundo lugar, analizaremos el posicionamiento crítico de Johann Gottfried Herder y de Karl Philipp Moritz frente a las perspectivas psicológico-efectuales y, en tercer término, nos concentra- remos en la redefinición del concepto de belleza que proponía Immanuel Kant en la Crítica de la facultad de Juzgar.11 En este punto resulta de nuestro interés advertir que la concep- ción estética kantiana de la belleza no solo colocaba a la subjetividad en un lugar central sino que modificaba, a su vez, el estatuto y las funciones del ámbito estético mismo. Con esto hacemos referencia al hecho de que Kant concibiera al placer estético a partir de la forma de un posible funcionamiento coherente de las facultades o disposiciones subjetivas. 11 Asumimos aquí la existencia de una continuidad entre el juicio teleológico y el juicio estético no solo en tanto ambos intentan dar respuesta a un mismo problema – que tiene su origen en el proceso de temporalización- sino también porque lo hacen por medio de un desplazamiento análogo más allá del ámbito de la representación. Una lectura de la obra kantiana que enfatiza, en cambio, las diferen- cias existentes entre los dos momentos de la Crítica de la facultad de juzgar puede encontrarse en Lewis White Beck y Paul Guyer. Cf. Lewis White Beck. Early German Philosophy. Cambridge: Belknap Press, 1969, pp. 496-498; Paul Guyer. Kant and the Claims of Taste. Cambridge: Harvard Univ. Press, 1979, pp. 213-18. No obstante, estas posturas han sido particularmente discutidas en los últimos años. En este sentido pueden ser consultadas las interpretaciones de Rachel Zuckert y de Hannah Ginsborg. Cf. Rachel Zuckert. Kant on Beauty and Biology. Cambridge: Cambridge Univ. Press, 2007 y Hannah Ginsborg. “Kant on Aesthetic and Biological Purposiveness”. Andrews Reath et al. (Eds.) Reclaiming the History of Ethics: Essays for John Rawls. Cambridge: Cambridge Univ. Press, 1997, pp. 329-360. Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 12 Una lectura opuesta a la que proponemos aquí ha sido desarrollada durantes los años 70 por Rüdiger Bubner. Este autor establece un hiato entre la estética kantiana y las filosofías idealistas de arte. Desde su perspectiva, la estética kantiana se concentraría en la experiencia estética, y no en la obra de arte, y resignaría el contenido de verdad de esta última. Cf. Rüdiger Bubner. “Über einige Bedingungen gegenwärtiger Ästhetik“. Neue hefte für Philosophie. Göttingen, Vandenhoeck & Ruprecht, V. 5, 1973, p. 65ss. 1. Introducción En su estudio sobre la tercera crítica kantiana, la autora sostiene que Kant habría introducido allí una nueva concepción de la subjetividad que tensionaba los márgenes del pensamiento crítico. Según Zuckert, este movimiento respondía a la necesidad de explicar “cómo podemos comprender lo 135 Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 María Verónica Galfione La promesa de lo bello: consideraciones acerca de la... esta forma contra la autonomía estética, recientemente conquistada, es algo sobre lo cual deberemos reflexionar a partir del recorrido propuesto. 2. Teleología natural y belleza artística En el marco de la crisis epistémica a la que hacíamos referencia más atrás, la recons- trucción del orden natural pasó a depender de la posibilidad de postular un principio o una fuerza oculta que estuviese en condiciones de superar el carácter aparentemente caótico de las formas orgánicas visibles. De hecho, el propio Kant había llegado a sugerir la necesidad de construir una historia de la naturaleza que permitiese ligar la apariencia actual de los seres naturales con sus formas ancestrales y ordenar, de esta forma, la dispersión existente en el mundo natural14. Sin embargo, la referencia a este orden natural oculto no fue consi- derada por Kant como una estrategia adecuada en orden a salvar la legitimidad de la repre- sentación artística. A esta alternativa se oponía, antes que nada, la propia imposibilidad epis- témica de afirmar el carácter objetivo de aquellas conclusiones a la que arribaba la mentada historia natural. Pues, como era puesto en evidencia en la Crítica de la facultad de juzgar, los resultados de esta última hacían referencia a un ordenamiento natural que no resultaba concebible a partir de ninguna de las formas causales que se hallaban al alcance de nuestra comprensión. Dicho con mayor precisión, la organización profunda de los seres naturales no podía ser deducida a partir de causas mecánicas,15 pero tampoco podía ser entendida como el producto de una acción de tipo intencional.16 Desde un punto de vista mecánico, el orden dado empíricamente, lo particular, lo contingente en cuanto tal –esto es, precisamente aquello que es marginal o se halla más allá de las formas universales, de los conceptos o de las leyes de las cuales se ocupa la filosofía kantiana (y muchas otras).” Rachel Zuckert. Op. cit. p. 6. Sin embargo, Zuckert no especifica las condiciones epistémicas que hicieron posible la emergencia del problema de lo particu- lar, es decir, que tornaron insuficientes las respuestas que había formulado la modernidad temprana frente al mismo. Tampoco entra dentro del campo de intereses de la autora el análisis de las conse- cuencias estéticas que traía aparejada la remisión del placer estético al ideal regulativo de una subje- tividad unificada. 14 Immanuel Kant. Kritik der Urteilskraft. Kants gesammelte Schriften. Tomo V. Berlin: Gruyter, 1913, p. 428. Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 14 Immanuel Kant. Kritik der Urteilskraft. Kants gesammelte Schriften. Tomo V. Berlin: Gruyter, 1913, p. 428. 2. Teleología natural y belleza artística Desde este punto de vista, la naturaleza podía ser renciación kantiana entre organismos y máquinas que ofrecen Peter McLaughlin, Henry Allison o Guyer. Cf. Peter McLaughlin. Kant’s Critique of Teleology in Biological Explanation: Antinomy and Teleology. New York: Mellen, 1990, p. 153; Henry Allison. “Kant’s Antinomy of Teleological Judgment”. Southern Journal of Philosophy, supl. 30, 1991, p. 27; Paul Guyer. “Organisms and the Unity of Science”. Eric Watkins (Ed.) Kant and the Sciences, p. 264. Oxford: Oxford University Press, 2001, p. 264. Aunque con algunos matices, los intérpretes mencionados coinciden en reducir dicha diferencia a la imposibilidad/posibilidad de que la totalidad sea explicada en función de la mera inte- racción de las partes. Como ponen en evidencia Ginsborg y Förster, esta lectura pasa por alto el hecho de que tampoco la explicación de los artefactos es posible a partir del mero análisis de las partes. Estos últimos solo pueden ser comprendidos si se introduce la referencia a un propósito o una intención y este, justamente, el punto en el cual se diferencian de los organismos naturales. Cf. Nota 18. 17 Immanuel Kant. Op. cit. p. 375. 18 Según la lectura de Ginsborg o de Förster, Kant habría definido a los organismos como aquellas entidades en el marco de las cuales las partes no solo se articulaban en función de una totalidad, como sucedía en el caso de un reloj, por ejemplo, sino que establecían entre ellas, además, una relación de acción recíproca: “Un cuerpo que en sí y según su posibilidad interna es juzgado como fin natural exige que sus partes sean producidas mutuamente tanto en lo que concierne a su forma como a su conexión, y que así produzcan un todo a partir de la propia causalidad.” Ibidem. p. 373. Como resul- ta comprensible, esta definición de los organismos en términos de acción recíproca entre las partes y el todo imposibilitaba toda conceptualización de los mismos, en la medida en que eran las partes las que, pese a su dependencia con respecto al todo, debían dar cuenta de la producción del mismo. Si bien Kant asumía la diferencia entre organismos y artefactos como un dato evidente –“Un ser orgánico no es una mera máquina, pues esta tiene solo fuerza motriz, sino que posee en sí fuerza formadora, es decir, una fuerza que él trasmite a materiales que no la tienen (los organiza). Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 2. Teleología natural y belleza artística 15 Esto es, a diferencia de otros fenómenos naturales, como por ejemplo el movimiento de los astros, la particular articulación de las leyes empíricas que se registraba en los cuerpos orgánicos no podía deducirse a priori a partir de las leyes generales de la naturaleza. “Si se menciona, por ejemplo, la constitución de un pájaro, la concavidad de sus huesos, la posición de las alas para el movimiento y de la cola para maniobrar, entonces se debe asumir que, si uno no se vale de un tipo especial de cau- salidad, es decir, la de los fines (nexus finalis), todo esto resulta completamente contingente según el mero nexus effectivus de la naturaleza; es decir, que la naturaleza, como mero mecanismo, podría había formado de mil maneras distintas sin dar con la unidad de dicho principio”. Ibídem p. 360. 16 Con respecto a este tema, se pueden consultar los trabajos de Hannah Ginsborg y de Eckart Förster. Cf. Hannah Ginsborg. “Two Kinds of Mechanical Inexplicability in Kant and Aristotle. Journal of the History of Philosophy, vol. 42 (1), 2004, pp. 33-39 y Eckart Förster. “Die Bedeutung von §§76, 77 der ‘Kritik der Urteilskraft’ für die Entwicklung der nachkantischen Philosophie“. Zeitschrift für philo- sophische Forschung, vol. 56, 2002, pp. 169-190. En ambos casos, la referencia a la imposibilidad de explicar los organismos por medio de un propósito se orienta a discutir la interpretación de la dife- 136 Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 La promesa de lo bello: consideraciones acerca de la... María Verónica Galfione de las formas naturales debía ser considerado como un producto azaroso, mientras que si se lo concebía como el resultado de una acción intencional, el mismo dejaba de presentarse como un orden de carácter natural17. Esto es, la postulación de una intencionalidad objetiva obligaba a introducir un concepto exterior y anterior con respecto a las partes que compo- nían el orden natural, y esto último tenía como consecuencia la destrucción de la relación de acción recíproca entre las partes y el todo que diferenciaba a los productos orgánicos de los meros objetos de fabricación.18 Probablemente por este motivo, Kant se vio llevado a defi- nir la aparente “conformidad a fin” del mundo natural a partir de la introducción de princi- pios de carácter estrictamente subjetivo19. renciación kantiana entre organismos y máquinas que ofrecen Peter McLaughlin, Henry Allison o Guyer. Cf. Peter McLaughlin. Kant’s Critique of Teleology in Biological Explanation: Antinomy and Teleology. New York: Mellen, 1990, p. 153; Henry Allison. “Kant’s Antinomy of Teleological Judgment”. Southern Journal of Philosophy, supl. 30, 1991, p. 27; Paul Guyer. “Organisms and the Unity of Science”. Eric Watkins (Ed.) Kant and the Sciences, p. 264. Oxford: Oxford University Press, 2001, p. 264. Aunque con algunos matices, los intérpretes mencionados coinciden en reducir dicha diferencia a la imposibilidad/posibilidad de que la totalidad sea explicada en función de la mera inte- racción de las partes. Como ponen en evidencia Ginsborg y Förster, esta lectura pasa por alto el hecho de que tampoco la explicación de los artefactos es posible a partir del mero análisis de las partes. Estos últimos solo pueden ser comprendidos si se introduce la referencia a un propósito o una intención y este, justamente, el punto en el cual se diferencian de los organismos naturales. Cf. Nota 18. 17 Immanuel Kant. Op. cit. p. 375. 2. Teleología natural y belleza artística Por lo tanto, posee una fuerza se forma y procrea y que no puede ser explicada a partir de la mera capacidad del movimiento (el mecanismo).” Ibid. p. 374 –, resulta necesario recordar aquí que se trataba de una distinción rela- tivamente reciente. De hecho, la misma resultaba dependiente de las investigaciones embriológicas de Wolff a las que hemos hecho mención más arriba. Estas investigaciones llevaron a poner definitiva- mente en duda la idea de que los organismos hubiesen sido producidos por un plan o idea general de carácter divino al comienzo de los tiempos y obligaron a presuponer la existencia de algún tipo de fuerza inmanente que permitiera explicar la serie articulada de transformaciones que tenían lugar durante el desarrollo del embrión. Kant se hacía eco de tales cambios epistémicos al afirmar en la Crítica de la facultad de juzgar: “Se dice demasiado poco de la naturaleza y de su capacidad en los productos organizados si se los piensa por medio de la analogía con el arte. Pues entonces se piensa a un artista (un ser inteligente), fuera de ella. Pero la naturaleza se organiza ella misma y lo hace cier- tamente en cada especie de sus productos organizados… Quizás uno se acerca más a esta característi- ca insondable si la denomina un análogo de la vida.” Ibid. p. 374. 19 Ibid. p. 182. 137 La promesa de lo bello: consideraciones acerca de la... María Verónica Galfione contemplada como un producto del arte, pero solo en tanto se recordara que la perspectiva así ganada era el resultado de conjeturas y suposiciones que, pese a su urgencia subjetiva, no encontraban correlato alguno en el mundo natural.20 Pero el posicionamiento de Kant con respecto al problema del orden natural no solo lo obligaba a renunciar a la posibilidad de pensar en una estética de carácter mimético sino que le impedía, además, reemplazar aquella dimensión ontológica por medio de la propia repre- sentación artística. En este punto, las observaciones kantianas afectaban a tendencias tales como el Sturm und Drang o el clasicismo de Weimar, que, desde Alexander G. 2. Teleología natural y belleza artística Baumgarten en adelante, habían procurado garantizar el valor de verdad de la esfera estética frente a las estéticas de carácter efectual.21 En un primer momento, aquellas tendencias habían perse- guido fines eminentemente estéticos en la medida en que habían intentado superar la relati- vización de los juicios estéticos a la que conducía el abordaje psicológico-efectual. Sin embargo, en su búsqueda de una legitimación alternativa para el ámbito estético, las pers- pectivas de finales del siglo XVIII acabaron dando lugar a una concepción de la represen- tación artística que descargaba sobre ella funciones de carácter filosófico.22 Dicho en pocas 20 No podemos analizar aquí con detenimiento el conjunto de problemas provenientes de la historia natural clásica al que procuraba dar respuesta la propuesta kantiana de un uso regulativo de principios teleológicos. Concretamente la pretensión kantiana de considerar a la naturaleza como si la misma pro- cediese de acuerdo a determinada finalidad se hallaba orientada a conjurar el fantasma de los cambios morfológicos de carácter irreversible. Esto puede observarse con claridad en el tratamiento kantiano del problema de las razas. Pues, por medio de la idea de disposiciones germinales que se activaban de manera selectiva, Kant lograba retrotraer a los orígenes la totalidad de las transformaciones tempora- les que habían dado lugar a las diferentes razas de la especie humana. Para este tema, es necesario recurrir tanto a los escritos precríticos de Kant como a los nuevos análisis del problema que propone el autor desde mediados de los años 80. Cf. Immanuel Kant. “Von den verschiedenen Rassen der Menschen“. Kants gesammelte Schriften. Tomo II. Berlin: Gruyter, 1912, pp. 427-443; „Bestimmung des Begriffs einer Menschenrasse“. Kants gesammelte Schriften. Tomo VIII, Berlin: Gruyter, 1923, pp. 89–106; „Über den Gebrauch teleologischer Principien in der Philosophie. Kants gesammelte Schriften“. Tomo VIII, Berlin: Gruyter, 1923, pp. 157-184. 21 En Baumgarten esta tendencia se presentaba con cierta ambigüedad. Por una parte, Baumgarten encabezaba la reacción a la estética de escuela de Gottsched, que pretendía sostener aún el principio de la imitación. En este sentido, tanto Baumgarten como Johann J. Bodmer y Johann J. Breitinger enfatizaban el momento sensible de lo bello contra la focalización de Gottsched en la imitación de la naturaleza. Cf. Dieter Kliche. “Ästhetik“. Karlheinz Barck et al. (Eds.) Ästhetische Grundbegriffe Historisches Wörterbuch in sieben. Tomo I. Stuttgart/Weimar: Metzler, 2010, p. 331. Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 20 No podemos analizar aquí con detenimiento el conjunto de problemas provenientes de la historia natural clásica al que procuraba dar respuesta la propuesta kantiana de un uso regulativo de principios teleológicos. Concretamente la pretensión kantiana de considerar a la naturaleza como si la misma pro- cediese de acuerdo a determinada finalidad se hallaba orientada a conjurar el fantasma de los cambios morfológicos de carácter irreversible. Esto puede observarse con claridad en el tratamiento kantiano del problema de las razas. Pues, por medio de la idea de disposiciones germinales que se activaban de manera selectiva, Kant lograba retrotraer a los orígenes la totalidad de las transformaciones tempora- les que habían dado lugar a las diferentes razas de la especie humana. Para este tema, es necesario recurrir tanto a los escritos precríticos de Kant como a los nuevos análisis del problema que propone el autor desde mediados de los años 80. Cf. Immanuel Kant. “Von den verschiedenen Rassen der Menschen“. Kants gesammelte Schriften. Tomo II. Berlin: Gruyter, 1912, pp. 427-443; „Bestimmung des Begriffs einer Menschenrasse“. Kants gesammelte Schriften. Tomo VIII, Berlin: Gruyter, 1923, pp. 89–106; „Über den Gebrauch teleologischer Principien in der Philosophie. Kants gesammelte Schriften“. Tomo VIII, Berlin: Gruyter, 1923, pp. 157-184. 20 No podemos analizar aquí con detenimiento el conjunto de problemas provenientes de la historia natural clásica al que procuraba dar respuesta la propuesta kantiana de un uso regulativo de principios teleológicos. Concretamente la pretensión kantiana de considerar a la naturaleza como si la misma pro- cediese de acuerdo a determinada finalidad se hallaba orientada a conjurar el fantasma de los cambios morfológicos de carácter irreversible. Esto puede observarse con claridad en el tratamiento kantiano del problema de las razas. Pues, por medio de la idea de disposiciones germinales que se activaban de manera selectiva, Kant lograba retrotraer a los orígenes la totalidad de las transformaciones tempora- les que habían dado lugar a las diferentes razas de la especie humana. Para este tema, es necesario recurrir tanto a los escritos precríticos de Kant como a los nuevos análisis del problema que propone el autor desde mediados de los años 80. Cf. Immanuel Kant. “Von den verschiedenen Rassen der Menschen“. Kants gesammelte Schriften. Tomo II. Berlin: Gruyter, 1912, pp. 427-443; „Bestimmung des Begriffs einer Menschenrasse“. Kants gesammelte Schriften. Tomo VIII, Berlin: Gruyter, 1923, pp. 89–106; „Über den Gebrauch teleologischer Principien in der Philosophie. Kants gesammelte Schriften“. Tomo VIII, Berlin: Gruyter, 1923, pp. 157-184. 21 En Baumgarten esta tendencia se presentaba con cierta ambigüedad. Por una parte, Baumgarten encabezaba la reacción a la estética de escuela de Gottsched, que pretendía sostener aún el principio de la imitación. En este sentido, tanto Baumgarten como Johann J. Bodmer y Johann J. Breitinger enfatizaban el momento sensible de lo bello contra la focalización de Gottsched en la imitación de la naturaleza. Cf. Dieter Kliche. “Ästhetik“. Karlheinz Barck et al. (Eds.) Ästhetische Grundbegriffe Historisches Wörterbuch in sieben. Tomo I. Stuttgart/Weimar: Metzler, 2010, p. 331. Pero, por otra parte, Baumgarten llamaba la atención acerca del hecho de que la sensibilidad, a diferencia del enten- dimiento disociador, tenía que ver con representaciones en las cuales la experiencia se presentaba de manera unificada. En este sentido, la apariencia estética permitía hacer presente el todo del mundo en la medida en que la misma se hallaba organizada a partir de una conexión interna. Su rendimiento era así la fundamentación de la unidad. Cf. Eberhard Ostermann. Das Fragment. Geschichte einer ästhe- tischen Idee. München: Wilhelm Fink, 1991, pp. 26-7. 22 Joachim Ritter concibe estas funciones en términos compensatorios al interpretar el surgimiento de la estética y de la autonomía del arte como una respuesta a la pérdida de realidad a la que condujo el proceso moderno de objetivación de la naturaleza. A partir este proceso, la realidad dejaba de presen- tarse como un cosmos y el arte asumía la tarea de revivir el lenguaje extinguido del mismo. El surgi- miento de este interés en el ámbito de la estética es descripto por Ritter en los siguientes términos: 3. La obra de arte como naturaleza La apreciación con la que concluimos el parágrafo anterior resulta comprensible si se tiene en cuenta hasta qué punto la crisis de la historia natural clásica privó a la representa- ción artística de aquel objeto en relación con el cual la misma había definido su valor y legi- timado su existencia. De hecho, con la temporalización de las formas naturales y el desmo- ronamiento del acuerdo preformista entre causalidad mecánica y final, se tornó necesaria una completa redefinición del concepto de belleza. Pues, a partir de tales transformaciones epistémicas dejaba de ser posible identificar la belleza con ciertas propiedades de carácter objetivo y se volvía inminente el giro de la reflexión estética hacia el ámbito de la subjeti- vidad. Este hecho, que fue puesto en evidencia de manera paradigmática por David Hume, tuvo como resultado la invención de la estética, en tanto disciplina independiente, y el flo- recimiento de las perspectivas psicológico-efectuales. En consonancia con la propia defini- ción baumgarteniana de la estética como ciencia del conocimiento sensible24, las teorías efectuales procuraron sustituir la referencia de la obra a la belle nature por un análisis de carácter psicológico. En este sentido, afirmaba Friedrich Just Riedel que lo bello no era una característica intrínseca de las cosas que se llamarían bellas: “A diferencia de la perfección, la belleza no les corresponden de por sí a los objetos contemplados, sin relación a una enti- “Baumgarten adhiere a la definición de Chr. Wolff, según la cual lo bello es lo que place (quod placet dicitur pulchrum). Pero Baumgarten amplía a su vez la estética como teoría del arte bello y la trans- forma en “ciencia de lo bello”. Lo que se hace presente de manera subjetiva en el bello pensamiento, es el mundo en su perfección (perfectio)”. Joachim Ritter. „Ästhetik, ästhetisch“. Joachim Ritter at al. (Eds.) Historisches Wörterbuch der Philosophie. Tomo I. Darmstadt: Wissenschaftliche Buchgesellschaft, 1971, p. 569. En el contexto de la metafísica de Baumgarten, la perfección desig- naba la armonía y el orden en la composición de las partes que hacían posible la comprensión del mundo como aquella unidad por medio de la cual dios daba a conocer su perfección. Ritter atribuye un sentido positivo a esta compensación por la unidad perdida entre el sujeto y el mundo que se da en el terreno artístico en la medida en que el autor sostiene la necesidad del proceso de modernización. 2. Teleología natural y belleza artística Pero, por otra parte, Baumgarten llamaba la atención acerca del hecho de que la sensibilidad, a diferencia del enten- dimiento disociador, tenía que ver con representaciones en las cuales la experiencia se presentaba de manera unificada. En este sentido, la apariencia estética permitía hacer presente el todo del mundo en la medida en que la misma se hallaba organizada a partir de una conexión interna. Su rendimiento era así la fundamentación de la unidad. Cf. Eberhard Ostermann. Das Fragment. Geschichte einer ästhe- tischen Idee. München: Wilhelm Fink, 1991, pp. 26-7. 22 Joachim Ritter concibe estas funciones en términos compensatorios al interpretar el surgimiento de la estética y de la autonomía del arte como una respuesta a la pérdida de realidad a la que condujo el proceso moderno de objetivación de la naturaleza. A partir este proceso, la realidad dejaba de presen- tarse como un cosmos y el arte asumía la tarea de revivir el lenguaje extinguido del mismo. El surgi- miento de este interés en el ámbito de la estética es descripto por Ritter en los siguientes términos: 138 Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 María Verónica Galfione La promesa de lo bello: consideraciones acerca de la... palabras, las nuevas tendencias estéticas colocaban a la propia dimensión artística en el lugar de la antigua naturaleza perdida. Puesto que legitimaban la representación artística por medio de cualidades que la convertían de suyo en una instancia capaz de superar la contin- gencia23 a la que había conducido la crisis de la historia natural. Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 “Baumgarten adhiere a la definición de Chr. Wolff, según la cual lo bello es lo que place (quod placet dicitur pulchrum). Pero Baumgarten amplía a su vez la estética como teoría del arte bello y la trans- forma en “ciencia de lo bello”. Lo que se hace presente de manera subjetiva en el bello pensamiento, es el mundo en su perfección (perfectio)”. Joachim Ritter. „Ästhetik, ästhetisch“. Joachim Ritter at al. (Eds.) Historisches Wörterbuch der Philosophie. Tomo I. Darmstadt: Wissenschaftliche Buchgesellschaft, 1971, p. 569. En el contexto de la metafísica de Baumgarten, la perfección desig- naba la armonía y el orden en la composición de las partes que hacían posible la comprensión del mundo como aquella unidad por medio de la cual dios daba a conocer su perfección. Ritter atribuye un sentido positivo a esta compensación por la unidad perdida entre el sujeto y el mundo que se da en el terreno artístico en la medida en que el autor sostiene la necesidad del proceso de modernización. Como Schiller, Ritter considera: “que la escisión... no sólo es corregible estéticamente sino también una condición necesaria y persistente de la cultura” y que “no es adecuado que la sociedad existente de las necesidades y de la utilidad se interrumpa en algún momento”. Ibídem pp. 568s. 23 El término contingencia remite, en este contexto, a “un modo deficitario de sentido, coherencia y orden final” que es caracterizado “en función de determinaciones negativas como no necesidad, irre- gularidad, dependencia, fugacidad, mensurabilidad, previsibilidad.” Werner Frick. Providenz und Kontingenz. Untersuchungen zur Schicksalssemantik im deutschen und europäischen Roman des 17. und 18. Jahrhunderts I. Tübingen: Niemeyer, 1988, p. 11. 24 Alexander G. Baumgarten. Aesthetica, Hildesheim: Georg Olms, 1970, par. 14; Cf. Ma. Jesús Soto Bruna. “La Aesthetica de Baumgarten y sus antecedentes leibnicianos”. Anuario Filosófico, vol. 20, 1987, pp. 181–190. 3. La obra de arte como naturaleza Como Schiller, Ritter considera: “que la escisión... no sólo es corregible estéticamente sino también una condición necesaria y persistente de la cultura” y que “no es adecuado que la sociedad existente de las necesidades y de la utilidad se interrumpa en algún momento”. Ibídem pp. 568s. 23 El término contingencia remite, en este contexto, a “un modo deficitario de sentido, coherencia y orden final” que es caracterizado “en función de determinaciones negativas como no necesidad, irre- gularidad, dependencia, fugacidad, mensurabilidad, previsibilidad.” Werner Frick. Providenz und Kontingenz. Untersuchungen zur Schicksalssemantik im deutschen und europäischen Roman des 17. und 18. Jahrhunderts I. Tübingen: Niemeyer, 1988, p. 11. 24 Alexander G. Baumgarten. Aesthetica, Hildesheim: Georg Olms, 1970, par. 14; Cf. Ma. Jesús Soto Bruna. “La Aesthetica de Baumgarten y sus antecedentes leibnicianos”. Anuario Filosófico, vol. 20, 1987, pp. 181–190. 139 Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 María Verónica Galfione La promesa de lo bello: consideraciones acerca de la... dad que los percibe. La belleza es de naturaleza relativa y solo una relación en la cual algo nos gusta.”25 En un primer momento esta nueva perspectiva fue duramente criticada por la antigua estética de escuela, la cual acusaba a las tendencias efectuales de incorporar al ámbito artís- tico elementos que resultaban irreductibles a la dimensión racional –esto es, elementos tales como la expresión, la sensibilidad o la imaginación. En este sentido, al presentar su traduc- ción de Las artes reducidas a un único principio (1754) de Batteaux, Johann Christoph Gottsched señalaba que la difusión de esta obra se hallaba orientada a combatir “la corrien- te de doctrinas equivocadas que no quieren buscar la naturaleza del arte poético en la imi- tación, sino en las meras expresiones salvajes de una imaginación sin reglas, que llaman estética.”26 Pero no fueron estas las únicas críticas de las que fueron objeto los nuevos desarrollos de la estética efectual. Pues, también las generaciones más jóvenes miraron con desagrado las tendencias instrumentales que se hallaban contenidas en la consideración de la repre- sentación artística desde el punto de vista del espectador. Para tales perspectivas, ya no era la confusión que introducía el primado de la imaginación lo que resultaba preocupante, sino más bien la posibilidad de rebajar las formas artísticas al estatuto de entretenimiento o de reorientarlas hacia una formación de carácter moral. 25 Friedrich Just Riedel. Philosophische Bibliothek. Tomo 1. Wien/Jena: Gebauer, 1769, p. 116. 26 Johann Christoph Gottsched. Das Neueste aus der anmuthigen Gelehrsamkeit (Auszug aus der Herrn Batteux schönen Künsten aus dem einzigen Grundsatze der Nachahmung), Leipzig: Breithopf, 1754, p. 464. 27 Cf. John H. Zammito. „Herder, Kant, Spinoza und die Ursprünge des deutschen Idealismus“. Herder und die Philosophie des deutschen Idealismus. Marion Heinz. (Ed.) Amsterdam y Atlanta: Rodopi, 1997, pp. 107–145; John H. Zammito. The Genesis of Kant’s Critique of Judgment. Chicago: University of Chicago Press, 1992, pp. 189–99. Friedrich Just Riedel. Philosophische Bibliothek. Tomo 1. Wien/Jena: Gebauer, 1769, p. 116. 27 Cf. John H. Zammito. „Herder, Kant, Spinoza und die Ursprünge des deutschen Idealismus“. Herder und die Philosophie des deutschen Idealismus. Marion Heinz. (Ed.) Amsterdam y Atlanta: Rodopi, 1997, pp. 107–145; John H. Zammito. The Genesis of Kant’s Critique of Judgment. Chicago: University of Chicago Press, 1992, pp. 189–99. 3. La obra de arte como naturaleza Sin embargo, las nuevas perspectivas estéticas ya no contaban con ninguna referencia externa a la hora de fundamentar la rele- vancia de la esfera estética. Por este motivo, las mismas debieron concentrarse en los momentos no miméticos de la representación artística a los fines de sostener la amenazada artisticidad de la representación artística. En este sentido es posible leer tanto la tendencia del Sturm und Drang a enfatizar el carácter poiético de la creación artística como la focali- zación del clasicismo de Weimar en los aspectos formales de la representación artística. p p En esta oportunidad no podemos analizar con detenimiento la significación histórico- filosófica que asumió el arte en tanto expresión de una naturaleza de carácter poiético o pro- ductivo. Al respecto, baste realizar aquí una breve referencia al particular empleo de las teo- rías antipreformistas que es posible encontrar en el marco de la obra temprana de Herder. Dicho en pocas palabras, las nuevas concepciones del fenómeno de la generación –tales como las de Wolff– le ofrecieron al joven filósofo un modelo adecuado a los fines de dar cuenta tanto de la objetividad de la representación artística como de las consecuencias esté- ticas del proceso de temporalización27. Por medio de la referencia a una naturaleza de carác- ter productivo, Herder podía desechar aquellas interpretaciones clasicistas que establecían un lazo estático y representativo entre las formas artísticas y la apariencia visible de la natu- raleza devenida. Puesto que, así consideradas, estas últimas asumían un carácter secundario o meramente superficial, mientras que las formas artísticas originales se presentaban como 140 Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 María Verónica Galfione La promesa de lo bello: consideraciones acerca de la... expresión directa de la propia instancia productiva. Al igual que las formas orgánicas natu- rales, ellas eran “miembros de este cuerpo y todos los caracteres y modos de pensar, rasgos para este espíritu.”28 De esta manera, era posible resguardar la verdad de la representación artística y garan- tizar, a su vez, la irreductibilidad de las obras particulares a criterios normativos generales. Puesto, liberado de las reglas abstractas y superficiales de la poética clásica y de toda mediación de naturaleza reflexiva, el sentimiento del artista enlazaba de manera inmediata con la plasticidad infinita del principio originario: “¡Afortunado hijo de los dioses por su empresa! 28 Johann Gottfried Herder. Shakespeare. Trad. Juan Probst Universidad Nacional de Buenos Aires. Facultad de Filosofía y Letras. Instituto de Literatura Anglogermánica, Buenos Aires, 1949, p. 41. El interés de Herder por las nuevas ciencias naturales se acentúa algunos años más tarde, en el marco de Ideas. Cf. Johann Gottfried Herder. Ideen zur Philosophie der Geschichte der Menschheit. Ed. W. Pross. München/Wien: Carl Hanser, 2002, pp. 172 /4. Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 30 Karl Philipp Moritz. Schriften zur Ästhetik und Poetik. Kritische Aufgaben. Tübingen: Walter de Gruyter, 1962, p. 3. 29 Herder, Johann Gottfried. Shakespeare. Op. Cit., p. 25. Ä 3. La obra de arte como naturaleza 1 (2014): 131-153 La promesa de lo bello: consideraciones acerca de la... María Verónica Galfione va mimética sin renunciar, por ello, al carácter absoluto de las mismas. Esto es, la conside- ración de la obra de arte como “conformidad a fin interna”31 ofrecía la posibilidad de fun- damentar la objetividad de la representación artística en su propia independencia estructu- ral. Ya que, a diferencia de los artefactos, la estructura interna de la obra arte no resultaba dependiente de un concepto que preexistiese a la propia instancia de la representación.32 Entre las partes y el todo se establecía así una relación de acción recíproca, de tal manera que la perfección de la obra ya no se desprendía de finalidades externas y podía ser consi- derada, en este sentido, de carácter absoluto. Ahora bien, por medio de la identificación de la estructura de la obra con la consuma- ción interna (in sich selbst vollendet), Moritz lograba atribuirle a la representación artística una función mediadora entre el hombre y la naturaleza y concederle, así, no solo un valor artístico sino también una significación particular de carácter filosófico. Pues en tanto tota- lidad cerrada sobre sí misma, la obra de arte se presentaba como una imagen sensible “del gran todo de la naturaleza que nos circundaba”; el cual, pese a “tener en sí mismo su fin final y existir para su propia gloria”, no resultaba inmediatamente perceptible para nosotros. En este contexto, la determinación de la obra en tanto consumación interna permitía liberar a las formas naturales de su contingencia empírica –la Hülle der Existenz, según afirma Moritz en “Über die bildende Nachahmung des Schönen”33– e integrarlas en una totalidad inmediatamente visible que, en cuanto tal, se convertía en un medio capaz de incentivar nuestro sentido para la totalidad. “Considerado de esta forma”, concluía Moritz en “Bestimmung des Zwecks einer Theorie der schönen Künste”, lo bello “puede ser verdade- ramente útil en la medida en que afina nuestra capacidad de percepción del orden y la armo- nía y eleva nuestro espíritu por sobre las pequeñeces porque ella nos permite mirar clara- mente todo en el todo y en relación al todo.”34 La obra de arte, señalaba Moritz en “Über die bildende Nachahmung des Schönen”, “puede reflejar libremente las relaciones del gran 33 Ibid. p. 74. 32 A los fines de explicar esta idea, Moritz recurría a la mitología antigua. Haciendo un uso despecti- vo del término alegoría, Moritz había cuestionado en 1791 la tendencia a interpretar las figuras mito- lógicas como meras representaciones figurativas de conceptos abstractos y generales. Del mismo modo en que en el terreno de la fantasía, señalaba Moritz, Júpiter significaba simplemente Júpiter, las obras de arte verdaderas no podían significar algo que se encontrase más allá de sí mismas. Ellas “son algo acabado y perfecto en sí mismo, que existe por su propia voluntad, y cuyo valor radica en sí mismo y en la relación armónica de sus partes” y no, como pretendía la decadente tradición del arte moderno, un conglomerado de elementos sensibles que servía de ocasión para la manifestación de un pensamiento de carácter conceptual. De ser así, las obras se asimilarían a meros jeroglíficos o letras de imprenta que “pueden estar conformados como quieran, siempre y cuando designen aquello en lo que haya de pensarse”, como sostiene Moritz en “Götterlehre oder Mythologische Dichtungen der Alten”. Ibid. p. 196. 31 Ibídem p. 6. 32 A los fines de explicar esta idea, Moritz recurría a la mitología antigua. Haciendo un uso despecti- vo del término alegoría, Moritz había cuestionado en 1791 la tendencia a interpretar las figuras mito- lógicas como meras representaciones figurativas de conceptos abstractos y generales. Del mismo modo en que en el terreno de la fantasía, señalaba Moritz, Júpiter significaba simplemente Júpiter, las obras de arte verdaderas no podían significar algo que se encontrase más allá de sí mismas. Ellas “son algo acabado y perfecto en sí mismo, que existe por su propia voluntad, y cuyo valor radica en sí mismo y en la relación armónica de sus partes” y no, como pretendía la decadente tradición del arte moderno, un conglomerado de elementos sensibles que servía de ocasión para la manifestación de un pensamiento de carácter conceptual. De ser así, las obras se asimilarían a meros jeroglíficos o letras de imprenta que “pueden estar conformados como quieran, siempre y cuando designen aquello en lo que haya de pensarse”, como sostiene Moritz en “Götterlehre oder Mythologische Dichtungen der Alten”. Ibid. p. 196. 33 Ibid. p. 74. En “Die metaphysische Schönheitslinie” Moritz concretiza esta idea en los siguientes términos: “el poeta corta los hilos por medio de los cuales los acontecimientos podrían tener una incli- nación más allá de sí, él omite lo que interviene en otra esfera de los acontecimiento, reúne causa y efecto como si ellas estuvieran en el curso habitual de las cosas. Vemos reunidos en un breve espacio de tiempo, una multiplicidad de acontecimientos que se suceden, que suceden a lo largo de siglos.” Ibid. p.153. 34 Ibid. p. 122. Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 3. La obra de arte como naturaleza –señala Herder refiriéndose a Shakespeare– ¡Precisamente lo nuevo, lo primero, lo totalmente distinto demuestra la fuerza primitiva de su vocación!”29 Sin embargo, no solo se aseguraba de esta forma el estatuto ontológico de la representación artística. Pues, al invertir la relación clasicista entre arte y naturaleza, para transformar a esta última en una instancia de carácter productivo, Herder dotaba a la creación artística de una significación filosófica de carácter singular. Ya que, en tanto manifestación directa de una fuerza genéti- ca originaria, la esfera artística hacía presente en la historia el recuerdo vivo de una antigua naturaleza perdida. p Pero no solo Herder convirtió a la representación artística en un recurso adecuado a las fines de superar la experiencia moderna de la contingencia. Ya que también el intento clasi- cista por depurar a los aspectos formales de la obra de arte de su carácter instrumental –y por ende relativo–, ubicó a estos últimos al límite de su existencia sensible y dio lugar, de esta forma, a una metafísica de la obra de arte autónoma. Esta última tendencia alcanzó su máxima expresión en el pensamiento de Moritz quien, en “Über den Begriff des in sich selbst Vollendeten”, contraponía el valor relativo de los objetos útiles al carácter absoluto de los objetos bellos. En clara referencia a las estéticas efectuales, Moritz señalaba allí: El objeto meramente útil no es un todo o algo en sí mismo completo, sino que lo llega a ser recién cuando alcanza su fin en mí o se completa en mí. Pero en la contemplación de lo bello, saco el fin de mí y lo coloco en el objeto mismo: lo contemplo como algo que no se completa en mí, sino que es en sí mismo completo; como algo, por ende que constituye en sí mismo un todo y me con- cede placer en aras de sí mismo.30 Como puede constatarse a partir del pasaje citado, la autonomización de la obra de arte con respecto a toda posible utilidad o referencia subjetiva se ofrecía, para Moritz, como una estrategia adecuada para sostener la dignidad de la belleza en un contexto en el cual la tota- lidad de los fines concebibles habían devenido relativos. Puesto que la contraposición entre lo bello y lo útil permitía desligar las cualidades estético-representativas de toda perspecti- 141 Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 31 Ibídem p. 6. 32 33 Ibid. p. 74. En “Die metaphysische Schönheitslinie” Moritz concretiza esta idea en los siguientes términos: “el poeta corta los hilos por medio de los cuales los acontecimientos podrían tener una incli- nación más allá de sí, él omite lo que interviene en otra esfera de los acontecimiento, reúne causa y efecto como si ellas estuvieran en el curso habitual de las cosas. Vemos reunidos en un breve espacio de tiempo, una multiplicidad de acontecimientos que se suceden, que suceden a lo largo de siglos.” Ibid. p.153. 3. La obra de arte como naturaleza En “Die metaphysische Schönheitslinie” Moritz concretiza esta idea en los siguientes términos: “el poeta corta los hilos por medio de los cuales los acontecimientos podrían tener una incli- nación más allá de sí, él omite lo que interviene en otra esfera de los acontecimiento, reúne causa y efecto como si ellas estuvieran en el curso habitual de las cosas. Vemos reunidos en un breve espacio de tiempo, una multiplicidad de acontecimientos que se suceden, que suceden a lo largo de siglos.” Ibid. p.153. 34 Ibid. p. 122. 142 Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 María Verónica Galfione La promesa de lo bello: consideraciones acerca de la... todo de la naturaleza en su completa extensión”35 y “comprende en sí todo lo que la reali- dad hubiera debido destruir si ella no hubiese tenido el poder de desprenderlo de sí y pre- sentarlo fuera de sí de manera figurativa.”36 35 Ibid. p. 77. 36 Ibid. p. 90. 37 Kant había negado ya en la Crítica de la razón pura (1781) la posibilidad de una ciencia de lo bello: “Los alemanes son los únicos que emplean hoy la palabra estética para designar lo que otros denomi- nan crítica del gusto. Tal empleo se basa en una equivocada esperanza concebida por el destacado crí- tico Baumgarten. Esta esperanza consistía en reducir la consideración crítica de lo bello a principios racionales y en elevar al rango de ciencia las reglas de dicha consideración crítica. Pero este empeño es vano, ya que las mencionadas reglas o criterios son, de acuerdo con sus fuentes, meramente empí- ricas y, consiguientemente, jamás pueden servir para establecer leyes a priori por las que debería regir- se nuestro juicio del gusto.” Immanuel Kant. Crítica de la razón pura. Trad. Ribas, P. Madrid: Alfaguara, 1998, A 22/ B36. Sin embargo, un tratamiento acabado del problema se haría esperar hasta la publicación de la Crítica de la facultad de juzgar. También es establecida en la Crítica de la razón pura la distinción fundamental entre estética en el sentido de teoría de la sensibilidad y estética en el sentido de crítica del gusto. Cf. Ibídem A 21/ B35/6. 38 De este punto de vista, no solo se cerraba la posibilidad de remitir el placer que deparaba el arte al objeto imitado sino también de interpretarlo como el resultado de carácter técnicamente logrado de la imitación. Cf. Dieter Henrich. „Kunst und Natur in der idealistischen Ästhetik“. H. Robert Jauß (Ed.) Nachahmung und Ilussion. München: Wilhelm Fink, 1969, p. 129. 39 Moritz postulaba la existencia de una fuerza activa (tätige Kraft o Tatkraft) que subyacía tanto a la imaginación como a la sensibilidad estética. Karl Philipp Moritz. Op. Cit. pp. 75-87. 38 De este punto de vista, no solo se cerraba la posibilidad de remitir el placer que deparaba el arte al objeto imitado sino también de interpretarlo como el resultado de carácter técnicamente logrado de la imitación. Cf. Dieter Henrich. „Kunst und Natur in der idealistischen Ästhetik“. H. Robert Jauß (Ed.) Nachahmung und Ilussion. München: Wilhelm Fink, 1969, p. 129. 39 Moritz postulaba la existencia de una fuerza activa (tätige Kraft o Tatkraft) que subyacía tanto a la imaginación como a la sensibilidad estética. Karl Philipp Moritz. Op. Cit. pp. 75-87. 37 Kant había negado ya en la Crítica de la razón pura (1781) la posibilidad de una ciencia de lo bello: “Los alemanes son los únicos que emplean hoy la palabra estética para designar lo que otros denomi- nan crítica del gusto. Tal empleo se basa en una equivocada esperanza concebida por el destacado crí- tico Baumgarten. Esta esperanza consistía en reducir la consideración crítica de lo bello a principios racionales y en elevar al rango de ciencia las reglas de dicha consideración crítica. Pero este empeño es vano, ya que las mencionadas reglas o criterios son, de acuerdo con sus fuentes, meramente empí- ricas y, consiguientemente, jamás pueden servir para establecer leyes a priori por las que debería regir- se nuestro juicio del gusto.” Immanuel Kant. Crítica de la razón pura. Trad. Ribas, P. Madrid: Alfaguara, 1998, A 22/ B36. Sin embargo, un tratamiento acabado del problema se haría esperar hasta la publicación de la Crítica de la facultad de juzgar. También es establecida en la Crítica de la razón pura la distinción fundamental entre estética en el sentido de teoría de la sensibilidad y estética en el sentido de crítica del gusto. Cf. Ibídem A 21/ B35/6. 4. El escepticismo estético kantiano La desconfianza de Kant con respecto a los proyectos estético-culturales mencionados hasta aquí remitía a motivos similares a los que habían resultado determinantes a la hora de rechazar la existencia de una intencionalidad objetiva en el plano natural.37 Desde el punto de vista kantiano, la “conformidad a fin” de las formas artísticas solo podía ser explicada en términos objetivos a partir de la postulación de una subjetividad creadora que dispusiera las partes en función de un fin preconcebido. Pero entonces, señalaba Kant, ya no era posible establecer una distinción entre las obras de arte y los demás productos del arte humano pues- to que en ambas oportunidades se establecía una articulación de las partes y el todo que res- pondía a un concepto preexistente con respecto a los propios elementos constitutivos. Esto es, en tanto productos intencionales, las obra de arte debían ser juzgadas en función de obje- tivos externos a la instancia de la representación y de acuerdo con procedimientos similares a aquellos que eran utilizados en el caso de los artefactos mecánicos. Un juicio semejante, sin embargo, solo podía remitir a la posible utilidad o perfección de las obras de arte y care- cía de interés en el momento de establecer el valor específicamente estético de las mismas.38 Ciertamente, existía la posibilidad de neutralizar la intencionalidad subjetiva por medio de su remisión a la fuerza productiva natural. Esto era lo que había hecho en cierta forma el propio Moritz, por medio de la “teoría del genio”39, y antes que él, el joven Herder y los demás integrantes del Sturm und Drang. Sin embargo, dicha estrategia no hacía más que 39 Moritz postulaba la existencia de una fuerza activa (tätige Kraft o Tatkraft) que subyacía tanto a la imaginación como a la sensibilidad estética. Karl Philipp Moritz. Op. Cit. pp. 75-87. 143 Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 La promesa de lo bello: consideraciones acerca de la... María Verónica Galfione proyectar el problema más atrás, puesto que entonces era necesario dar cuenta del modo en que la propia naturaleza producía “conforme a fin”. Desde el punto de vista de Kant, Herder simplemente evadía este problema por medio de la identificación de la naturaleza con una fuerza generativa que era capaz de manifestarse en infinitas formas singulares. 40 Elías Palti. Op. cit; John H. Zammito. „Herder, Kant, Spinoza und die Ursprünge des deutschen Idealismus“. Op. Cit. pp. 107 – 145. 41 Cf. Immanuel Kant. „Recensionen von J.G. Herders Ideen zur Philosophie der Geschichte der Menschheit“. Kants gesammelte Schriften. Tomo VIII. Berlin: Walter de Gruyter, 1923, pp. 62-3 (Utilizo la traducción de Roldan Panadero, Concha y Rodríguez Aramayo, Roberto. Cf. Kant, Immanuel. “Recensiones sobre la obra de Herder Ideas para una filosofía de la historia de la humani- dad”. Ideas para una historia universal en clave cosmopolita y otros escritos sobre Filosofía de la Historia. Madrid: Tecnos, 1994, p. 51-2). También en Sobre el uso de principios teleológicos Kant cri- ticaba esta tendencia a unificar aquellas fuerzas que gozaban de algún tipo de correlato empírico en una fuerza general: “Algunos han creído que tenía que suponerse una única fuerza fundamental para explicar la unidad de la sustancia e incluso han pretendido conocerla tan solo con nombrarla con el título común de diversas fuerzas fundamentales…” Immanuel Kant. „Über den Gebrauch teleologi- scher Principien in der Philosophie“. Op. cit. p. 180 (Utilizo la traducción de Sánchez Madrid, Nuria. Cf. Kant, Immanuel. “Sobre el uso de principios teleológicos en la filosofía“. Logos. Anales del Seminario de Metafísica. Vol. 37 (2004), pp. 7-47, p. 27). El pasaje se aplica perfectamente a la idea de fuerza generativa de Herder. Sin embargo, es posible suponer que el mismo se dirigía en verdad a cuestionar la teoría de la Representación de Reinhold, en la medida en que la misma reducía las fuer- zas del alma a “la fuerza de representación del mundo.” 42 l di i d l ibilid d d hi i Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 4. El escepticismo estético kantiano Pues, una perspectiva semejante introducía una serie de supuestos que frustraban el uso normal de nuestras facultades cognitivas y que reubicaban al hombre en el marco del orden natural. La primera de estas consecuencias se seguía, según sostenía Kant en su reseña de Ideas, de la plasticidad infinita que le era atribuida por Herder a la fuerza genética originaria. Pues, una vez asumida la existencia de una fuerza que era capaz de intervenir de manera directa en el ámbito de la historia, ya no resultaba posible sostener la necesidad de las clasificacio- nes científicas de los seres naturales ni asegurar la racionalidad intrínseca del mundo natu- ral40. En este registro es posible leer la afirmación kantiana con respecto a la conveniencia de limitar el poder creativo de la fuerza genética originaria: si la causa que organiza desde dentro estuviera limitada por su naturaleza a un cierto número y grado de diferencias en el desarrollo de su criatura (organización según la cual dicha causa no sería libre para modelar conforme a otro patrón en caso de modificarse las circunstancias), podría denominarse a esta determinación natural de la naturaleza configuradora de “gérmenes” o “dis- posiciones originarias”, sin considerar por ello a los primeros como dispositivos colocados en un principio que solo se despliegan por casualidad y aisladamente cual capullos (como en el sistema de la evolución), sino como meras limitaciones inexplicables de una facultad autoconfiguradora que tampoco podríamos explicar o hacer comprensible.41 si la causa que organiza desde dentro estuviera limitada por su naturaleza a un cierto número y grado de diferencias en el desarrollo de su criatura (organización según la cual dicha causa no sería libre para modelar conforme a otro patrón en caso de modificarse las circunstancias), podría denominarse a esta determinación natural de la naturaleza configuradora de “gérmenes” o “dis- posiciones originarias”, sin considerar por ello a los primeros como dispositivos colocados en un principio que solo se despliegan por casualidad y aisladamente cual capullos (como en el sistema de la evolución), sino como meras limitaciones inexplicables de una facultad autoconfiguradora que tampoco podríamos explicar o hacer comprensible.41 Sin embargo, la concepción herderiana de la naturaleza no solo abandonaba el mundo natural al imperio de fuerzas irracionales sino que tendía a negar, además, la independencia del juicio moral con respecto a las determinaciones naturales.42 Por cierto, no se trataba aquí Palti. Op. cit; John H. Zammito. 4. El escepticismo estético kantiano „Herder, Kant, Spinoza und die Ursprünge des deutschen mus“. Op. Cit. pp. 107 – 145. 42 Con respecto a la discusión que Kant sostuvo con Herder en torno a la posibilidad de una “historia natural de la humanidad”, se puede consultar Manfred Riedel. Cf. Manfred Riedel. “Historizismus und 144 Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 La promesa de lo bello: consideraciones acerca de la... María Verónica Galfione de que Herder postulase una concepción materialista de la historia ni del hecho de que el mismo redujese al hombre a su dimensión material. De hecho, su perspectiva histórica y antropológica se hallaba atravesada, más bien, por una fuerte tendencia hacia la espirituali- zación de la totalidad de las disposiciones y fuerzas naturales. Sin embargo, justamente por este hecho, Herder interpretaba el devenir histórico como manifestación de una fuerza tras- histórica y clausuraba, de esta forma, la posibilidad de acciones humanas que encontraran su fundamento en la incondicionalidad de la buena voluntad. Pero si la identificación de la historia empírica de la naturaleza con su historia en un sentido metafísico privaba de fundamento al conocimiento científico y anula la posibilidad de una moral de carácter prescriptivo, el simple abandono del concepto de lo bello tenía tam- bién su precio. Puesto que suponía la renuncia a la única experiencia que parecía confirmar la posibilidad de una aplicación concreta de las diversas fuerzas subjetivas.43 Pues, dejando de lado el problema de la racionalidad práctico-moral y limitándonos aquí al ámbito teóri- co, ¿no se derivaba el placer estético del misterioso acuerdo entre lo particular y lo general que era posible encontrar en ciertas configuraciones artísticas o naturales? Y este acuerdo inexplicable, ¿no alentaba enormes expectativas con respecto a la posibilidad de una con- cordancia secreta entre nuestras capacidades cognitivas y la disposición efectiva de las for- mas naturales? Más aun, ¿no indicaba la bella apariencia, en el caso de la belleza natural, la “disposición a fin” de la naturaleza en su conjunto? Como es sabido, Kant no planteó de manera explícita ninguna de estas preguntas ni se expresó a favor de la necesidad de incorporar un suplemento para el correcto funciona- miento del sistema crítico. Kritizismus. Kants Streit mit G. Forster und J.G. Herder“. Urteilskraft und Vernunft. Kants ursprüng- liche Fragestellung. Frankfurt am Main: Suhrkamp, 1989, pp. 148-170. 43 Un análisis semejante podría realizarse también en lo que respecta a los fines morales. Menke estu- dia este problema a partir del concepto de lo sublime. Cf. Christoph Menke. “Subjektivität“. Karlheinz Barck et al. (Eds.) Ästhetische Grundbegriffe Historisches Wörterbuch in sieben. Tomo V. Stuttgart/Weimar: Metzler, 2010, pp. 760s. 44 Cf. Ibídem pp. 760s. Un análisis levemente más radical presenta Szczepanski. Para este autor, el recurso a lo bello sería una muestra del fracaso del proyecto crítico en tanto que la necesidad de lo bello se desprendería de la incapacidad de la razón para fundar algo positivo en su relación crítica con- sigo misma. Cf. Jens Szczepanski. Subjektivität und Ästhetik. Gegendiskurse zur Metaphysik des Subjekts im ästhetischen Denken bei Schlegel, Nietzsche und de Man. Bielefeld: Transcript, 2007, p. 104. A partir de aquí, la empresa crítica se transformaría en el intento por reglar de manera racional el acceso racionalmente necesario a lo suprasensible. En este hecho podría vislumbrarse la posición intermedia que ocupa Kant entre la temprana modernidad y el idealismo. Pues, el funcionamiento de la razón kantiana resultaría dependiente de un reaseguro externo, como en el caso de los gérmenes pre- formados. Sin embargo, también Kant procuraría por momentos ir más allá de esta solución de com- Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 44 Cf. Ibídem pp. 760s. Un análisis levemente más radical presenta Szczepanski. Para este autor, el recurso a lo bello sería una muestra del fracaso del proyecto crítico en tanto que la necesidad de lo bello se desprendería de la incapacidad de la razón para fundar algo positivo en su relación crítica con- sigo misma. Cf. Jens Szczepanski. Subjektivität und Ästhetik. Gegendiskurse zur Metaphysik des Subjekts im ästhetischen Denken bei Schlegel, Nietzsche und de Man. Bielefeld: Transcript, 2007, p. 104. A partir de aquí, la empresa crítica se transformaría en el intento por reglar de manera racional el acceso racionalmente necesario a lo suprasensible. En este hecho podría vislumbrarse la posición intermedia que ocupa Kant entre la temprana modernidad y el idealismo. Pues, el funcionamiento de la razón kantiana resultaría dependiente de un reaseguro externo, como en el caso de los gérmenes pre- formados. Sin embargo, también Kant procuraría por momentos ir más allá de esta solución de com- f f p g liche Fragestellung. Frankfurt am Main: Suhrkamp, 1989, pp. 148-170. 43 Un análisis semejante podría realizarse también en lo que respecta a los fines morales. Menke estu- dia este problema a partir del concepto de lo sublime. Cf. Christoph Menke. “Subjektivität“. Karlheinz Barck et al. (Eds.) Ästhetische Grundbegriffe Historisches Wörterbuch in sieben. Tomo V. Stuttgart/Weimar: Metzler, 2010, pp. 760s. 44 Cf. Ibídem pp. 760s. Un análisis levemente más radical presenta Szczepanski. Para este autor, el recurso a lo bello sería una muestra del fracaso del proyecto crítico en tanto que la necesidad de lo bello se desprendería de la incapacidad de la razón para fundar algo positivo en su relación crítica con- sigo misma. Cf. Jens Szczepanski. Subjektivität und Ästhetik. Gegendiskurse zur Metaphysik des Subjekts im ästhetischen Denken bei Schlegel, Nietzsche und de Man. Bielefeld: Transcript, 2007, p. 104 A partir de aquí la empresa crítica se transformaría en el intento por reglar de manera racional el liche Fragestellung. Frankfurt am Main: Suhrkamp, 1989, pp. 148-170. 43 Un análisis semejante podría realizarse también en lo que respecta a los fines morales. Menke estu- dia este problema a partir del concepto de lo sublime. Cf. Christoph Menke. “Subjektivität“. Karlheinz Barck et al. (Eds.) Ästhetische Grundbegriffe Historisches Wörterbuch in sieben. Tomo V. Stuttgart/Weimar: Metzler, 2010, pp. 760s. Kritizismus. Kants Streit mit G. Forster und J.G. Herder“. Urteilskraft und Vernunft. Kants ursprüng- liche Fragestellung. Frankfurt am Main: Suhrkamp, 1989, pp. 148-170. 43 Un análisis semejante podría realizarse también en lo que respecta a los fines morales. Menke estu- dia este problema a partir del concepto de lo sublime. Cf. Christoph Menke. “Subjektivität“. Karlheinz Barck et al. (Eds.) Ästhetische Grundbegriffe Historisches Wörterbuch in sieben. Tomo V. Stuttgart/Weimar: Metzler, 2010, pp. 760s. 44 Cf Ibídem pp 760s Un análisis levemente más radical presenta Szczepanski Para este autor el 4. El escepticismo estético kantiano Pues, si esto era posible, también era factible interpretar el placer que deparaba la contemplación de lo bello como un indicio a favor de la “disposición a fin” de nuestras diversas capacidades cognitivas, sin que fuese necesario presuponer para ello la existencia de entidades bellas, que pudiesen poner en peli- gro la totalidad del edificio crítico.45 Reconstruir en detalle el análisis que realizaría Kant del juicio estético no se encuentra dentro de los objetivos fijados en este trabajo. Al respecto, nos limitamos a llamar la aten- ción aquí acerca de papel fundamental que desempeñaba en este contexto el concepto kan- tiano de “juicio reflexionante”. Pues, era justamente este tipo de juicios el que le permitía a Kant vincular el placer estético con la idea de una experiencia en la cual el sujeto se volvía capaz de experimentarse a sí mismo como poseedor de facultades efectivamente cogniti- vas.46 La argumentación de Kant en este punto consistía en mostrar que aquello que expe- promiso con el mundo teológico y garantizar así la autofundación de la razón. Desde otra perspectiva, también Ginsborg y Zuckert reconocen la función central que desempeña el principio a priori del jui- cio reflexionante en el marco del pensamiento crítico. Cf. Hannah Ginsborg. The role of taste in Kant´s Theory of cognition. New York: Garland, 1990, cap. 4; Rachel Zuckert. Kant on Beauty and Biology. Op. Cit.. Béatrice Longuenesse radicaliza esta postura en tanto sostiene que la propia aplicación de las categorías es inseparable de un proceso que tiene un aspecto reflexivo. Cf. Béatrice Longuenesse. Kant and the Capacity to Judge. Trad. Charles T. Wolfe. Princeton/Oxford: Princeton University Press, 2000, pp. 163s. Una posición contraria con respecto a este tema, puede encontrarse en Guyer. Guyer considera menor la relevancia cognitiva del principio de la conformidad a fin y sostiene que el mismo provee más bien una motivación racional en orden a alcanzar la sistematización de la naturaleza. Cf. Paul Guyer. Kant and the Claims of Taste. Op. cit. p. 57. 45 En este contexto puede explicarse la negativa de Kant a admitir la existencia de una ciencia de lo bello: “No hay ni una ciencia de lo bello, sino tan solo una crítica, ni una ciencia bella, sino tan solo arte bello”. Immanuel Kant. Kritik der Urteilskraft. Op. cit., p. 304. 4. El escepticismo estético kantiano No obstante, algunos indicios permiten suponer que el filósofo crítico advertía hasta qué punto el ejercicio efectivo de las facultades humanas, que habían sido descriptas en las dos primeras críticas, solo resultaba concebible si se demostraba la posibilidad de un acuerdo profundo entre las mismas. En este sentido, sería posible pensar que el abordaje del problema de la belleza, que emprendía Kant en el marco de la tercera crítica, encontraba su punto de partida en el reconocimiento implícito tanto de la necesidad de asegurar las condiciones del normal funcionamiento del juicio como de la imposibilidad de hacerlo dentro de los límites del pensamiento crítico.44 Desde esta perspectiva, de lo que 44 Cf. Ibídem pp. 760s. Un análisis levemente más radical presenta Szczepanski. Para este autor, el recurso a lo bello sería una muestra del fracaso del proyecto crítico en tanto que la necesidad de lo bello se desprendería de la incapacidad de la razón para fundar algo positivo en su relación crítica con- sigo misma. Cf. Jens Szczepanski. Subjektivität und Ästhetik. Gegendiskurse zur Metaphysik des Subjekts im ästhetischen Denken bei Schlegel, Nietzsche und de Man. Bielefeld: Transcript, 2007, p. 104. A partir de aquí, la empresa crítica se transformaría en el intento por reglar de manera racional el acceso racionalmente necesario a lo suprasensible. En este hecho podría vislumbrarse la posición intermedia que ocupa Kant entre la temprana modernidad y el idealismo. Pues, el funcionamiento de la razón kantiana resultaría dependiente de un reaseguro externo, como en el caso de los gérmenes pre- formados. Sin embargo, también Kant procuraría por momentos ir más allá de esta solución de com- 145 Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 La promesa de lo bello: consideraciones acerca de la... María Verónica Galfione se trataba era de determinar en qué medida la idea de Moritz de una finalidad sin fin podía ser vinculada a la revitalización de las fuerzas del ánimo que, según la tradición estético- efectual, tenía lugar en el marco de la experiencia estética. Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 4. El escepticismo estético kantiano Kant llegaba a esta conclusión una vez que comprobaba la imposibilidad de determinar conceptualmente la constitución de los objetos que nos resultan bellos: “No puede haber ninguna regla objetiva de gusto que determine por medio des conceptos lo que es bello. Pues todo juicio que surja a partir de allí es estético, es decir, su fundamento es el sentimiento del sujeto y no el concepto de un objeto. Buscar un principio del gusto, que fije por medio de conceptos determinados el criterio general de lo bello, es un esfuerzo infructuoso.” Ibídem p. 231. “Por un principio del gusto se entendería un principio bajo cuya condición se pudiera subsu- mir el concepto de un objeto y extraer así la conclusión de que es bello”. Ibid. p. 285. 46 En este contexto devienen significativos los esfuerzos de Kant por rechazar el relativismo estético aun cuando el mismo no admitiese la existencia de un principio objetivo de juicio. “Si los juicios de gusto tuviesen (al igual que los juicios de conocimiento) un principio objetivo determinado, entonces aquel que realizara tales juicios según este principio podría tener pretensiones de que su juicio fuese incondicionalmente necesario. Pero si los juicios de gusto carecieran de todo tipo de principio, como los meros juicios de los sentidos, entonces, no se podría pensar ninguna necesidad de los mismos. Por lo tanto, ellos deben tener un principio subjetivo que, solo por medio del sentimiento y no de los con- ceptos, pero con validez general, determine qué gusta y que no.” Ibid. p. 237. Sobre la importancia que adquiere en este punto la ejercitación, en tanto mecanismo de disciplinamiento de las fuerzas huma- nas se puede consultar el posicionamiento de Menke. Cf. Christoph Menke. Estética y negatividad. Trad. Gustavo Leyva. Buenos Aires: FCE, 2011, pp. 87s y 271s; Christoph Menke. Kraft, Frankfurt: Suhrkamp, 2008, p. 88. 146 Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 La promesa de lo bello: consideraciones acerca de la... María Verónica Galfione rimentábamos con placer durante la experiencia estética era la libre concordancia, es decir, la concordancia sin concepto, entre la imaginación y el entendimiento,47 para identificar luego esta concordancia con la condición subjetiva de todo conocimiento posible. 4. El escepticismo estético kantiano Se trata- ba, sostenía Kant, de aquel estado de ánimo en el cual se establecía una relación tal entre las fuerzas representativas que “estas remit[ían] una representación dada al conocimiento en general.”48 De esta manera, Kant podía continuar definiendo la belleza, como ya lo había hecho Moritz, en términos de finalidad sin fin.49 Sin embargo, lo que dicha expresión mentaba no era ya la existencia de un objeto efectivamente autónomo –la obra de arte–, sino más bien aquel armonioso juego que, en ausencia de todo concepto, se establecía entre las facultades cognoscitivas durante la contemplación de determinados objetos.50 De esta forma, se salva- 47 La diferencia entre juicio determinante y reflexionante consiste en que, en el primer caso, la imagi- nación forma una imagen sensible que unifica las representaciones de los elementos percibidos deter- minándolas por medio de una regla que corresponde a un concepto determinado. Durante la percep- ción habitual de objetos, uno se considera determinado por una regla general y esta relación con una regla general basta para explicar el acuerdo entre entendimiento e imaginación. En el caso del juicio estético, no existe ni un concepto ni una regla que guíe la actividad de la imaginación y sin embargo se produce el acuerdo de las facultades en la medida en que, el objeto en virtud de su estructura y dis- posición no se presenta como un producto de carácter contingente. No es posible determinar un con- cepto como causa de posibilidad del objeto, pero la apariencia del mismo se presenta no obstante como conforme a fin. Cf. Hannah Ginsborg. Op. Cit. pp. 45-97. 48 Immanuel Kant. Op. Cit. p. 217. “La condición subjetiva de todos los juicios es la propia capacidad para juzgar, o la facultad de juzgar. En vista a una representación por medio de la cual es dado un obje- to, aquella utiliza, exige, el acuerdo de dos facultades representativas: esto es, de la imaginación (para la intuición y la composición de lo múltiple) y el entendimiento (para el concepto de la representación de la unidad de esta composición). Puesto que aquí el juicio no tiene como fundamento ningún con- cepto del objeto, solo puede consistir en la subsunción de la propia imaginación (frente a una repre- sentación por medio de la cual me es dado el objeto), bajo la condición de que el entendimiento pueda llegar así de la intuición a los conceptos. Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 4. El escepticismo estético kantiano Esto quiere decir que, la libertad de tales juicios consiste jus- tamente en el hecho de que la imaginación esquematiza sin concepto. El juicio de gusto debe fundar- se así en el mero sentimiento de vivificación recíproca entre la imaginación, en su libertad, y el enten- dimiento, en su legalidad, esto es, fundarse en un sentimiento que permite juzgar al objeto según la adecuación de la representación (por medio de la cual es dado el objeto) para el fomento de las facul- tades del conocimiento en su libre juego.” Ibídem p. 287. 49 De esta forma se rechazaba la tentativa sensualista de identificar el placer estético con el sentimiento de agrado. En este punto, el planteo de Kant parece coincidir con el de Moritz en la medida en que en ambos casos se trataba de diferenciar belleza y utilidad por medio del concepto de Zweckmäßigkeit. Desde la perspectiva de Peter Szondi, sin embargo, Kant continuaba pensando esta “disposición a fin” como un fenómeno de la sensibilidad y situándose, por ende, dentro de los límites de la estética de los efectos. A la superioridad de la perspectiva de Moritz en este aspecto se refiere Szondi en los siguien- tes términos: “La fórmula en la obra misma es indisociable de un programa completo con el cual se despide el pensamiento estético del siglo (la estética de los efectos).” Peter Szondi. Poetik und Geschichtsphilosophie I. Frankfurt: Suhrkamp, 1974, p. 98. Desde nuestro punto de vista, no obstan- te, Szondi pierde de vista en qué medida la fundamentación kantiana del juicio estético se alejaba de la estética de los efectos e introducía tópicos que serían desarrollados de una manera acabada en el marco de la filosofía idealista del arte. 50 En este punto tomamos distancia de la lectura de Zuckert en la medida en que esta autora remite el sentimiento de belleza a cualidades objetivas. Al respecto Zuckert sostiene: “es la forma de un objeto 147 Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 La promesa de lo bello: consideraciones acerca de la... María Verónica Galfione ba tanto la validez de la explicación mecánico-causal como el carácter incondicionado de la libertad, esto es, aquellos dos elementos que eran puestos en duda por las posturas que remi- tían la creación artística a la potencia vital de una naturaleza productiva. lo que lo hace bello”. Cf. Rachel Zuckert. “The Purposiveness of Form: A Reading of Kant’s Aesthetic Formalism”. Journal of the History of Philosophy, 44.4, 2006, p. 600. 51 Christoph Menke. Op. cit. p. 33. 4. El escepticismo estético kantiano Pero gracias a la solución kantiana del problema se garantizaba también la posibilidad de un tránsito entre la subjetividad y las leyes del universo natural. Esto último resultaba particularmente impor- tante a la hora de dar cuenta del carácter práctico de nuestras facultades subjetivas, es decir, de demostrar que estas últimas se hallaban capacitadas para su efectiva aplicación. Pues, así entendido, el placer estético venía a confirmar el carácter enlazable de dichas facultades y, con él, el hecho de que las mismas resultaban adecuadas para el conocimiento en general. En este sentido, la experiencia estética iluminaba una zona oscura de la filosofía crítica en la medida en que suspendía el uso determinante de las facultades cognitivas a los fines de ofrecerle al hombre la posibilidad de experimentarse a sí mismo como sujeto, es decir, como poseedor de determinadas fuerzas subjetivas que era capaz de emplear con éxito en el plano cognoscitivo real51. 5. Consideraciones finales A lo largo de estas páginas hemos intentado reconstruir el contexto en el cual se produ- ce el giro hacia una estética de carácter idealista. Según hemos podido constatar, la reflexión estética no asumió desde un primer momento funciones de carácter filosófico sino que pro- curó dar respuesta, en principio, a la crisis de legitimación artística que se seguía de la corro- sión de los supuestos histórico-naturales clásicos. En este sentido, sería posible afirmar que el propio afán de los pensadores estéticos de finales del siglo XVIII por sostener sobre nue- vas bases la objetividad de la representación artística jugó un rol fundamental a la hora de convertir a la obra de arte misma en un recurso adecuado para afrontar el problema moder- no de la contingencia. Esto es, la temporalización de las formas orgánico-naturales limitó las pretensiones nomológicas al ámbito de los fenómenos inorgánicos y tornó impensable, de esta forma, toda posible fundamentación de la belleza artística que tomase como punto de referencia la racionalidad intrínseca del orden natural. Privadas de todo referente exter- no, las nuevas perspectivas estéticas debieron concentrarse en los aspectos no miméticos de la propia representación. Ya sea en la variante vitalista de Herder o en la deriva clasicista y formalista de Moritz, tales elementos fueron dotados así de un carácter incondicional que los habilitaba sin más para asumir la tarea filosófica de superar la historia en el marco de la propia temporalidad. Pero si de esta forma se convertía al arte en el recuerdo de una naturaleza perdida, no por ello se resolvía de manera adecuada el problema de la legitimidad de la belleza artísti- ca. Pues, las perspectivas mencionadas se hallaban obligadas a escoger entre la remisión de esta última a una fuerza absolutamente indeterminada o su derivación a partir de una ins- tancia de carácter intencional. Según analizamos a lo largo del presente artículo, la primera de estas opciones impedía dar cuenta de la necesidad de las configuraciones artísticas parti- 148 Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 La promesa de lo bello: consideraciones acerca de la... María Verónica Galfione culares,52 mientras que la segunda equiparaba la representación artística a la producción de artefactos de carácter instrumental. Por este motivo, a la hora de explicar el origen del fenó- meno estético, Kant hacía referencia a un juicio de naturaleza subjetiva. 52 En este sentido, la estética herderiana se hallaba atravesada por la misma dificultad que su teoría del conocimiento. Esto es justamente lo que le reprochaba Kant en su reseña de Ideas. Esto es, que el carácter absolutamente indeterminado de la fuerza genética originaria volvía imposible la explicación de las formas orgánicas naturales. 53 Ginsborg hace referencia a una “normatividad primitiva” al analizar la concepción kantiana del jui- cio de gusto. Sin embargo, lo que está en juego aquí para la autora es más la validez intersubjetiva del conocimiento empírico que la posibilidad de garantizar la unidad del sujeto. Hannah Ginsborg. “Thinking the Particular as Contained Under the Universal”. Rebecca Kukla (Ed.) Aesthetics and Cognition in Kant’s Critical Philosophy. Cambridge, Cambridge University Press, 2006, p. 35 y p. 58n. 5. Consideraciones finales Según pudimos advertir, el filósofo crítico no se limitaba a precisar así algunos conceptos generales de la tradición psicológico-efectual sino que introducía, más bien, una serie de elementos que devendrían fundamentales para el desarrollo de una estética de carácter idealista. Concretamente, Kant vinculaba de esta forma el placer estético a la forma de una subjetivi- dad unificada.53 Pues, el estado en el cual se posicionaba el sujeto durante la experiencia estética se definía a partir del acuerdo espontáneo de sus facultades cognitivas. A esta con- cordancia remitía la idea kantiana de un “libre juego” de la sensibilidad y del entendimien- to, esto es, a un tipo de correspondencia entre ambas disposiciones que, a diferencia de lo que sucedía en los juicios determinantes, no se desprendía de la presencia de un concepto de carácter general54. Como ya mencionamos, la ausencia de toda determinación conceptual le permitía a Kant diferenciar al estado estético de aquella esfera en la cual tenían lugar los procesos de carácter cognoscitivo. En este sentido, sería posible sostener que la reflexión kantiana se orientaba a fundamentar la autonomía del juicio de gusto con respecto a los diversos usos determinantes de las facultades o disposiciones subjetivas. Sin embargo, por medio de esta diferenciación, el estado que posibilitaba la experiencia estética obtenía una función inédi- ta en relación al plano específicamente cognoscitivo. Pues, en la medida en que dicho esta- do aludía a la libre concordancia entre el entendimiento y la imaginación, el juicio estético se presentaba como un mecanismo adecuado para experimentar la disposición a fin de las fuerzas subjetivas para un uso cognitivo en general. De esta manera se establece una tensión entre la fundamentación kantiana de la autonomía de la dimensión estética, por una parte, y su caracterización del placer estético en términos de un estado de libre concordancia entre las facultades subjetivas, por la otra. Pues, la interrupción de las funciones subjetivas habi- tuales, que tenía lugar en la esfera estética, no introducía una ruptura real en el terreno de la praxis determinante, sino que apuntalaba, más bien, el uso extra-estético de las propias fuer- zas subjetivas, allí donde esto ya no resultaba posible en términos estrictamente objetivos. Sin embargo, no fue Kant, sino Schiller, el encargado de vincular la figura de una sub- jetividad reconciliada con el problema de la legitimación de la belleza artística. Anales del Seminario de Historia de la Filosofía Vol. 31 Núm. 1 (2014): 131-153 54 Immanuel Kant. Op. Cit. p. 219. 5. Consideraciones finales De esta manera, la elevación de la obra artística por encima de la contingencia histórica, que anti- guamente había sido garantizada en función de una referencialidad de carácter ontológico, 149 La promesa de lo bello: consideraciones acerca de la... María Verónica Galfione pasaba a presentarse como un “préstamo” de la propia subjetividad. Pero Schiller no se limi- taba a concebir la representación artística bajo la forma de la libertad subjetiva, sino que avanzaba hasta convertir al ámbito estético mismo en una condición necesaria para la exis- tencia de la propia subjetividad55. Así, quedaba definitivamente consolidado el vínculo entre estética y subjetividad que, en términos indirectos, había afirmado Kant al remitir la belle- za al libre juego de las facultades subjetivas. No obstante, tampoco eran pocas las dificulta- des que se hallaban contenidas en este modelo estético. Pues, si la objetividad de la obra de arte era salvada por medio de la referencia a una subjetividad reconciliada, esta última resul- taba dependiente del proceso formativo que tenía lugar por intermedio de la propia expe- riencia estética. En este punto era posible advertir la confusión de terrenos que suponía la equiparación idealista del placer estético con el juego armónico de las facultades subjetivas. Pues, en la medida en que la naciente sociedad burguesa solo dejaba aparecer la libre con- cordancia de las facultades humanas bajo la forma de un imperativo, la sujeción de la esfe- ra estética al principio del juego –esto es, al modelo de la “finalidad sin fin”- debía traer apa- rejado el sometimiento del arte a funciones y objetivos de carácter extra-estético. Este des- cubrimiento se encontraría a cargo del joven escritor Friedrich Schlegel quien, en algunas notas de mediados de los años 90, tacharía de dogmática a la interpretación de Schiller del escepticismo estético kantiano56. Pero el análisis del debate en torno a la herencia de la esté- tica kantiana, que tendría lugar en Alemania desde finales del siglo XVIII, se encuentra más allá de los límites del presente trabajo. 56 Friedrich Schlegel. „Von der Schönheit in der Dichtkunst“. Kritische Friedrich Schlegel-Ausgabe. Ed. E. Behler, H. Einchner, J.J. Anstett et al. München, Paderborn, Wien, Schöningh, 1981, p. 11. 55 Para el primer momento se puede consultar Kallias. Para el segundo, en cambio, las Cartas sobre la educación estética del hombre. Cf. Friedrich Schiller. Kallias. Cartas sobre la educación estética del hombre. Trad. Feijóo, J. y Seca, J. Barcelona, Anthropos, 2005, p. 19, p. 241. Referencias bibliográficas ALLISON, H.: “Kant’s Antinomy of Teleological Judgment”. Southern Journal of Philosophy, vol. 30, 1991, pp. 25-42. BAUMGARTEN, A. G.: Aesthetica. Hildesheim: Georg Olms, 1970. BECK, L.W.: Early German Philosophy. Cambridge: Belknap Press, 1969. Ä BIERBRODT, J.: Naturwissenschaft und Ästhetik, 1750-1810. Würzburg: Königshausen und Neumann, 2000. BLUMENBACH, J.F.: Beiträge zur Naturgeschichte. Göttingen: Johann Christian Dieterich, 1790. BUBNER, R.: „Über einige Bedingungen gegenwärtiger Ästhetik“. Neue hefte für Philosophie. Göttingen, Vandenhoeck & Ruprecht 1973, V. 5, 1773, pp. 38-73. BUFFON, G.-L. 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F.: „Die stete und feste Gang der Natur zur Organisation“. Breidbach, Olaf y Paul Ziche (Eds.) Naturwissenschaften um 1800. Weimar: Hermann Böhlaus Nachfolger, 2001, pp. 27-46. GALFIONE, M.V.: “Historia Natural y temporalización: Algunas consideraciones sobre la his- toria natural de Buffon”. História, Ciências, Saúde – Manguinhos. Vol.20/n.°3, pp. 813- 829. GINSBORG, H.: “Kant on Aesthetic and Biological Purposiveness”. Reath, Andrews et al. (Eds.) Reclaiming the History of Ethics: Essays for John Rawls. Cambridge: Cambridge Press, 1997, pp. 329-360. BORG, H.: The role of taste in Kant´s Theory of cognition. New York: Garland, 1990. GINSBORG, H.: “Thinking the Particular as Contained Under the Universal”. Aesthetics and Cognition in Kant’s Critical Philosophy. Kukla, Rebecca (Ed.) Cambridge: Cambridge University Press, 2006, pp. 35-60. GINSBORG, H.: “Two Kinds of Mechanical Inexplicability in Kant and Aristotle”. Journal of the History of Philosophy, 42(1), 2004, pp. 33-65. 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Instituto de Literatura Anglogermánica, Buenos Aires, 1949. KANT, I.: „Bestimmung des Begriffs einer Menschenrasse“. Kants gesammelte Schriften. Tomo VIII. Hrsg. von der Königlich Preußischen Akademie der Wissenschaften, Berlin: Walter de Gruyter, 1923, pp. 89-106. KANT, I.: Crítica de la razón pura. Trad. Ribas, P. Madrid: Alfaguara, 1998. KANT, I.: Kritik der Urteilskraft. Kants gesammelte Schriften. Tomo V. Hrsg. von der Königlich Preußischen Akademie der Wissenschaften, Berlin: Walter de Gruyter, 1913. KANT, I.: „Recensionen von J.G. Herders Ideen zur Philosophie der Geschichte der Menschheit“. Kants gesammelte Schriften. Tomo VIII. Hrsg. von der Königlich Preußischen Akademie der Wissenschaften, Berlin: Walter de Gruyter, 1923, pp. 43-66. Traducción al español de Roldan Panadero, Concha y Rodríguez Aramayo, Roberto. 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Optimizing Administrative Datasets to Examine Acute Kidney Injury in the Era of Big Data: Workgroup Statement from the 15<sup>th</sup> ADQI Consensus Conference
Canadian journal of kidney health and disease
2,016
cc-by
7,862
Siew et al. Canadian Journal of Kidney Health and Disease (2016) 3:12 DOI 10.1186/s40697-016-0098-5 REVIEW Open Access Optimizing administrative datasets to examine acute kidney injury in the era of big data: workgroup statement from the 15th ADQI Consensus Conference Edward D. Siew1,2*, Rajit K. Basu3, Hannah Wunsch4, Andrew D. Shaw5, Stuart L Goldstein6, Claudio Ronco7, John A. Kellum8, Sean M. Bagshaw9 and on behalf of the 15th ADQI Consensus Group Abstract Purpose of review: The purpose of this review is to report how administrative data have been used to study AKI, identify current limitations, and suggest how these data sources might be enhanced to address knowledge gaps in the field. Objectives: 1) To review the existing evidence-base on how AKI is coded across administrative datasets, 2) To identify limitations, gaps in knowledge, and major barriers to scientific progress in AKI related to coding in administrative data, 3) To discuss how administrative data for AKI might be enhanced to enable “communication” and “translation” within and across administrative jurisdictions, and 4) To suggest how administrative databases might be configured to inform ‘registry-based’ pragmatic studies. Source of information: Literature review of English language articles through PubMed search for relevant AKI literature focusing on the validation of AKI in administrative data or used administrative data to describe the epidemiology of AKI. Setting: Acute Dialysis Quality Initiative (ADQI) Consensus Conference September 6-7th, 2015, Banff, Canada Patients: Hospitalized patients with AKI Key messages: The coding structure for AKI in many administrative datasets limits understanding of true disease burden (especially less severe AKI), its temporal trends, and clinical phenotyping. Important opportunities exist to improve the quality and coding of AKI data to better address critical knowledge gaps in AKI and improve care. Methods: A modified Delphi consensus building process consisting of review of the literature and summary statements were developed through a series of alternating breakout and plenary sessions. (Continued on next page) * Correspondence: edward.siew@vanderbilt.edu 1 Tennessee Valley Health System (TVHS), Nashville Veterans Affairs Hospital, Nashville, TN, USA 2 Vanderbilt University Medical Center, Department of Medicine, Division of Nephrology and Hypertension, Vanderbilt Center for Kidney Diseases (VCKD), 1161 21st Avenue South, MCN S3223, Nashville, TN 37232, USA Full list of author information is available at the end of the article © 2016 Siew et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Siew et al. Canadian Journal of Kidney Health and Disease (2016) 3:12 Page 2 of 12 (Continued from previous page) Results: Administrative codes for AKI are limited by poor sensitivity, lack of standardization to classify severity, and poor contextual phenotyping. These limitations are further hampered by reduced awareness of AKI among providers and the subjective nature of reporting. While an idealized definition of AKI may be difficult to implement, improving standardization of reporting by using laboratory-based definitions and providing complementary information on the context in which AKI occurs are possible. Administrative databases may also help enhance the conduct of and inform clinical or registry-based pragmatic studies. Limitations: Data sources largely restricted to North American and Europe Implications: Administrative data are rapidly growing and evolving, and represent an unprecedented opportunity to address knowledge gaps in AKI. Progress will require continued efforts to improve awareness of the impact of AKI on public health, engage key stakeholders, and develop tangible strategies to reconfigure infrastructure to improve the reporting and phenotyping of AKI. Why is this review important?: Rapid growth in the size and availability of administrative data has enhanced the clinical study of acute kidney injury (AKI). However, significant limitations exist in coding that hinder our ability to better understand its epidemiology and address knowledge gaps. The following consensus-based review discusses how administrative data have been used to study AKI, identify current limitations, and suggest how these data sources might be enhanced to improve the future study of this disease. What are the key messages?: The current coding structure of administrative data is hindered by a lack of sensitivity, standardization to properly classify severity, and limited clinical phenotyping. These limitations combined with reduced awareness of AKI and the subjective nature of reporting limit understanding of disease burden across settings and time periods. As administrative data become more sophisticated and complex, important opportunities to employ more objective criteria to diagnose and stage AKI as well as improve contextual phenotyping exist that can help address knowledge gaps and improve care. ABRÉGÉ Article synthèse: Cette revue vise à rendre compte de la manière dont les données administratives ont été utilisées jusqu’à maintenant pour l’étude de l’insuffisance rénale aiguë (IRA). On a également voulu définir les limites actuelles et suggérer une manière dont les sources de données pourraient être améliorées pour pallier les lacunes des connaissances dans ce domaine. Objectifs de la revue: Cette revue visait plusieurs objectifs: 1) Répertorier les données probantes sur la manière dont l’IRA est codée dans les ensembles de données administratives. 2) Identifier les limites, les lacunes au niveau des connaissances et les principaux obstacles aux avancées de la science relevant de la codification de l’IRA dans les données administratives. 3) Discuter de la façon dont les données administratives relatives à l’IRA pourraient être bonifiées afin de favoriser leur transmission au sein des différents secteurs de compétences. 4) Suggérer une nouvelle façon de configurer les bases de données administratives afin qu’elles puissent servir de source d’information pour la tenue d’études pragmatiques fondées sur la consultation de registres. Sources: L’information a été colligée à la suite d’un passage en revue des articles pertinents publiés en anglais sur PubMed. Les articles portant sur la validation de l’IRA au sein des données administratives ou sur l’usage de celles-ci pour rendre compte de l’épidémiologie de l’IRA ont été retenus. Cadre de la revue: La revue a eu lieu dans le cadre de la 15e réunion annuelle de concertation de l’Acute Dialysis Quality Initiative (ADQI) qui s’est tenue les 6 et 7 septembre 2015 à Banff, au Canada. Population observée: Les cas retenus pour la revue sont ceux de patients hospitalisés atteints d’IRA. (Continued on next page) Siew et al. Canadian Journal of Kidney Health and Disease (2016) 3:12 Page 3 of 12 (Continued from previous page) Points saillants: La façon dont l’IRA est codée au sein des ensembles de données limite la compréhension réelle du fardeau que représente cette maladie, particulièrement pour les patients atteints moins sévèrement. Il semble également que l’on ne puisse identifier correctement les tendances temporelles ni le phénotype clinique de l’IRA parmi ces données. On a aussi constaté qu’il existe d’importantes possibilités d’amélioration de la codification de l’IRA dans les ensembles de données pour arriver à mieux cerner les lacunes dans la connaissance de la maladie et bonifier les soins prodigués aux patients. Méthodologie: Une version adaptée de la méthode Delphi pour l’atteinte d’un consensus a été utilisée pour produire le compte-rendu. La revue de la littérature et des récapitulatifs ont été élaborés à la suite d’une série de discussions alternant entre des ateliers en petits groupes et des séances plénières. Résultats: Les codes administratifs concernant l’IRA sont limités en raison du faible taux de rappel des documents pertinents lors des recherches, du manque de normalisation dans le classement de la sévérité de la maladie et de la faible identification de son phénotype. Ces limites sont de plus freinées par un manque de sensibilisation des fournisseurs de soins de santé face à l’IRA et par le caractère subjectif d’un signalement volontaire des cas observés. Bien qu’une définition idéalisée de l’IRA soit difficile à mettre en œuvre, améliorer la normalisation des signalements en se basant sur un consensus scientifique et fournir de l’information quant au contexte où se manifeste l’IRA sont possibles. Les bases de données administratives peuvent améliorer les pratiques entourant la conduite d’études cliniques ou d’études pragmatiques basées sur la consultation de registres, et servir de sources d’informations pour celles-ci. Limites de la revue: La provenance des sources consultées pour cette revue se limite en grande partie à l’Amérique du Nord et à l’Europe. Conclusions: La croissance et l’évolution rapide des bases de données administratives représentent une occasion unique de s’attaquer aux lacunes dans les connaissances sur l’IRA. L’avancement de ces connaissances demandera le déploiement d’efforts constants pour favoriser la prise de conscience des répercussions de cette maladie sur la santé publique. Cela demandera aussi l’engagement des principaux intervenants ainsi que la mise en place de stratégies concrètes visant la reconfiguration des infrastructures existantes et l’amélioration de la collecte de données et de l’identification du phénotype de l’IRA. Background The clinical study of acute kidney injury (AKI) has been facilitated in recent years by the increasing availability of administrative data. The housing of vast amounts of information in accessible data warehouses and the relatively low cost of procurement suggest potential usefulness. However, as usually not collected for conducting clinical research, concerns over the quality of the data and its ability to fill knowledge gaps and evaluate quality of care have been raised [1, 2]. On September 6th, 2015, the Acute Dialysis Quality Initiative (ADQI) convened a panel of experts in nephrology, critical care, pharmacology, pediatrics, epidemiology, health services research and data analytics from five countries from North American and Europe to examine how rapidly evolving clinical data infrastructures in the era of ‘big data’ can be leveraged to enhance scientific progress and improve outcomes in patients with AKI. Here, we review how administrative data have been used to study AKI, identify current limitations, and suggest how these data sources might be enhanced to address knowledge gaps in the field. We present four key questions regarding the use of administrative data for research and quality improvement; and then a corresponding series of consensus statements developed through reviewing the literature and iterative discussion. Methods The ADQI methodology has been previously detailed [3]. In brief, the consensus-building process was informed by pre-conference, conference, and postconference review of English language articles through PubMed search for relevant AKI literature. We selected articles if they focused on the validation of AKI in administrative data or used administrative data to describe the epidemiology of AKI. We conducted a 2-day conference in September 2015 in Banff, Canada, where summary statements were developed through a series of alternating breakout and plenary sessions. Panelists were assigned to 3–5 person workgroups. In each breakout session, the workgroups refined the key questions, identified the supporting evidence, and generated consensus statements. Workgroup members presented their findings during the plenary sessions and then revised their drafts as needed until a Siew et al. Canadian Journal of Kidney Health and Disease (2016) 3:12 Page 4 of 12 final version was agreed upon. A writing committee assembled the individual reports from the workgroups and each report was edited to conform to a uniform style and for length. The final reports were provided to each participant for comment. For this manuscript, we will define health care-related administrative data as information collected and stored for patient disease registration, to inform transactions, or perform other record keeping (http://www.adls.ac.uk/ adls-resources/guidance/introduction/). Major producers of administrative data include, but are not limited to, governmental agencies (e.g., Federal, State/Provincial, and Veterans Affairs), insurers, and healthcare systems. Most literature using administrative data to study AKI have applied the 9th and 10th iterations of the International Classification of Disease, Clinical Modification (ICD-9-CM, ICD-10-CM) and procedure codes and the American Medical Association’s Current Procedural Terminology (CPT) codes to capture the diagnosis of AKI and renal replacement therapy (RRT) [2, 4]. Within these classification systems, AKI is most commonly coded as Acute Kidney Failure with various histologic descriptors (Table 1). The latter are generally based on clinical impression as confirmatory histologic diagnosis requires kidney biopsy, something rarely pursued in clinical practice. Further these histologic diagnoses have traditionally been non-exhaustive and include distinctions of less clear relevance (e.g. tubular versus medullary necrosis). While subsequent iterations (ICD-10) have broadened to capture additional lesions of interest (e.g. acute tubulointerstitial nephritis), they remain independent codes that do not readily link to major AKI codes and little data is available reflecting their performance against either an Review Question #1: What is the existing evidence-base for how AKI is coded across various administrative datasets? Consensus Statement #1A: Administrative data are defined as information collected and stored for patient or disease registration, to inform transactions, or to promote other record keeping. The coding of AKI using these data sources has utilized billing/claims, limited laboratory-based definitions in diseasespecific registries, and population-based health registries. Consensus Statement #1B: Commonly used administrative codes for AKI generally demonstrate poor sensitivity and high specificity. Table 1 Examples of Administrative codes for AKI ICD-9-CM ICD-10 Name 584 N17 Acute kidney failure 584.5 N17.0 Acute kidney failure with lesion of tubular necrosis 584.6 N17.1 Acute kidney failure with lesion of renal cortical necrosis 584.7 N17.2 Acute kidney failure of renal medullary (papillary) necrosis 584.8 N17.8 Acute kidney failure with other specified pathological lesion in the kidney 584.9 N17.9 Acute kidney failure, unspecified 593.9 N28 Unspecified disorder of kidney and ureter N28.0 Ischemia and infarction of the kidney N28.9 Disorder of kidney and ureter 583.00-90 N00.xN01.xN05.2,5,8,9 ICD- 9: Nephritis and nephropathy, not specified as acute or chronic with lesion of ….ICD-10: Acute Nephritic syndrome (N00) with…Rapidly progressive nephritic syndrome (N01) withUnspecified nephritic syndrome (N05) with… 669.30,32,34 O09.4 ICD-9 Acute kidney failure following labor and delivery… N99.8 Postprocedural (acute)(chronic) kidney failure O04.82 Renal failure following (induced) termination of pregnancy O07.32 Renal Failure following failed attempted termination of pregnancy N10 Acute tubulo-interstitial nephritis N14.0-4 Drug- and heavy-metal-induced tubule-interstitial and tubular conditions (acuity not specified) The most commonly studied and used codes are bolded Siew et al. Canadian Journal of Kidney Health and Disease (2016) 3:12 Page 5 of 12 adjudicated or histologic standard. Lastly, most administrative coding datasets do not provide a standardized approach to staging of severity outside of recording dialysis. Disease or procedural registries also report data on renal failure, occasionally collecting serum creatinine data. However, definitions for AKI in such registries tend to focus on the most severe phenotypes, may not distinguish between acute or chronic disease, or harmonize with modern consensus definitions. For example, the National Surgical Quality Improvement Program (NSQIP) and American College of Surgeons Committee on Trauma have traditionally defined AKI as a rise in serum creatinine above 2 mg/ dl (177 μmol/l) or dialysis and a serum creatinine above 3.5 mg/dl (309 μmol/l), respectively [5–7]. This heterogeneity may hinder comparisons and underestimate disease burden. Recent efforts to recalibrate renal failure definitions, however, may indicate greater acceptance of standardized definitions in some of these data sources. For example, the Society of Thoracic Surgeon database recently changed from a threshold creatinine of > 2 mg/dl (177 μmol/l) to define AKI to incorporate the RIFLE classification system (http://www.sts.org/sites/default/files/docu ments/Training%20Manual%20for%20website_2.pdf). Coding performance has been examined in several studies by comparing them to a reference standard of creatinine change or manual chart review [8–11]. Vlasschaert et al. performed a systematic review examining the diagnostic accuracy of ICD-9 and ICD-10 codes in US and Canadian datasets between 1987 and 2004 [8]. Despite variations in the reference standards used (varying creatinine-based definitions of AKI or chart review), sensitivity was generally low (median 29 %; range 15-81 %) though specificity remained high (>94 % in all datasets examined). This performance translated to high negative predictive values with variable positive predictive values depending on the cohort examined and underlying prevalence of AKI (between 0.5-52 %). Despite having a lower prevalence of chronic kidney disease, sensitivity in children for AKI is also poor. A recent study found that administrative data had poor sensitivity (21-23 %) for detecting nephrotoxin-associated AKI in children who had serum creatinine monitored daily [12]. Performance between US and Canadian datasets were similar, although sensitivity was slightly higher in Canadian datasets. Restriction to dialysis-requiring AKI generally improved validity, but not always. For example, Waikar et al. found that the combination of any Acute Kidney Failure code (574.5-9) and hemodialysis procedure codes produced high sensitivity (90.4 %) and specificity (93.8 %) for detecting dialysis-requiring AKI as verified by chart review in Boston-area teaching hospitals (USA) between 1988–2002 [13]. However, more recently, Grams et al. used a similar approach to examine the performance of administrative codes for dialysis- requiring AKI in a select cohort of participants of The Atherosclerosis Risk in Communities (ARIC) study hospitalized in Washington County, Maryland between 1996–2008 [11]. In this study, specificity for the dialysisrequiring AKI coding algorithm was high (99.9 %) but had lower sensitivity (36.5 %). Lastly, there is evidence that performance can change over time. For example, several studies have demonstrated that the sensitivity of billing codes has improved in recent years but generally remains modest. [10, 11, 13] The collective performance of administrative codes for detecting AKI, particularly when not directly linked to serum creatinine, has important implications. For example, poor or changing sensitivities limit value in understanding true disease burden; provide overly conservative estimates of disease prevalence, skew temporal trends due to changes in the sensitivity of diagnostic codes, and contain uncertainty in distinguishing acute from chronic disease. The relative high specificities may make current administrative data more amenable to examining outcomes of patients identified with AKI. However, positive predictive value may vary depending on the underlying risk of the population studied and the severity detected may also vary across regions or time. Question #2: What are the limitations, gaps in knowledge, and major barriers to scientific progress in AKI related to how AKI is coded? Consensus Statement 2a: Major limitations of current administrative data include poor awareness of AKI and the subjective nature of its assessment, lack of information on severity, and the use of non-consensus criteria for defining and staging AKI. Consensus Statement 2b: Major knowledge gaps in AKI using current administrative data sources include, but are not limited to, lack of contextual phenotyping, information on complications including dialysisdependence or other indicators of recovery, and coding structure and performance in non-North American registries. Limitations of administrative data Figure 1 shows the current strengths, limitations, opportunities, and threats to optimal use of administrative datasets for examining AKI. In addition to strengths listed, we identified several factors contributing to the limited performance of current administrative coding. Siew et al. Canadian Journal of Kidney Health and Disease (2016) 3:12 Page 6 of 12 Fig. 1 Strength, Weaknesses, Opportunities, and Threat (SWOT) Analysis of Current Administrative Codes for AKI. Source: Acute Dialysis Quality Initiative 15 www.adqi.org; used with permission One major limitation likely contributing to the low sensitivities observed is poor provider awareness. Accurate and sensitive reporting of AKI depends on recognition that 1) AKI has occurred and 2) it was clinically relevant. As most AKI does not require dialysis and not linked to a procedure code, coding becomes wholly dependent on provider recognition. AKI is generally well-recognized by nephrologists or other experts, however, primary diagnosis codes for patients requiring acute care are most often entered by providers or coding personnel with less expertise or interest in the area. While consensus criteria have likely improved awareness, overall sensitivity remains modest. [11] The resulting discrepancy between exposure and awareness contributes to underreporting of disease (Fig. 2), underscoring the need for improved dissemination of data regarding the short- and long-term clinical implications of AKI to a broader audience [14]. Subjective coding may also be influenced by external forces, including patient related factors (i.e. patient complexity), variations in practice and coding patterns across medical disciplines, geography, and practice settings. For example, capturing AKI in a critically ill patient with prolonged hospitalization, multi-system organ failure, and contributing comorbidities requires overcoming substantial coding ‘inertia’. In addition, while sensitivity has improved, the resulting changes make interpreting temporal trends challenging. For example, uncertainty exists as to whether increases in disease incidence and improvement in survival observed are attributable to better reporting of less severe AKI, changes in case-mix (e.g. more acute on chronic disease), or true improvements in care [13, 15]. Similarly, variations in treatment may affect underlying assumptions in classifying AKI. For example, dialysis-requiring AKI has traditionally been considered the most severe form of AKI. However, thresholds to initiate dialysis may vary depending on situation or over time that can violate that assumption. For example, a mildly volume depleted patient dialyzed for lithium intoxication may not have severe AKI and evidence for earlier application of RRT in recent years may indicate a trend toward dialyzing less severe injury [16]. A lack of readily accompanying information (e.g., indication or stage) in these situations often hinders the ability to make these important distinctions. How improving upon coding limitations may help address current knowledge gaps in AKI It remains possible to enhance administrative data to address critical knowledge gaps in AKI. For example, an increased understanding of the changing case-mix of AKI and its relative impact in different settings is needed [17]. While novel biomarkers may eventually redefine phenotyping, opportunities exist to provide additional contextual information. For example, as AKI most often occurs due to underlying illness, it is most often phenotyped based on the setting in which it occurs. Thus, linking the diagnosis of AKI as a complication of conditions Siew et al. Canadian Journal of Kidney Health and Disease (2016) 3:12 Page 7 of 12 Fig. 2 Relative Differences in Scope of AKI Cared and Coded for by Provider Type. The pyramid on the left represents the full burden of AKI seen by non-nephrologists with the relative prevalences of mild, moderate-severe, and dialysis-requiring AKI indicated by shading. In contrast, while nephrologists are more likely to code for less severe AKI, they will encounter a more select population of AKI patients favored by highly severe stages. Source: Acute Dialysis Quality Initiative 15 www.adqi.org; used with permission including sepsis (e.g. ‘sepsis complicated by AKI’, ‘contrast/chemotherapy-associated AKI’), might help overcome the inertia of coding AKI as a separate entity and harmonize with how most AKI is conventionally phenotyped (i.e. by clinical context rather than histology). Further phenotyping of AKI severity and pre-/posthospitalization kidney function may better understanding of the role of AKI on the natural history of kidney health and disease progression. However, lack of quality information on pre-morbid kidney function, inpatient trajectory, and post-AKI kidney function in current administrative datasets introduce uncertainty into analyses. Integration or linkage of data from advancing electronic health records would enable use of unified diagnostic criteria, distinguishing relative contributions from AKI and CKD, improve further critical phenotyping of AKI (e.g. community-versus-nosocomial acquired AKI, creatinine trajectory, duration, recovery trends), and examine future outcomes. Barriers to improving administrative coding Integrating unified diagnostic criteria and providing contextual information will require some reconfiguration of existing infrastructure. Key stakeholders including payers, government, providers, and coders will need to be engaged and convinced that doing so will improve costs and quality by providing a fuller understanding of disease burden, help to identify patients at high risk for the sequelae of AKI, and opportunities to examine care. Meaningful changes in coding infrastructure in the kidney disease space are not without precedent. In 2005, the Kidney Disease Outcomes Quality Initiative (KDOQI) staging system for CKD was incorporated into the ICD-10 coding structure. The impetus for these changes in the US was provided by the National Center for Health Statistics, National Kidney Foundation, and the Renal Physicians Association whose goals were to improve understanding of disease burden, follow patients longitudinally, understand treatments provided, and assess the quality of care being delivered. More recent examples of mutual engagement include ongoing initiatives between the critical care community in Canada and the Canadian Institute for Health Information (CIHI) to develop a standardized structure for reporting critical care capacity, operations, quality, and outcomes data. This collaboration will enable a national description of critical care utilization, quality indicators and outcomes aimed at driving health systems improvements. Another current example that model such improvements include the National Health Services ‘Think Kidneys’ program in the United Kingdom [18]. By coupling electronic alerting embedded in all hospitals to improve the recognition of AKI and reduce variability in ascertainment and reporting, developing this critical infrastructure has enabled the building of a prospective Siew et al. Canadian Journal of Kidney Health and Disease (2016) 3:12 Page 8 of 12 registry to capture data on all patients with AKI in a standardized fashion and link to a larger National Renal Registry. A non-diagnosis related barrier is the deliberate transition of medical records to digital filing. Although the American Reinvestment and Recovery Act of 2009 mandated use of EHRs, integration across institutions in the United States has taken longer than anticipated leading to variability in the extractability of information (as some data remains in paper charting). Additionally, the coding structure and performance for capturing AKI in systems outside of the US, Canada, and UK are underreported. These inconsistencies negatively impact the reliability of administrative datasets across registries and limit the ability to do cross-institutional/-country comparisons. Finally, a down-stream concern related to changes in coding is the potential for negative reimbursement for ‘nosocomially-acquired AKI’. As value based purchasing and quality-based reimbursements by both insurance agencies and governmental emerge (e.g., US), granularity in AKI diagnosis with regards to the etiology of injury could actually dissuade clinicians from coding for AKI (e.g., contrast nephropathy induced AKI may be deemed a nosocomial AKI and therefore not reimbursed). Avoidance of this problem will require involvement of relevant stakeholders to ensure appropriate quality metrics that do not penalize accurate diagnostic coding. improvement initiatives [22, 23], widespread evidence of its penetration into administrative datasets is lacking. An ideal “administrative” definition of AKI would incorporate factors that describe the onset, duration, severity, context, histology, progression, and recovery of AKI (Fig. 3). Limitations in current clinical phenotyping and the practicality of standardizing all of these elements as discussed above place some feasibility restrictions on this idealized goal for the near future. However, these limitations do not preclude attainable progress. Two areas identified where improvements are currently possible include improving coding of AKI severity and providing contextual information for the setting in which AKI occurs (Fig. 3 red oval). Introduction of the KDIGO classification system for AKI including “mild” (stage 1), “moderate” (stage 2) and “severe” (stage 3) AKI would enhance capture and phenotyping of AKI beyond the binary system currently in place. As AKI diagnosis relies heavily on laboratory data, the fidelity of these systems will be improved greatly and lend themselves to true standardization through automated inclusion of laboratory data as research networks evolve to integrate large volumes of EHR, administrative, and registry data sources. The latter would also enhance the ability to distinguish between chronicity, onset (community versus hospital-acquired), and permit longitudinal phenotyping including recovery. Similarly, current AKI coding systems should be modified to provide additional contextual information in which AKI occurs. A natural extension would be to include AKI as a complication of known major precipitants (e.g., sepsis, contrast, cirrhosis, cardiac surgery) as is currently the case for CKD coding algorithms (e.g., diabetes mellitus with diabetic chronic kidney disease, hypertensive chronic kidney disease). As most cases will not be seen by nephrologists, this structure may also have the added benefit of improving sensitivity as clinicians are more likely to consider AKI if linked to the primary disease rather than overcoming the effort to code it as a separate entity. In summary, limitations in the current clinical phenotyping of AKI should not prevent tangible improvements to the current coding of AKI. The reliance of physiologic and laboratory data for AKI diagnosis should prompt progressive consideration for automatic capture and inclusion as administrative datasets evolve. Questions #3: What would be the ideal minimal definition for AKI in administrative databases to enable “communication” and “translation” within and across administrative jurisdictions (i.e., institutional, regional, national, international)? Consensus Statement #3: The ideal definition of AKI in administrative databases that would enable crossregistry communication would include descriptors of the onset, duration, severity, context, histology, progression, and recovery of AKI. Minimum essential elements include a standardized, objective description of AKI severity (i.e., stage) accompanied by complementary information on the context in which AKI occurs. Definitions of AKI have evolved over the past 20 years as stakeholders have begun to appreciate the benefits of consensus, both for clinical management and for research. The achievement of standardized definitions (e.g., RIFLE, AKIN, KDIGO) has advanced the field by providing a platform for communication and comparison [19–21]. While emerging evidence indicates that clinicians are beginning to use these consensus criteria in quality Question #4: How can administrative databases be configured to inform “registry-based” pragmatic studies? Consensus Statement #4A: Administrative databases can be used to identify and enrich target populations Siew et al. Canadian Journal of Kidney Health and Disease (2016) 3:12 Page 9 of 12 Fig. 3 Elements of an “Ideal” Definition of AKI Potentially Captured in Future Administrative Data. An idealized future definition of AKI would include descriptive elements able to identity cause, severity, chronicity, type, timing, and context. Given current limitations in clinical phenotyping and coding structure, severity of injury and the context in which it occurs (e.g. cardiac surgery, sepsis, contrast) may be the best initial targets to pursue. Source: Acute Dialysis Quality Initiative 15 www.adqi.org; used with permission of interest and inform the feasibility and power of trials. Consensus Statement #4B: Patients enrolled in clinical trials can be potentially linked to administrative databases to enhance outcome ascertainment, reduce costs, and examine additional outcomes of interest if patient identifiers are available. Administrative databases, particularly if enhanced by improvements in coding and accuracy, can be leveraged to inform clinical studies or facilitate registry-based pragmatic studies (Fig. 4). First, administrative databases can help inform study design by refining inclusion and exclusion criteria. Analysis of characteristics that identify patients with, or who are likely to develop AKI and its sequelae, can refine the approach to targeting of Fig. 4 Schematic Illustrating Potential Leveraging of Administrative Data for Clinical Trials. Given limitations in time of reporting and sensitivity, administrative data may be best suited to facilitate the planning and follow-up stages of a putative randomized or pragmatic trial, though disease or procedure-specific registries may be able to be used as a ‘real-time’ tool to guide enrollment or intervention. Source: Acute Dialysis Quality Initiative 15 www.adqi.org; used with permission Siew et al. Canadian Journal of Kidney Health and Disease (2016) 3:12 Page 10 of 12 patients, estimate study feasibility, and project event rates and sample size. Poor recruitment can hamper trials [24]. Identifying sites with high volumes of patients meeting inclusion criteria allows for targeted collaboration across locations and could improve recruitment. Assessment of event rates can also reduce the need for mid-study adjustments in sample size [25]. The use of large databases for such practices is well illustrated by the design of the PROMISE trial; this trial of early-goal directed therapy for severe sepsis patients in the United Kingdom accurately predicted in-hospital mortality in the control arm based on preliminary assessment of the Intensive Care National Audit and Research Centre (ICNARC) Case Mix Programme Database, a registry of intensive care patients in the United Kingdom [26]. It is important to note that this use of administrative data to inform studies related to AKI requires a clear knowledge of the quality of the data. Datasets with poor sensitivity but high specificity for AKI may be better suited to examine outcomes in populations with moderate to severe AKI rather than those at risk or with mild AKI. Such limitations do not preclude the use of administrative data for these purposes, but do limit the ways they may be used. Another potential benefit of administrative data in clinical studies is the ability to enhance follow-up of patients already enrolled in studies [27]. Administrative data that provide longitudinal information or can be readily linked through available identifiers to other outcome registries (death, ESRD) can be a powerful and cost-saving tool to augment long-term follow up to determine basic outcome information such as mortality, or dialysis. Administrative data can also provide information on other important outcomes such as long-term healthcare resource use by individuals following an intervention as well as cost data. The latter may be an important tool as larger trials are proposed and follow-up of individuals becomes prohibitively expensive, particularly among studies where patients may be more difficult to track using traditional means such as telephone interviews or surveys. Notably, some administrative databases may be “locked” in terms of the data available in them (e.g. Medicare) that may preclude use for tracking specific outcomes. For example, some select adverse events used as surrogate or part of a composite outcome may require more detailed information for adjudication (e.g. post-operative myocardial infarction) that available. However, other administrative databases may have some flexibility in terms of adding data fields for the purposes of a clinical study. An example of this might be the addition of the type and duration of dialysis in a registry of ICU patients that normally only requires inclusion of a binary variable for dialysis. Given the current time lag of data availability in most administrative datasets, some may be less well-suited to guide real-time enrollment or randomization of individual patients during the acute phase of illness. This would be especially true where information on short-term events (e.g. hospital mortality) or a greater degree of granularity to accurately capture process-oriented variables such as select adverse events or complications are required in a timely fashion. However, administrative or registry data may be potentially used in certain situations. For example, in a cluster-randomized study where a process of care such as quality improvement or compliance with care bundles (e.g. use of intravenous volume expansion prior to contrast exposure) is an outcome, the use of administrative data may be feasible. More recently developed disease and procedure registries, such as the APPROACH database, a Canadian registry of patients undergoing cardiac catheterization and cardiac surgery, are being used to capture the majority of process and outcome data for the trial. (ClinicalTrials.gov Identifier: NCT02096406). Including research stakeholders within administrative or diseaseregistry development or oversight teams may facilitate their configuration to integrate research and quality improvement aims. Conclusions In summary, administrative datasets have facilitated the study of AKI on a population-level and are being leveraged by an expanding user base. Major limitations currently include low sensitivity, the subjective nature of assessment, and limited clinical phenotyping of AKI. These limitations highlight important opportunities to improve the quality and coding of data to better address critical knowledge gaps in AKI and improve care. Refinements in coding and the potential to link to increasingly sophisticated EMR data represent key opportunities to advance current administrative data. While an idealized, comprehensive definition of AKI is not currently tenable, tangible progress can currently be made and can help refine how AKI is captured and described within administrative data. We propose the integration of standardized classification schemes and the improvement of linkage of AKI to the clinical context in which it occurs as feasible, substantial improvements on current coding. Progress will require continued efforts to improve awareness of the impact of AKI on public health, engagement of key stakeholders, and tangible strategies to reconfigure infrastructure and improve the reporting and phenotyping of AKI. Competing interests EDS, RKB, HW, and ADS report no relevant disclosures. SMB has consulted for and received speaker fees and unrestricted educational grants from Baxter and Spectral. JAK has received grant support and/or consulting fees from: Astute Medical, Baxter, Fresenius, Spectral and Premier. SLG receives grant funding from Baxter Healthcare and consulting fees from: Baxter Healthcare, Akebia, La Jolla Pharmaceuticals, Bellco, and AM Pharma. Siew et al. Canadian Journal of Kidney Health and Disease (2016) 3:12 Page 11 of 12 Authors’ contributions EDS was primarily responsible for drafting and revising the manuscript and providing intellectual input. RB, HW, ADS, and SMB all drafted portions of the manuscript and provided intellectual input. SLG, CR, and JAK were responsible for the conception of the ADQI, its methodologic approach, the identification of key clinical questions, providing intellectual input, and revising the manuscript. All authors read and approved the final manuscript. Faculty of Medicine and Dentistry, and the Faculty of Medicine and Dentistry, University of Alberta (Edmonton, AB, Canada); Center for Acute Care Nephrology, Cincinnati Children's Hospital Medical Center (Cincinnati, OH, USA); Astute Medical (San Diego, CA, USA); Baxter Healthcare Corp (Chicago, IL, USA); Fresenius Medical Care Canada (Richmond Hill, ON, Canada); iMDsoft Inc (Tel Aviv, Israel); La Jolla Pharmaceutical (San Diego, CA, USA); NxStage Medical (Lawrence, MA, USA); Premier Inc (Charlotte, NC, USA); Philips (Andover, MA, USA); Spectral Medical (Toronto, ON, Canada). Acknowledgements EDS is supported by the Veterans Affairs HSR&D Merit Award IIR 13-073-3, NIDDK U01 DK92192-07 (The ASSESS-AKI Study), and the Vanderbilt Center for Kidney Disease (VCKD). HW’s research is supported by the Canadian Institutes of Health Research and the Sunnybrook Research Institute. SMB is supported by a Canada Research Chair in Critical Care Nephrology. ADQI 15 consensus group contributors Sean M. Bagshaw, Division of Critical Care Medicine, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada; Rajit Basu, Division of Critical Care and the Center for Acute Care Nephrology, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; Azra Bihorac, Division of Critical Care Medicine, Department of Anesthesiology, University of Florida, Gainesville, FL, USA; Lakhmir S. Chawla, Departments of Medicine and Critical Care, George Washington University Medical Center, Washington, DC, USA; Michael Darmon, Department of Intensive Care Medicine, Saint-Etienne University Hospital, Saint-Priest-En-Jarez, France; R.T. Noel Gibney, Division of Critical Care Medicine, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada; Stuart L. Goldstein, Department of Pediatrics, Division of Nephrology &Hypertension, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; Charles E. Hobson, Department of Health Services Research, Management and Policy, University of Florida, Gainesville, FL, USA; Eric Hoste, Department of Intensive Care Medicine, Ghent University Hospital, Ghent University, Ghent, Belgium, and Research Foundation – Flanders, Brussels, Belgium; Darren Hudson, Division of Critical Care Medicine, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada; Raymond K. Hsu, Department of Medicine, Division of Nephrology, University of California San Francisco, San Francisco, CA, USA; Sandra L. Kane-Gill, Departments of Pharmacy, Critical Care Medicine and Clinical Translational Sciences, University of Pittsburgh, Pittsburgh, PA, USA; Kianoush Kashani, Divisions of Nephrology and Hypertension, Division of Pulmonary and Critical Care Medicine, Department of Medicine, Mayo Clinic, Rochester, MN, USA; John A. Kellum, Center for Critical Care Nephrology, Department of Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, USA; Andrew A. Kramer, Prescient Healthcare Consulting, LLC, Charlottesville, VA, USA; Matthew T. James, Departments of Medicine and Community Health Sciences, Cumming School of Medicine, University of Calgary, Calgary, Canada; Ravindra Mehta, Department of Medicine, UCSD, San Diego, CA, USA; Sumit Mohan, Department of Medicine, Division of Nephrology, College of Physicians & Surgeons and Department of Epidemiology Mailman School of Public Health, Columbia University, New York, NY, USA; Hude Quan, Department of Community Health Sciences, Cumming School of Medicine, University of Calgary, Calgary, Canada; Claudio Ronco, Department of Nephrology, Dialysis and Transplantation, International Renal Research Institute of Vicenza, San Bortolo Hospital, Vicenza, Italy; Andrew Shaw, Department of Anesthesia, Division of Cardiothoracic Anesthesiology, Vanderbilt University Medical Center, Nashville, TN, USA; Nicholas Selby, Division of Health Sciences and Graduate Entry Medicine, School of Medicine, University of Nottingham, UK; Edward Siew, Department of Medicine, Division of Nephrology and Hypertension, Vanderbilt Center for Kidney Disease (VCKD), Vanderbilt University Medical Center, Nashville, TN, USA; Scott M. Sutherland, Department of Pediatrics, Division of Nephrology, Stanford University, Stanford, CA, USA; F. Perry Wilson, Section of Nephrology, Program of Applied Translational Research, Yale University School of Medicine, New Haven, CT, USA; Hannah Wunsch, Department of Critical Care Medicine, Sunnybrook Health Sciences Center and Sunnybrook Research Institute; Department of Anesthesia and Interdepartmental Division of Critical Care, University of Toronto, Toronto, Canada Continuing medical education The 15th ADQI Consensus Conference held in Banff, Canada on September 6-8th, 2015 was CME accredited Continuing Medical Education and Professional Development, University of Calgary, Calgary, Canada. Financial support Funding for the 15th ADQI Consensus Conference was provided by unrestricted educational support from: Division of Critical Care Medicine, Author details 1 Tennessee Valley Health System (TVHS), Nashville Veterans Affairs Hospital, Nashville, TN, USA. 2Vanderbilt University Medical Center, Department of Medicine, Division of Nephrology and Hypertension, Vanderbilt Center for Kidney Diseases (VCKD), 1161 21st Avenue South, MCN S3223, Nashville, TN 37232, USA. 3Cincinnati Children’s Hospital, Department of Pediatrics, Division of Critical Care Medicine and the Center for Acute Care Nephrology, The University of Cincinnati, Cincinnati, OH, USA. 4Department of Critical Care Medicine, Sunnybrook Health Sciences Center Center and Sunnybrook Research Institute; Department of Anesthesia and Interdepartmental Division of Critical Care, University of Toronto, Toronto, ON, Canada. 5Vanderbilt University Medical Center Department of Anesthesiology, Nashville, TN, USA. 6 Center for Acute Care Nephrology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA. 7Department of Nephrology, Dialysis and Transplantation, International Renal Research Institute (IRRIV), San Bortolo Hospital, Vicenza, Italy. 8Center for Critical Care Nephrology, Department of Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, USA. 9Division of Critical Care Medicine, Faculty of Medicine and Dentistry, University of Alberta, Alberta, Canada. Received: 23 October 2015 Accepted: 14 January 2016 References 1. Iezzoni LI. Assessing quality using administrative data. Annals of internal medicine. 1997;127(8 Pt 2):666–74. PubMed. 2. Manaker S. Time to Get Off the diagnosis dime onto the 10th revision of the international classification of diseases. Annals of internal medicine. 2015; 8. PubMed. 3. Kellum JA, Mehta RL, Angus DC, Palevsky P, Ronco C, Workgroup A. The first international consensus conference on continuous renal replacement therapy. Kidney international. 2002;62(5):1855–63. PubMed. 4. Moriyama I, Loy, RM, Robb-Smith AHT. History of the Statistical Classification of Diseases and Causes of Death (edited by Rosenberg, HM, Hoyert, DL). National Center fo Health Statistics; 2011. Accessed at www.cdcgov/nchs/ data/misc/classification_diseases2011pdf. 2011. 5. Bihorac A, Delano MJ, Schold JD, Lopez MC, Nathens AB, Maier RV, et al. Incidence, clinical predictors, genomics, and outcome of acute kidney injury among trauma patients. Annals of surgery. 2010;252(1):158–65. PubMed Pubmed Central PMCID: 3357629. 6. Bihorac A, Brennan M, Ozrazgat-Baslanti T, Bozorgmehri S, Efron PA, Moore FA, et al. National surgical quality improvement program underestimates the risk associated with mild and moderate postoperative acute kidney injury. Critical care medicine. 2013;41(11):2570–83. PubMed Pubmed Central PMCID: 3812338. 7. American College of Surgeions National Surgical Quality Improvement Program: User Guide for the 2010 Participant Use Data Faile. Chicago I, American College of Surgeons. 2010. 8. Vlasschaert ME, Bejaimal SA, Hackam DG, Quinn R, Cuerden MS, Oliver MJ, et al. Validity of administrative database coding for kidney disease: a systematic review. American journal of kidney diseases : the official journal of the National Kidney Foundation. 2011;57(1):29–43. PubMed. 9. Waikar SS, Wald R, Chertow GM, Curhan GC, Winkelmayer WC, Liangos O, et al. Validity of international classification of diseases, ninth revision, clinical modification codes for acute renal failure. Journal of the American Society of Nephrology : JASN. 2006;17(6): 1688–94. PubMed. 10. Hwang YJ, Shariff SZ, Gandhi S, Wald R, Clark E, Fleet JL, et al. Validity of the International Classification of Diseases, Tenth Revision code for acute kidney injury in elderly patients at presentation to the emergency department and at hospital admission. BMJ open. 2012;2(6). PubMed Pubmed Central PMCID: 3533048. Siew et al. Canadian Journal of Kidney Health and Disease (2016) 3:12 11. Grams ME, Waikar SS, MacMahon B, Whelton S, Ballew SH, Coresh J. Performance and limitations of administrative data in the identification of AKI. Clinical journal of the American Society of Nephrology : CJASN. 2014; 9(4):682–9. PubMed Pubmed Central PMCID: 3974361. 12. Schaffzin JK, Dodd CN, Nguyen H, Schondelmeyer A, Campanella S, Goldstein SL. Administrative data misclassifies and fails to identify nephrotoxin-associated acute kidney injury in hospitalized children. Hospital pediatrics. 2014;4(3):159–66. PubMed. 13. Waikar SS, Curhan GC, Wald R, McCarthy EP, Chertow GM. Declining mortality in patients with acute renal failure, 1988 to 2002. J Am Soc Nephrol. 2006;17(4):1143–50. PubMed Epub 2006/02/24. eng. 14. Lewington AJ, Cerda J, Mehta RL. Raising awareness of acute kidney injury: a global perspective of a silent killer. Kidney international. 2013;84(3):457–67. PubMed Pubmed Central PMCID: 3758780. 15. Hsu RK, McCulloch CE, Dudley RA, Lo LJ, Hsu CY. Temporal changes in incidence of dialysis-requiring acute kidney injury. J Am Soc Nephrol. 2013; 24(1):37–42. 16. Siddiqui NF, Coca SG, Devereaux PJ, Jain AK, Li L, Luo J, et al. Secular trends in acute dialysis after elective major surgery–1995 to 2009. CMAJ. 2012; 184(11):1237–45. PubMed Pubmed Central PMCID: 3414596. 17. Siew ED, Davenport A. The growth of acute kidney injury: a rising tide or just closer attention to detail? Kidney international. 2014. PubMed. 18. Selby NM, Hill R, Fluck RJ, Programme NHSETKA. Standardizing the early identification of acute kidney injury: the NHS England national patient safety alert. Nephron. 2015;10. PubMed. 19. Bellomo R, Ronco C, Kellum JA, Mehta RL, Palevsky P. Acute renal failure definition, outcome measures, animal models, fluid therapy and information technology needs: the Second International Consensus Conference of the Acute Dialysis Quality Initiative (ADQI) Group. Critical care. 2004;8(4):R204–12. PubMed. 20. Mehta RL, Kellum JA, Shah SV, Molitoris BA, Ronco C, Warnock DG, et al. Acute Kidney Injury Network: report of an initiative to improve outcomes in acute kidney injury. Crit. care. 2007;11(2):R31. PubMed Epub 2007/03/03. eng. 21. Kellum JA, Lameire N, for the KAKIGWG. Diagnosis, evaluation, and management of acute kidney injury: a KDIGO summary (Part 1). Crit care. 2013;17(1):204. PubMed. 22. Kolhe NV, Staples D, Reilly T, Merrison D, McIntyre CW, Fluck RJ, et al. Impact of compliance with a care bundle on acute kidney injury outcomes: a prospective observational study. PloS one. 2015;10(7), e0132279. PubMed Pubmed Central PMCID: 4498890. 23. Wilson FP, Shashaty M, Testani J, Aqeel I, Borovskiy Y, Ellenberg SS, et al. Automated, electronic alerts for acute kidney injury: a single-blind, parallelgroup, randomised controlled trial. Lancet. 2015;385(9981):1966–74. PubMed Pubmed Central PMCID: 4475457. 24. Joannes-Boyau O, Honore PM, Perez P, Bagshaw SM, Grand H, Canivet JL, et al. High-volume versus standard-volume haemofiltration for septic shock patients with acute kidney injury (IVOIRE study): a multicentre randomized controlled trial. Intensive care medicine. 2013;39(9):1535–46. PubMed. 25. Pro CI, Yealy DM, Kellum JA, Huang DT, Barnato AE, Weissfeld LA, et al. A randomized trial of protocol-based care for early septic shock. The New England journal of medicine. 2014;370(18):1683–93. PubMed Pubmed Central PMCID: 4101700. 26. Mouncey PR, Osborn TM, Power GS, Harrison DA, Sadique MZ, Grieve RD, et al. Trial of early, goal-directed resuscitation for septic shock. The New England journal of medicine. 2015;372(14):1301–11. PubMed. 27. Noah MA, Peek GJ, Finney SJ, Griffiths MJ, Harrison DA, Grieve R, et al. Referral to an extracorporeal membrane oxygenation center and mortality among patients with severe 2009 influenza A(H1N1). Jama. 2011;306(15): 1659–68. PubMed. 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Lumbar Radiculitis as a Complication of Vaccination against Tick-Borne Encephalitis: A Differential Diagnosis of Low Back Pain and Nerve Root Compression
Case reports in medicine
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Hindawi Case Reports in Medicine Volume 2020, Article ID 6130364, 4 pages https://doi.org/10.1155/2020/6130364 Hindawi Case Reports in Medicine Volume 2020, Article ID 6130364, 4 pages https://doi.org/10.1155/2020/6130364 Craig Kingston ,1 G¨unther Zech,1 Caroline Pauli,2 and Ulrich Walker 1 1Basel University Hospital, Department of Rheumatology, Basel, Switzerland 2Praxis Pauli AG, Reinach, Switzerland Correspondence should be addressed to Ulrich Walker; ulrich.walker@usb.ch Received 27 August 2019; Accepted 17 February 2020 Academic Editor: Walter Zidek Copyright © 2020 Craig Kingston 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. Serious adverse reactions following immunisation with adult tick-borne encephalitis (TBE) vaccines are rare, but when they occur, they most frequently involve the nervous system. We present a case of a female patient who developed a sensory and motor L4 monoradiculopathy following self-injection of an inactivated vaccine against TBE in the ipsilateral quadriceps muscle. The motor and sensory L4 dysfunction vanished after 12 months. TBE vaccine-induced radiculopathy should be considered as a mimic of spinal root compression. . Discussion According to the latest consensus, TBE vaccination is rec- ommended for all age groups above 1 year in highly endemic areas (≥5 cases/100,000/year) and also for individuals at risk in areas with a lower incidence [2]. Many European countries are reporting an increasing incidence of TBE, the highest being in Slovenia, Lithuania, and Latvia [2] with foci now occurring as far north as Norway, Sweden, and Finland [4]. New endemic foci have recently been documented in Siberia, Mongolia, northern China, the Korean peninsula, Kyrgyzstan, Armenia, Azer- baijan, Uzbekistan, and Kazakhstan, including areas at al- titudes of up to 2000 m. Austria is one of the few countries which has recently seen a fall in TBE cases due to a robust vaccination program, but the risk for the unvaccinated re- mains high [2].h In 2004, a case report of acute disseminated en- cephalomyelitis (ADEM) following the third vaccination against TBE was published from Germany. ADEM manifested as recurrent retrobulbar neuritis, grand mal seizures, and a single focal tonic-clonic episode. The causality was interpreted as “definite” by the author. A similar case was presented as a poster at the annual conference of the European Society of Neurology in 1998 [13]. In 1998, there was another case report from Germany of diverse psychomotor deficits (left faciobrachial sensory deficits, a fluctuating slowing of psychomotor function, disorientation, and memory deficits) in a patient fol- lowing TBE vaccination, all of which had completely subsided 8 weeks after exposition [14]. From a publication in 1992, there were a total of 72 registered cases of neurological side effects following TBE vaccination [15]. From these, only three were considered definite or probable, and all presented as some form of neuritis without permanent sequelae. In a small randomized controlled trial of patients with multiple sclerosis, how- ever, there was no significant worsening of symptoms of demyelination in the vaccinated group in comparison to the placebo group [16]. The prevalence of infected ticks can also vary greatly within countries, with certain areas of Switzerland having a prevalence of over 14% in a single focus [1]. This trend towards an increasing number of cases per year is typical for many northern European countries. 1. Introduction A 40-year-old female patient presented to our service with a 5-day history of progressive right-sided low back pain, which radiated over the right buttock into the anterior thigh in an L4 distribution. The pain was nonpositional and exacerbated by coughing. She also complained of new bifrontal head- aches, difficulty in concentrating, stool retention, and dif- ficulty in passing urine. Two weeks previously, she had commenced an immunisation against adult tick-borne en- cephalitis (TBE) by self-injecting a vaccine (Encepur™) into the ipsilateral quadriceps muscle. Her medical history was otherwise unremarkable. Symptoms of a viral infection such as fever, rash, and oral or genital lesions were not present, and recent exposure to tics was denied. The TBE virus is a member of the genus Flavivirus, family Flaviviridae, and is the causative agent implicated in the disease TBE. In Western Europe, it is primarily transmitted via infected Ixodes ricinus ticks and has in recent years been observed at increasingly higher latitudes [1]. The total number of annual cases is estimated to be up to 13,000 in the Eurasian northern hemisphere [2]. In addition to physical barrier measures such as pro- tective clothing, vaccination is the most effective method for preventing TBE. Two preparations are available for adults in Europe, namely, FSME-Immune™and Encepur™. These vaccines have been robustly tested and are considered to be safe and effective [1]. f Side effects are common but generally mild and transient and include mainly headache (12%), fever (1–10%), muscle pain (12%), and local irritation (46%). Transient paraesthesia occurs rarely (<0.01%). Isolated cases of serious adverse neurological events, including transient encephalitis, myelitis, oculomotor paresis, and Guillain–Barr´e syndrome, have been reported [3].h The patient was afebrile, her blood pressure was 170/110, and her heart rate was 92. The alignment of the spinal column was normal. On the right leg, there was a positive straight-leg raising test, weakness of knee extension, and a diminished patellar reflex. There was also slight hypaesthesia over the knee and the anterior aspect of the thigh. The Babinski sign was negative, and there was no evidence of meningism such as photophobia, neck-stiffness, or a positive Brudzinski sign. The following case describes a unique neurological side effect simulating a unilateral lumbar radiculopathy. 2 Case Reports in Medicine The ESR was 7 mm/hr, her CRP was 8.9 mg/L, and the blood differential was normal. The remaining blood chemistry was negative, including tests for paraproteins, ANA, and HIV. 1. Introduction Abdominal ultrasound excluded urine retention and other intraabdominal pathology. On con- trast-enhanced MRI, there was no compression of the spinal roots or the cauda equina and no involvement of the cranial meninges. The patient refused a lumbar puncture. demonstrating economic saving related to decreased in- cidence, especially of severe cases [8]. In vivo and in vitro studies have shown that the European vaccines protect against all TBEV subtypes circulating in endemic areas of Europe and Asia, indicating equally potent cross-pro- tection [2]. Due to a decline in antibody production following vaccination in old age, an additional priming dose of the vaccine has been implemented in Sweden in those over 60 years of age [9]. Serious adverse reactions are extremely rare and most frequently involve the nervous system. The mechanism by which the vaccine may induce neurological deficits is not well understood. The most likely mode of damage is mo- lecular mimicry, in which a conformational homology between a vaccine antigen and self-antigen directs T cells and/or humoral immunity towards host tissue. Molecular mimicry has been demonstrated in multiple animal models and most notably in experimental allergic encephalomy- elitis [10]. Both TBE vaccine preparations have been im- plicated in nervous system adverse reactions [11, 12]. Swissmedic (formerly the Swiss Drug Monitoring Centre) received notifications between 1987 and 2000 regarding 33 patients with 39 adverse neurological reactions following TBE vaccination. Twelve of these patients were hospital- ised, and all of the reactions were completely reversible after a maximal period of one month. The latency period ranged from a few hours to 30 days, with the majority of reactions after the first vaccination. This included three cases with radicular symptoms and five cases with men- ingeal symptoms [12]. We diagnosed a sensorimotor radiculitis of the fourth lumbar root secondary to TBE vaccination. A 5-day course of prednisolone (1 mg/kg) did not influence symptoms and was thus discontinued. Three months later, the patient still required analgesics (NSAIDS, tramadol, and pregabalin) and had developed a pronounced weakness and atrophy of the quadriceps muscle and a foot drop of the affected leg. The motor and sensory function had started to return to normal at 6 months, and by 12 months, the deficit had vanished. In contrast to this, the headaches and difficulties to concentrate persisted at the one-year mark. . Discussion Until 2019, vaccination was only indicated for specific endemic regions within Switzerland but due to increasing incidence (see Figure 1), there is now a recommendation to vaccinate the population in the whole country, except the counties of Geneva and Tessin [5]. The seroprevalence rate in high-risk areas of Western European countries such as France remain however lower than those of other tick-borne diseases such as bor- reliosis or tularaemia [6]. The vaccine is normally administered in 3 doses over a period of 6 months. A Cochrane review summarized 11 vaccine trials, and amongst them, four randomized studies were on licensed European vaccines (Encepur and FSME-Immune) with a total of 5063 participants: the vaccines reached seroconversion rates of 92%–100% [7]. Cost-effectiveness studies on TBE vaccines have been performed in Slovenia, Sweden, Finland, and Estonia, As these case reports show, motor, sensory, and memory deficits are known but are rare complications of vaccination. Our case is the first to demonstrate a prolonged neurological deficit as a result of TBE vaccination. Adverse reactions to TBE vaccines may be an underdiagnosed mimic of spinal root compression. 2000 2002 2004 2006 2008 2010 2012 2016 2018 2014 Year 400 300 200 100 0 Number of cases Figure 1: Annual number of TBE cases in Switzerland, adapted from [5] with permission. Case Reports in Medicine 3 Case Reports in Medicine 3 Figure 1: Annual number of TBE cases in Switzerland, adapted from [5] with permission. 4. Conclusions borne diseases. Guidelines from the French scientific societies (I): prevention, epidemiology , diagnosis,” M´edecine et mal- adies infectieuses, vol. 49, no. 5, pp. 318–334, 2019, in Press.f Neurological complications are one of the more common side effects of TBE vaccines, though most take the form of transient headaches. Serious neurological complications are exceedingly rare and to date, completely reversible. Tick- borne encephalitis vaccines can cause a radiculopathy that mimics spinal root compression, which can cause long-term functional deficits. The safety profile of the available TBE vaccines remains however excellent, and TBE vaccines should therefore continue to be administered in endemic areas, as this is associated with a dramatic fall in the number of TBE cases [17]. [7] V Demicheli, P. Graves, M. Pratt, and T. Jefferson, Vaccines for preventing tick-borne encephalitis,” Cochrane Database of Systematic Reviews, vol. 1, 2003. [8] R. ˇSmit and M. J. Postma, “Vaccines for tick-borne diseases and cost effectiveness of vaccination: a public health challenge to reduce the diseases burden,” Expert Review of Vaccines, vol. 15, no. 1, pp. 5–7, 2016. [9] K. E. Hansson, A. Rosdahl, M. Insulander et al., “Tick-borne encephalitis vaccine failures: a 10-year retrospective study supporting the rationale for adding an extra priming dose in individuals starting at age 50 years,” Clinical Infectious Dis- eases, vol. 70, no. 2, pp. 245–251, 2019. Conflicts of Interest [10] M. B. A. Oldstone, “Molecular mimicry, microbial infection, and autoimmune disease: evolution of the concept,” in Mo- lecular Mimicry: Infection-Inducing Autoimmune Disease. Current Topics in Microbiology and Immunology, M. B. Oldstone, Ed., vol. 296, Berlin, Heidelberg, Springer, 2005. The authors declare that they have no conflicts of interest. [17] C. Kunz, “TBE vaccination and the Austrian experience,” Vaccine, vol. 21, no. 1, pp. S50–S55, 2003. References [11] C. Schattenfroh, “Acute disseminated encephalomyelitis after active immunisation against early summer encephalitis,” Nervenarzt, vol. 75, no. 8, pp. 776–779, 2004. [1] K. L. Mansfield, N. Johnson, L. P. Phipps, J. R. Stephenson, A. R. Fooks, and T. Solomon, “Tick-borne encephalitis virus—a review of emerging zoonosis,” Journal of General Virology, vol. 90, no. 8, pp. 1781–1794, 2009. [12] A. Doser, K. Hartmann, F. Fleisch, and M. Kuhn, “Suspected neurological side effects of tick-borne meningoencephalitis vaccination: experiences of the swiss adverse drug reaction reporting centre,” Schweizerische Rundschau fur Medizin Praxis, vol. 91, pp. 159–162, 2002. gy [2] P. Taba, E. Schmutzhard, P. Forsberg et al., “EAN consensus review on prevention, diagnosis and management of tick- borne encephalitis,” European Journal of Neurology, vol. 24, no. 10, pp. 1214–e61, 2017. [13] G. Roob, “Acute disseminated encephalomyelitis following acitve immunisation for European spring-summer menin- goencephalitis,” Journal of Neurology, vol. 245, pp. 245–434, 1998. pp [3] https://compendium.ch/prod/encepur-n-inj-susp/de. [4] T. Smura, E. Tonteri, A. J¨a¨askel¨ainen et al., “Recent estab- lishment of tick-borne encephalitis foci with distinct viral lineages in the Helsinki area, Finland,” Emerging Microbes & Infections, vol. 8, no. 1, pp. 675–683, 2019. [14] M. Schabet, H. Wiethoelter, W. Grodd et al., “Neurological complications after simultaneous immunisation against tick- borne encephalitis and tetanus,” The Lancet, vol. 333, no. 8644, pp. 959-960, 1989. [5] Bundesamt f¨ur Gesundheit Schweiz, Abteilung ¨Ubertragbare Krankheiten, Fr¨uhsommer-Meningoenzephalitis (FSME): Aus- weitung der Risikogebiete, 2019, https://www.bag.admin.ch/dam/ bag/de/dokumente/mt/i-und-b/richtlinien-empfehlungen/neue- empfehlungen-2019/zeckenenzephalitis-impfung-risikogebiet.pdf. download.pdf/zeckenenzephalitis-impfung-risikogebiet-de.pdf. [15] R. Gold, H. Wieth¨olter, I. Rihs, J. L¨ower, and L. Kappos, “Fr¨uhsommer-meningoenzephalitis-impfung–indikation und kritische beurteilung neurologischer impfkomplikationen,” Deutsche Medizinische Wochenschrift, vol. 117, pp. 112–116, 1992. g g [6] J. Figoni, C. Chirouze, Y. Hansmann, C. Lemogne, V. Hentgen, and A. Saunier, “Lyme borreliosis and other tick- Case Reports in Medicine 4 [16] U. Baumhackl, C. Franta, J. Retzl, E. Salomonowitz, and G. Eder, “A controlled trial of tick-borne encephalitis vac- cination in patients with multiple sclerosis,” Vaccine, vol. 21, pp. S56–S61, 2003. [17] C. Kunz, “TBE vaccination and the Austrian experience,” Vaccine, vol. 21, no. 1, pp. S50–S55, 2003.
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Dexmedetomidine Alleviates Lipopolysaccharide-Induced Hippocampal Neuronal Apoptosis via Inhibiting the p38 MAPK/c-Myc/CLIC4 Signaling Pathway in Rats
Molecular neurobiology
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Research Article Research Article Keywords: Dexmedetomidine, Lipopolysaccharide, Hippocampal neuronal apoptosis, p38 MAPK/c- Myc/CLIC4 signaling pathway, Alzheimer's disease, Depression Posted Date: June 2nd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-538490/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Keywords: Dexmedetomidine, Lipopolysaccharide, Hippocampal neuronal apoptosis, p38 MAPK/c- Myc/CLIC4 signaling pathway, Alzheimer's disease, Depression Posted Date: June 2nd, 2021 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/22 Page 1/22 Version of Record: A version of this preprint was published at Molecular Neurobiology on August 7th, 2021. See the published version at https://doi.org/10.1007/s12035-021-02512-9. Version of Record: A version of this preprint was published at Molecular Neurobiology on August 7th, 2021. See the published version at https://doi.org/10.1007/s12035-021-02512-9. Version of Record: A version of this preprint was published at Molecular Neurobiology on August 7th, 2021. See the published version at https://doi.org/10.1007/s12035-021-02512-9. Page 2/22 Abstract Dexmedetomidine (DEX) reportedly possessed multiple bioactivities. Here, we mainly investigated the neuroprotective role and detailed molecular mechanism of DEX against lipopolysaccharide (LPS)-induced hippocampal neurons apoptosis. In vivo, Sprague Dawley rats were administered with LPS (10 mg/kg) and/or DEX (30 µg/kg). We found that DEX improved LPS-induced hippocampal microstructure (necrosis and number reduction of neurons in the CA1 and CA3 regions) and ultrastructure (mitochondrial damage) lesions. DEX also attenuated LPS-induced hippocampal apoptosis by down-regulating the expression of mitochondrial apoptosis pathway-related proteins. Moreover, DEX prevented the activation of c- Myc/chloride intracellular channel 4 (CLIC4) pathway induced by LPS. Notably, DEX inhibited p38 MAPK pathway, not JNK and ERK. To further clarify whether DEX alleviated LPS-induced neuronal apoptosis through the p38 MAPK/c-Myc/CLIC4 pathway, PC12 cells were treated with p38 MAPK inhibitor SB203582 (10µM). As expected, DEX had the same effect as SB203582 in reducing the protein and mRNA expression of c-Myc and CLIC4. Furthermore, DEX and SB203582 diminished LPS-induced apoptosis, showing decreased Bax and Tom20 fluorescent double-stained positive cells, reduced Annexin V-FITC/PI apoptosis rate, and lessened protein expression levels of Bax, cytochrome C, cleaved caspase-9 and cleaved caspase-3. Taken together, DEX attenuates LPS-induced hippocampal neuronal apoptosis by regulating the p38 MAPK/c-Myc/CLIC4 signaling pathway, which will provide new insights into the mechanism research and drug development of Alzheimer's disease and depression. 1. Introduction Alzheimer's disease (AD) and depression pose a huge threat to human health and are major causes of increasing morbidity and mortality worldwide. According to incomplete statistics, approximately, 5.4 million people in the United States alone have AD [1]. Beyond that, more than 350 million victims in the world are currently affected by depression [2], which is predicted to be the world's second largest disability-inducing factor by 2030 [3]. Notably, hippocampal injury (loss of neuronal populations [4], reduced hippocampal volume [5], and massive neuronal death [6]) is regarded as an important pathological hallmark of AD and depression. Numerous studies have corroborated that apoptosis is implicated in hippocampal injury. Clearly, it is necessary to find safe, side-effect-free antiapoptotic drugs to prevent and treat AD and depression. Lipopolysaccharide (LPS) was found to be abundant in hippocampal lysates of AD brain [7]. It is well known that LPS can give rise to depression [8]. Li et al. revealed that LPS induces hippocampal neuronal apoptosis by activating the c-Myc/chloride intracellular channel 4 (CLIC4) pathway [9]. Notably, the mitogen-activated protein kinase (MAPK) pathway is implicated in hippocampal apoptosis [10]. The MAPK family consists of stress-activated protein kinases (JNK), extracellular signal-regulated kinase (ERK) and p38 MAPK. The JNK signaling pathway has been reported to be involved in the apoptosis process of hippocampal neuronal regulated by oxidative stress [11]. ERK participates in the pathogenesis of children's learning disabilities by activating hippocampal apoptosis [12]. In addition, p38 MAPK have been demonstrated to mediate apoptosis via multiple mechanisms, such as increased c-Myc expression Page 3/22 Page 3/22 [13], Bax transposition, and caspase-3 activation [14]. However, whether the MAPK pathway mediates LPS-induced hippocampal apoptosis is unknown. Moreover, which member of the MAPK family is involved in the process of LPS-induced hippocampal apoptosis through the regulation of c-Myc/CLIC4 pathway is unknown. Dexmedetomidine (DEX), as a neurological drug, possesses multiple biological activities such as anti- inflammation [15], anti-oxidative stress [16] and anti-apoptosis [17]. Recently, it has been reported that DEX exerts neuroprotective efficacy in various brain injury models [18, 19]. However, the specific underlying molecular mechanism is extremely complex and elusive. Importantly, DEX can reverse LPS- induced neuronal apoptosis [20]. Additionally, DEX has been proven to weaken apoptosis by inhibiting MAPK pathway activation [21]. DEX can also alleviate apoptosis through regulating p38 MAPK/ERK pathway and ROS/JNK pathway, respectively [22, 23]. 2.1. Animals and Treatments Forty-eight male Sprague Dawley rats (6 weeks old, 190-210g) were provided by the Central Hospital of Harbin Medical University (Harbin, China). Rats were maintained under standard conditions and access to water and standard rodent pellet food ad libitum [24]. All animal procedures in the present study were conducted according to the Animal Ethics Committee of the Northeast Agricultural University (SRM-11, China). After a week of acclimatization, rats were divided at random into four groups (n = 12): CON, LPS, LPS +  DEX and DEX. LPS group was given LPS (Escherichia coli 0111: B4; Sigma-Aldrich, San Francisco, USA) dissolved in 0.9% physiological saline at 10 mg/kg by intraperitoneally (i.p.) injected. LPS + DEX group rats were i.p. with DEX (30 µg/kg, Wuhan Belka Biomedical Co., Ltd, Wuhan, China) 0.5 h before LPS administration. DEX group was treated with DEX (30 µg/kg, i.p.). In the CON group, rats were administered with an equal volume of physiological saline by i.p. injected. The doses of DEX and LPS refer to previous reports [9, 25, 26]. 1. Introduction However, the detailed molecular mechanism of the protective effect of DEX on LPS-induced hippocampal apoptosis remains unclear. Accordingly, in vivo, we focused on investigating the effects of DEX on MAPK family and c-Myc/CLIC4 pathway in LPS-induced hippocampal apoptosis. In vitro, further elucidate whether the p38 MAPK/c- Myc/CLIC4 pathway is involved in the protective mechanism of DEX against LPS-induced hippocampal apoptosis by using the p38 MAPK inhibitor (SB203582). 2.3. Histopathology and Ultrastructural Observation Brain tissue samples fixed with 10% formalin for 24 h were dehydrated and then embedded in paraffin. Next, they were cut into 4–5 µm sections and stained with hematoxylin and eosin (H&E, Wuhan Biotechnology Ltd. Co., Wuhan, China). After 5–10 min, all sections were observed and photographed under an optical microscope (TE2000, Nikon, Japan). The number of neurons in hippocampal CA1 and CA3 region was counted by ImageJ software (400x magnification). After fixation with 3% glutaraldehyde for 48 h, the hippocampus blocks (1 mm3) were exposed to 1% osmium tetroxide for 2 h. Next, the blocks were dehydrated, embedded, sectioned (60 nm), and then stained with lead citrate. All samples were captured by transmission electron microscope (Tecnai-G212, FEI Company, Netherlands). Based on the morphology and integrity of the mitochondrial membrane and cristae, the mitochondria in six discontinuous fields of each sample were scored. The criteria for judging mitochondrial damage are as follows: 0, well-defined and organized membranes and cristae; 1, minor distortions and/or swellings, but general organization retained; 2, major distortions and/or swellings and discontinuous membranes and cristae; 3, membranes and cristae dissociated into particulates to produce diffuse mitochondrial ghosts; 4, only a few mitochondrial remnants in cells. 2.2. Sample Collection Four hours after the final treatment, all rats were anesthetized with isoflurane (Yipin Pharmaceutical Co., Ltd, Hebei, China) and sacrificed. Blood was quickly collected by cardiac puncture, and centrifuged at 3000 rpm for 10 min at 4°C to take the supernatant for inspection of inflammatory indicators. The whole Page 4/22 brains of three rats in each group were immediately fixed in 10% formalin solution for histopathological observation and immunohistochemical detection. Three hippocampal tissues per group were used for ultrastructural observation. The remaining four hippocampal tissues in each group were rapidly frozen in liquid nitrogen and then stored at − 80°C for Real-time PCR and Western blot analysis. 2.4. Immunohistochemistry Analysis Immunohistochemical analysis was performed as described previously [27]. Briefly, paraffin-embedded brain tissue slices were dewaxed with xylene, dehydrated with gradient alcohol, incubated with hydrogen peroxide, and sealed with goat serum. After that, they were incubated with primary and secondary antibodies and labeled with horseradish enzyme. DAB was used for color development. Finally, all slices were observed and photographed under a DP73 type microscope (OLYMPUS, Japan). 2.8. Dual Immunofluorescence and Annexin V-FITC/PI Staining Assay 2.8. Dual Immunofluorescence and Annexin V-FITC/PI Staining Assay Cells in each group were fixed with 4% paraformaldehyde for 30 min and then permeated with 0.3% Triton X-100 for 10 min. Next, they were incubated overnight with Bax (Wanlei, Shenyang, China) at 4°C. The cells slides were incubated with CY3-labelled anti-rabbit IgG (Goodbio Technology, Wuhan, China) in the dark for 1.5 h at 37°C, followed by washing 3 times with PBS. Afterwards, the cells were incubated overnight with Tom20 (ABclonal, Wuhan, China) at 4°C. Finally, they were incubated with 488-anti-rabbit IgG (Goodbio Technology, Wuhan, China) in the dark for 1.5 h at 37°C. Images were captured using a fluorescent inverted microscope (Nikon, Japan). The resuspended cells were incubated with 5 µL Annexin V-FITC and 10 µL propidium iodide (Bioss, Beijing, China) for 15 min. Then, cell apoptosis was detected by the Attune NxT flow cytometer (Thermo Fisher Scientific). 2.5. Cell Culture and Drug Treatments PC12 cells were obtained from the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM medium (Gibco, Waltham, MA, USA) containing 10% FBS and 1% penicillin-streptomycin in a humidified incubator (37°C, 95% relative humidity, 5% CO2). The experimental groups were as follows: CON, LPS, LPS + DEX and LPS + SB. CON group: the cells were cultured in untreated medium. LPS group: the cells were cultured in medium supplemented with LPS. LPS + DEX group: the cells were treated with DEX for 30 min and then cultured in medium supplemented with LPS. LPS + SB group: after 30 min of p38 MAPK inhibitor SB203582 (10µM, Selleck.cn, Shanghai, China) administration, the cells were cultured in medium with LPS. The concentration of LPS and DEX was determined by CCK-8 assay. Page 5/22 2.6. Cell Viability Assay and Observation of Cell Morphology Page 5/22 Page 5/22 Cells were seeded into 96-well plates (4 × 103 cells per well) for 12 h, and then treated with/without DEX in the presence/absence of LPS. After 24 h, the cell viability was determined with the CCK-8 kit (Beyotime Institute of Biotechnology, Suzhou, China) according to the manufacturer's instructions. The absorbance was read at 450 nm by a Bio-Tek Epoch microplate reader (Bio-Tek, Winooski, VT, USA). Cell morphology of each group was observed and captured by inverted microscope (Leica, Germany). 2.7. LDH and ATP Release Assay Released LDH, ATP content and total ATPase activity were determined by LDH assay kit, ATP content assay kit and ultra-micro total ATPase kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), respectively 2.11. Statistical analysis All data were represented as mean ± SD and analyzed using IBM SPASS Statistics 23 software (SPASS, IL, USA). Statistical analysis was conducted via one-way ANOVA, followed by Tukey’s post hoc test. Values with P < 0.05 was considered statistically significant, P < 0.01 were considered extremely significant. 2.10. Western blot analysis Hippocampal tissue and PC12 cells were lysed in RIPA buffer with PMSF and phosphatase inhibitor (Beyotime Biotechnology, Shanghai, China). After the protein concentration was determined using the BCA assay kit, the equivalent amount of protein samples was separated by SDS-PAGE gel electrophoresis, and transferred to the PVDF membrane. Then, membranes were blocked in 5% skim milk for 2 h at room temperature. Subsequently, membranes were incubated with primary antibodies adding Primary Antibody Dilution Buffer (Leagene Biotechnology, Beijing, China) overnight at 4℃. The primary antibodies include Bax, cleaved caspase-3, cleaved caspase-9 (1:1000, Cell Signaling Technology, USA); Bcl-2, cytochrome C, P-JNK, JNK, P-ERK, ERK, P-p38, p38 (1:750, Wanlei, Shenyang, China); c-Myc, CLIC4 (1:1000, Santa Cruz Biotechnology Inc, USA). Next, they were incubated with appropriate combination of secondary antibody for 2 h at 37°C. The immunoreactive protein bands were visualized using the enhanced chemiluminescence kit (Beyotime Biotechnology, Shanghai, China), and were captured by Amersham Imager 600 software (GE, USA). Finally, all protein bands were quantified with ImageJ software. 2.9. Real-time PCR analysis Total RNA was isolated from hippocampus tissue and PC12 cells by using Total RNA Extraction Kit (Promega Biotech Co, Ltd, Beijing, China). According to the corresponding procedures, superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) was used for reverse transcription to obtain cDNA. Primer sequences of c-Myc, CLIC4 and GAPDH are shown in Table 1. RT-PCR analysis was performed using LightCycler480 (Roche, Basel, Switzerland). The relative quantification of the target gene expression was calculated according to 2−ΔΔCt method. Page 6/22 Table 1 Primer sequence for real-time PCR detection. Gene Accession number Primer sequence (5’-3’) GAPDH XM_216453 Forward: AGTGCCAGCCTCGTCTCATA Reverse: GATGGTGATGGGTTTCCCGT c-Myc NM_012603 Forward: GGAGAAACGAGCTGAAGCGTAG Reverse: CAGCCAAGGTTGTGAGGTTAGG CLIC4 NM_031818 Forward: GTCACCACCGTTGACCTGAA Reverse: TTGGGTGGGCACAAGACTTC 2.10. Western blot analysis Hippocampal tissue and PC12 cells were lysed in RIPA buffer with PMSF and phosphatase inhibitor (Beyotime Biotechnology, Shanghai, China). After the protein concentration was determined using the BCA assay kit, the equivalent amount of protein samples was separated by SDS-PAGE gel electrophoresis, and transferred to the PVDF membrane. Then, membranes were blocked in 5% skim mil for 2 h at room temperature. Subsequently, membranes were incubated with primary antibodies adding Primary Antibody Dilution Buffer (Leagene Biotechnology, Beijing, China) overnight at 4℃. The primary antibodies include Bax, cleaved caspase-3, cleaved caspase-9 (1:1000, Cell Signaling Technology, USA); Bcl-2, cytochrome C, P-JNK, JNK, P-ERK, ERK, P-p38, p38 (1:750, Wanlei, Shenyang, China); c-Myc, CLIC4 (1:1000, Santa Cruz Biotechnology Inc, USA). Next, they were incubated with appropriate combination of secondary antibody for 2 h at 37°C. The immunoreactive protein bands were visualized using the enhanced chemiluminescence kit (Beyotime Biotechnology, Shanghai, China), and were captured by Amersham Imager 600 software (GE, USA). Finally, all protein bands were quantified with ImageJ software. 2.11. Statistical analysis Table 1 Primer sequence for real-time PCR detection. Gene Accession number Primer sequence (5’-3’) GAPDH XM_216453 Forward: AGTGCCAGCCTCGTCTCATA Reverse: GATGGTGATGGGTTTCCCGT c-Myc NM_012603 Forward: GGAGAAACGAGCTGAAGCGTAG Reverse: CAGCCAAGGTTGTGAGGTTAGG CLIC4 NM_031818 Forward: GTCACCACCGTTGACCTGAA Reverse: TTGGGTGGGCACAAGACTTC Table 1 3.3. Effects of DEX on the MAPK/c-Myc/CLIC4 Signaling Pathway To investigate whether the MAPK pathway mediated LPS-induced hippocampal apoptosis, JNK, ERK, and p38 protein expressions were examined (Fig. 3a). Interestingly, the protein expression levels of P-JNK (Fig. 3b), JNK (Fig. 3c), P-JNK/JNK (Fig. 3d), P-ERK (Fig. 3e), ERK (Fig. 3f), P-ERK/ERK (Fig. 3g) and p38 (Fig. 3i) in the LPS group were not significantly different compared with the CON group, except for P-p38 and P-p38/p38. Immunohistochemical staining showed that the expression of P-P38 protein in the LPS group was evidently higher than that in the CON group (Fig. 3o). Notability, DEX effectively reversed these alterations wrought by LPS upon P-p38 and P-p38/p38 (P < 0.01, Fig. 3h and j). Moreover, DEX weakened the LPS-induced increase in protein (P < 0.05, Fig. 3k and l) and mRNA (P < 0.01, Fig. 3m and n) expression levels of c-Myc and CLIC4. 3. Results 3.1. DEX Improved LPS-induced Hippocampal Structural Lesion Page 7/22 H&E staining visually revealed the histopathological changes of hippocampus (Fig. 1a and b). In the CON and DEX groups, neurons in hippocampal CA1 and CA3 regions were arranged neatly and tightly, with round or oval nuclei and clearly visible nucleoli. In the LPS group, the neuronal arrangement was disordered, the intercellular space increased, nuclear pyknosis, nucleolus disappearance, partial cell lysis, and a large number of apoptotic and necrotic cells were observed. However, LPS-induced microstructural damage in hippocampal CA1 and CA3 regions was markedly ameliorated by DEX. Moreover, DEX significantly reversed the LPS-induced decrease in the number of neurons in hippocampal CA1 and CA3 regions (P < 0.01, Fig. 1c and d). Ultrastructural observation results (Fig. 1f) showed that no abnormalities were captured in the mitochondria, nuclear membrane and other organelles of hippocampal neurons in the CON and DEX group. In contrast, most of the mitochondria in the LPS group were severely distorted and swollen, the mitochondrial membrane was discontinuous, the mitochondrial cristae dissolved and disappeared, and mitochondrial ghosts were occasionally seen. However, DEX treatment obviously reversed the aforementioned LPS-induced mitochondrial damage (P < 0.01, Fig. 1e). 3.2. DEX Attenuated LPS-Induced Hippocampal Apoptosis via Mitochondrial Pathways As shown in Fig. 2, compared with the CON and DEX groups, the protein expression levels of Bax, Bax/Bcl-2, cytochrome C, cleaved caspase-9 and cleaved caspase-3 in the LPS group were markedly elevated (P < 0.01). Nevertheless, DEX effectively suppressed the upregulation of mitochondrial apoptosis-related protein expression (P < 0.01). 3.4. DEX Reduced LPS-induced PC12 cell Viability Inhibition, LDH and ATP Release Page 8/22 After treating PC12 cells with gradient concentrations of LPS (0, 50, 100, 150, 200, 250, 300, 350 and 400 µg/mL) for 24 h, the cell viability was tested to screen the optimal drug concentration of LPS. As shown in Fig. 4a, the cell viability of 250 µg/mL LPS was 52.54 ± 5.031, which was the suitable concentration for LPS modeling. Notably, to varying degrees, different concentrations of DEX (1, 5, 10, 25, 50, 100 and 200 µM) attenuated the inhibition of PC12 cell viability induced by LPS (250 µg/mL). 50 µM DEX contributed to ∼100% viability and was therefore selected as the therapeutic drug concentration in this study (Fig. 4b). The observation results of cell morphology (Fig. 4c) showed that the CON group cells were spindle- shaped or polygonal with a large number. In contrast, the cells in the LPS group were round and the number was obviously reduced. After treatment with DEX and SB203582 (p38 MAPK inhibitor), the number of cells increased, most of them were fusiform or polygonal, and a few were round. Compared with CON group, LDH level was significantly increased, ATP content and total ATPase activity were markedly decreased in LPS group (P < 0.01, Fig. 4d-f). However, DEX and SB203582 dramatically reversed all these changes induced by LPS (P < 0.01). 3.6. DEX Ameliorated LPS-induced Apoptosis in PC12 Cells To authenticate that DEX attenuates LPS-induced apoptosis by regulating the p38 MAPK/c-Myc/CLIC4 signaling pathway, Bax and mitochondria co-localize, Annexin V-FITC/PI staining assay and mitochondrial apoptosis pathway-related protein expression detection were performed. As shown in Fig. 6, Bax and Tom20 positive cells in the LPS group were significantly more than those in the CON group. In contrast, these positive cells were obviously reduced in the LPS + DEX and LPS + SB groups. Annexin V-FITC/PI staining assay was used to evaluate PC12 cell apoptosis. Incubation with LPS significantly increased the rate of apoptosis, which was remarkably reduced in the LPS + DEX and LPS +  SB groups (P < 0.01, Fig. 7a and b). The protein expression of Bax, Bax/Bcl-2, cytochrome C, cleaved caspase-9 and cleaved caspase-3 was significantly increased by incubation with LPS, whereas pretreatment with DEX and SB203582 effectively adjusted these alterations (P < 0.01, Fig. 7c-i). 3.5. DEX Suppressed LPS-induced Activation of the p38 MAPK/c-Myc/CLIC4 Pathway in PC12 Cells The expression of P-p38, c-Myc and CLIC4 was markedly elevated by incubation with LPS, whereas pretreatment with DEX and SB203582 strongly blocked these increases (P < 0.01, Fig. 5). 4. Discussion LPS-induced animal models are widely used to study AD and depression. AD and depression are both disorders characterized by hippocampal dysfunction [28]. The clinical symptoms of AD are memory disturbance, behavioral derangement and cognitive impairment [29]. Late-life depression has been Page 9/22 Page 9/22 reported to be related to cognitive impairment [30]. Notably, the hippocampus, especially the CA1 and CA3 regions, represents a vital neural control center for behavior, emotion, learning and memory, and cognition [31]. Alterations in hippocampal structure are implicated in cognitive decline [32]. Fenghui Guo et al. have demonstrated that DEX improves LPS-induced cognitive decline. In the current study, DEX ameliorated the LPS-induced hippocampal microstructural and ultrastructural lesion, presenting as the decreased necrotic neurons, increased neuron density in CA1 and CA3 regions and improved mitochondrial damage. Thus, DEX may be a promising drug for the prevention and treatment of AD and depression. Most reports of AD and depression simulated with LPS have focused on neuroinflammation. Therefore, the understanding of the pathogenesis of AD and depression is limited, which hinders the development of effective therapeutic drugs without side effects. Our previous studies have demonstrated that apoptosis is involved in the pathological process of LPS-induced neurodegenerative diseases [9]. Nanhui Yu et al. revealed that inhibition of neuronal apoptosis can alleviate cognitive impairment in AD model mice [33]. In addition, reducing apoptosis can improve depression-like behavior in rats [34]. The current results revealed that DEX attenuated the apoptosis of hippocampal neurons induced by LPS, which may be a potential neuroprotective mechanism of DEX against AD and depression. Numerous studies have corroborated that the MAPK pathway is a major signal transduction pathway that regulates apoptosis [35]. Deviation from strictly controlled MAPK signaling pathways contributes to the driving of many human diseases, including AD [36]. The MAPK family of mammals consists of JNK, ERK and p38. It has been reported that JNK is an important potential therapeutic target for depression [37]. Suppression of JNK attenuated hippocampal neuronal apoptosis and improved depression-like behavior in mice [38]. In addition, dephosphorylation of ERK leads to neuronal apoptosis, which contributes to depression [39]. Recent studies have shown that LPS can promote the phosphorylation of JNK and ERK in a variety of models in vitro and in vivo [40, 41]. Surprisingly, our results showed no significant increase in the protein expressions of P-JNK, JNK, P-ERK, and ERK in the hippocampus of rats treated with LPS. 4. Discussion This may be related to different modeling methods. The p38 signaling pathway is susceptible to being triggered by various signals including LPS [42]. Activated p38 MAPK cascade can induce neuronal apoptosis and lead to hippocampal neuronal damage [43]. Consistent with previous reports, in the current study, phosphorylated p38 is activated and participates in the process of LPS-induced neuronal apoptosis. However, the non-phosphorylated protein of p38 did not change significantly. Notably, DEX pretreatment markedly inhibited the phosphorylation of p38. These results suggest that inhibition of p38 phosphorylation may be a protective mechanism for DEX to improve LPS-induced hippocampal apoptosis. Furthermore, we found that DEX inhibited the LPS-induced increase in the protein and mRNA expressions of c-Myc and CLIC4 in the rat hippocampus. Recent studies have reported that p38 can promote the activation of c-Myc [44]. c-Myc, an effective apoptosis inducer, binds to the CLIC4 promoter and contributes to the activation of its transcription [45]. CLIC4 is a key mediator of c-Myc inducing apoptosis. Inhibition of CLIC4 activation can suppress c-Myc-induced apoptosis under various stress conditions. Notably, the c-Myc/CLIC4 signaling pathway is involved in LPS-induced apoptosis of hippocampal Page 10/22 Page 10/22 neurons. Therefore, we guessed that DEX exerts neuroprotective and anti-apoptotic effects by regulating the p38/c-Myc/CLIC4 signal pathway. This hypothesis was confirmed by observing the effect of the p38 inhibitor SB203582 in LPS-treated PC12 cells. Like DEX, SB203582 significantly reduced the protein and mRNA expressions of c-Myc and CLIC4. Moreover, as assessed by flow cytometry, SB203582 protects PC12 cells from LPS-induced apoptosis. Overall, our findings revealed that the p38 MAPK/c-Myc/CLIC4 signaling pathway was involved in the neuroprotective mechanism of DEX against LPS-induced apoptosis. CLIC4, enriched in mitochondrial outer membrane, is implicated in mitochondrial pathway apoptosis. Overexpression of CLIC4 decreased mitochondrial membrane potential, causing cytochrome C to be released into the cytoplasm, thereby activating caspase [46]. Studies have shown that CLIC4 and Bax synergistically induce apoptosis, but their direct physical interaction have not been detected [47]. Bax, a pro-apoptotic protein, is present in the cytoplasm as a monomer in an inactive state. Under stress conditions, Bax is activated and translocated to mitochondria, forming heterodimer with Bcl2, leading to the release of cytochrome C. Thus, Bax/Bcl2 is regarded as a switch that triggers apoptosis [48]. 4. Discussion Our results showed that DEX and SB203582 blocked the translocation of Bax to mitochondria and down- regulated the protein expressions of Bax/Bcl2, cytochrome C, cleaved caspase-9 and cleaved caspase-3. Collectively, DEX inhibited LPS-induced apoptosis in the mitochondrial pathway by regulating p38 MAPK/c-Myc/CLIC4. 5. Conclusion In summary, our results demonstrated that DEX inhibited the activation of LPS-induced p38 MAPK/c- Myc/CLIC4 signaling pathway, down-regulated the expression of mitochondrial apoptosis pathway- related proteins, thereby reducing neuronal apoptosis. This study elucidated the neuroprotective mechanism of DEX in LPS-induced hippocampal injury from the perspective of anti-apoptosis, which provides support for the clinical application of DEX in the treatment of Alzheimer's disease and depression. Consent for Publication Not applicable. Abbreviations AD, Alzheimer's disease; LPS, Lipopolysaccharide; DEX, Dexmedetomidine; CLIC4, Chloride intracellular channel 4; MAPK, Mitogen-activated protein kinase; JNK, Stress-activated protein kinases;ERK, extracellular signal-regulated kinase; H&E, Hematoxylin and eosin. Authors’ contributions Yongping Chen: Conceptualization, Methodology, Writing – original draft, Writing - review & editing, Supervision, Resources, Funding acquisition, Project administration. Lin Li: Investigation, Visualization, Validation. Jiuyan Zhang: Data curation, Formal analysis, Software. Hailin Cui, Jiucheng Wang: Investigation, Visualization, Validation. Chuqiao Wang, Mingxian Shi: Data curation, Formal analysis, Software. Honggang Fan: Conceptualization, Methodology, Writing - original draft, Writing - review & editing, Supervision, Resources, Funding acquisition, Project administration. Acknowledgements This work was supported by the National Natural Science Foundation of China Grant (grant number 31772806), the Natural Science Foundation of Heilongjiang Province Grant (grant number C2017022) and the Heilongjiang Key Laboratory for Laboratory Animals and Comparative Medicine. Consent for Participate Not applicable. Consent for Participate Not applicable. Declarations Funding This study was supported by National Natural Science Foundation of China (Grant No. 31772806 and 31902337) and Heilongjiang Key Laboratory for Laboratory Animals and Comparative Medicine, China. Conflicts of Interest No conflict of interest. Data Availability All data used to support this study are included in the article. Data Availability All data used to support this study are included in the article. Code Availability Not applicable. Page 11/22 Page 11/22 Page 11/22 Ethics Approval All animal experiment protocols in this study were approved by the Animal Ethics Committee of Northeast Agricultural University (SRM-11, China). References 1. 2016 Alzheimer's disease facts and figures. Alzheimer's & dementia: the journal of the Alzheimer's Association, 2016. 12(4): p. 459–509 2. Lim GY et al (2018) Prevalence of Depression in the Community from 30 Countries between 1994 and 2014. Scientific reports 8(1):2861 3. 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Shiio Y et al (2006) Quantitative proteomic analysis of myc-induced apoptosis: a direct role for Myc induction of the mitochondrial chloride ion channel, mtCLIC/CLIC4. J Biol Chem 281(5):2750–2756 46. Suh KS et al (2004) The organellular chloride channel protein CLIC4/mtCLIC translocates to the nucleus in response to cellular stress and accelerates apoptosis. J Biol Chem 279(6):4632–4641 46. Suh KS et al (2004) The organellular chloride channel protein CLIC4/mtCLIC translocates to the nucleus in response to cellular stress and accelerates apoptosis. J Biol Chem 279(6):4632–4641 47. Figures Page 15/22 Figure 1 DEX improved LPS-induced hippocampal structural lesion. a Histopathological observation of hippocampal CA1 and CA3 regions in CON group, LPS group, LPS + DEX group, DEX group, (magnification 400x, scale bars = 20 μm). Black and green arrows indicate necrotic and apoptotic neurons, respectively. b The CA1 and CA3 regions of the hippocampus (magnification 40x, scale bars = 200 μm). c Number of neurons in hippocampal CA1 region. d Number of neurons in hippocampal CA3 region. e Mitochondrial damage score. f Ultrastructural observation of hippocampal CA1 and CA3 regions. Red arrow indicates damaged mitochondria. Data were presented as mean ± SD (n = 3). **P < 0.01 vs CON group. ##P < 0.01 vs LPS group. Figure 1 DEX improved LPS-induced hippocampal structural lesion. a Histopathological observation of hippocampal CA1 and CA3 regions in CON group, LPS group, LPS + DEX group, DEX group, (magnification 400x, scale bars = 20 μm). Black and green arrows indicate necrotic and apoptotic neurons, respectively. b The CA1 and CA3 regions of the hippocampus (magnification 40x, scale bars = 200 μm). c Number of neurons in hippocampal CA1 region. d Number of neurons in hippocampal CA3 region. e Mitochondrial damage score. f Ultrastructural observation of hippocampal CA1 and CA3 regions. Red arrow indicates damaged mitochondria. Data were presented as mean ± SD (n = 3). **P < 0.01 vs CON group. ##P < 0.01 vs LPS group. Page 16/22 Figure 2 DEX attenuated LPS-induced apoptosis of hippocampal neurons via mitochondrial pathways. a Protei expression of Bax, Bcl-2, cytochrome C, cleaved caspase-9 and cleaved caspase-3 in hippocampus. Protein quantitative analysis of (b) Bax, (c) Bax/Bcl-2, (d) cytochrome C, (e) cleaved caspase-9 and (f) cleaved caspase-3. Data were presented as mean ± SD (n = 3). **P < 0.01 vs CON group. ##P < 0.01 vs LPS group Figure 2 DEX attenuated LPS-induced apoptosis of hippocampal neurons via mitochondrial pathways. a Protein expression of Bax, Bcl-2, cytochrome C, cleaved caspase-9 and cleaved caspase-3 in hippocampus. Protein quantitative analysis of (b) Bax, (c) Bax/Bcl-2, (d) cytochrome C, (e) cleaved caspase-9 and (f) cleaved caspase-3. Data were presented as mean ± SD (n = 3). **P < 0.01 vs CON group. ##P < 0.01 vs LPS group. Figure 2 Figure 2 DEX attenuated LPS-induced apoptosis of hippocampal neurons via mitochondrial pathways. a Protein expression of Bax, Bcl-2, cytochrome C, cleaved caspase-9 and cleaved caspase-3 in hippocampus. Protein quantitative analysis of (b) Bax, (c) Bax/Bcl-2, (d) cytochrome C, (e) cleaved caspase-9 and (f) cleaved caspase-3. Data were presented as mean ± SD (n = 3). **P < 0.01 vs CON group. ##P < 0.01 vs LPS group. Page 17/22 Page 17/22 Figure 3 Effects of DEX on the MAPK/c-Myc/CLIC4 signaling pathway. a Protein exp ERK P-p38, p38, c-Myc and CLIC4 in hippocampus. Protein quantitative ana P-JNK/JNK, (e) P-ERK, (f) ERK, (g) P-ERK/ERK, (h) P-p38, (i) p38, (j) P-p38/p (m) c-Myc and (n) CLIC4 mRNA expression analysis. o Immunohistochemis Figure 3 Effects of DEX on the MAPK/c-Myc/CLIC4 signaling pathway. a Protein expression of P-JNK, JNK, P-ERK, ERK P-p38, p38, c-Myc and CLIC4 in hippocampus. Protein quantitative analysis of (b) P-JNK, (c) JNK, (d) P-JNK/JNK, (e) P-ERK, (f) ERK, (g) P-ERK/ERK, (h) P-p38, (i) p38, (j) P-p38/p38, (k) c-Myc and (l) CLIC4. (m) c-Myc and (n) CLIC4 mRNA expression analysis. o Immunohistochemistry of P-p38 protein in the Page 18/22 Page 18/22 Page 18/22 hippocampus (magnification 200x, scale bars = 50 μm). Data were presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01 vs CON group. #P < 0.05, ##P < 0.01 vs LPS group. hippocampus (magnification 200x, scale bars = 50 μm). Data were presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01 vs CON group. #P < 0.05, ##P < 0.01 vs LPS group. Figure 4 DEX reduced LPS-induced PC12 cell viability inhibition, LDH and ATP release. a Cell viability of PC12 cells treated with different concentrations of LPS. b Cell viability of PC12 cells treated with different concentrations of DEX with or without LPS. c Cell morphology observation in the CON, LPS, LPS + DEX and LPS + SB groups, at 200x magnification, bars = 50 μm. d LDH activity. e ATP content. f Total ATPase Figure 4 DEX reduced LPS-induced PC12 cell viability inhibition, LDH and ATP release. a Cell viability of PC12 cells treated with different concentrations of LPS. b Cell viability of PC12 cells treated with different i f DEX i h i h LPS C ll h l b i i h CON LPS LPS DEX Figure 4 DEX reduced LPS-induced PC12 cell viability inhibition, LDH and ATP release. a Cell viability of PC12 cells treated with different concentrations of LPS. b Cell viability of PC12 cells treated with different concentrations of DEX with or without LPS. c Cell morphology observation in the CON, LPS, LPS + DEX and LPS + SB groups, at 200x magnification, bars = 50 μm. d LDH activity. e ATP content. f Total ATPase activity. Data were presented as mean ± SD (n = 6). **P < 0.01 vs CON group. ##P < 0.01 vs LPS group. Page 19/22 Figure 5 DEX suppressed LPS-induced activation of the p38 MAPK/c-Myc/CLIC4 pathway expression of P-p38, p38, c-Myc and CLIC4. Protein quantitative analysis of (b) P ( ) d (f) ( ) d (h) Figure 5 DEX suppressed LPS-induced activation of the p38 MAPK/c-Myc/CLIC4 pathway in PC12 Cells. a Protein expression of P-p38, p38, c-Myc and CLIC4. Protein quantitative analysis of (b) P-p38, (c) p38, (d) P- p38/p38, (e) c-Myc and (f) CLIC4. (g) c-Myc and (h) CLIC4 mRNA expression analysis. Data were presented as mean ± SD (n = 3). **P < 0.01 vs CON group. ##P < 0.01 vs LPS group. Figure 5 DEX suppressed LPS-induced activation of the p38 MAPK/c-Myc/CLIC4 pathway in PC12 Cells. a Protein expression of P-p38, p38, c-Myc and CLIC4. Protein quantitative analysis of (b) P-p38, (c) p38, (d) P- p38/p38, (e) c-Myc and (f) CLIC4. (g) c-Myc and (h) CLIC4 mRNA expression analysis. Data were presented as mean ± SD (n = 3). **P < 0.01 vs CON group. ##P < 0.01 vs LPS group. Page 20/22 Page 20/22 Figure 6 DEX inhibited the translocation of apoptotic protein Bax to mitochondria. The co-localization of Bax (red) and mitochondria (Tom20, green) was measured by double-labeled immunofluorescence assay (n = 3). Merge is the combination of Bax, Tom20 and DAPI (nucleus, blue). All microphotographs were observed and captured at 400x magnification, bars = 20 μm. DEX inhibited the translocation of apoptotic protein Bax to mitochondria. The co-localization of Bax (red) and mitochondria (Tom20, green) was measured by double-labeled immunofluorescence assay (n = 3). Merge is the combination of Bax, Tom20 and DAPI (nucleus, blue). All microphotographs were observed and captured at 400x magnification, bars = 20 μm. Page 21/22 Figure 7 DEX ameliorated LPS-induced PC12 Cell apoptosis via the mitochondrial apoptosis was detected by flow cytometry. b Annexin-V/PI positive rate. c 2 cytochrome C cleaved caspase 9 and cleaved caspase 3 Protein quan Figure 7 Page 22/22 DEX ameliorated LPS-induced PC12 Cell apoptosis via the mitochondrial pathways. a PC12 cell apoptosis was detected by flow cytometry. b Annexin-V/PI positive rate. c Protein expression of Bax, Bcl- 2, cytochrome C, cleaved caspase-9 and cleaved caspase-3. Protein quantitative analysis of (d) Bax, (e) Bcl-2, (f) Bax/Bcl-2, (g) cytochrome C, (h) cleaved caspase-9 and (i) cleaved caspase-3. Data were presented as mean ± SD (n = 3). **P < 0.01 vs CON group. ##P < 0.01 vs LPS group. Page 22/22
https://openalex.org/W2056592804
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English
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Experimental analysis of reinforced concrete columns strengthened with Self-Compacting concrete
Revista IBRACON de Estruturas e Materiais
2,010
cc-by
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a School of Civil Engineering, Federal University of Goias, mohaomar@gmail.com, Goiânia, Brazil. b School of Civil Engineering, Federal University of Goias, rbggomes@gmail.com, Praça Universitária, s/nº – Setor Universitário – CEP 74605-220, Goiânia, Brazil; c School of Civil Engineering, Federal University of Goias, andrea.liserre@gmail.com, Praça Universitária, s/nº – Setor Universitário – CEP 74605-220, Goiânia, Brazil; Received: 14 Sep 2009 • Accepted: 16 Apr 2010 • Available Online: 10 Sep 2010 Experimental Analysis of Reinforced Concrete Columns Strengthened with Self-Compacting Concrete Análise Experimental de Pilares de Concreto Armado Reforçados usando Concreto Auto-Adensável Volume 3, Number 3 (September, 2010) p. 271 - 283 • ISSN 1983-4195 Experimental Analysis of Reinforced Concrete Columns Strengthened with Self-Compacting Concrete Análise Experimental de Pilares de Concreto Armado Reforçados usando Concreto Auto-Adensável Experimental Analysis of Reinforced Concrete Columns Strengthened with Self-Compacting Concrete Análise Experimental de Pilares de Concreto Armado Reforçados usando Concreto Auto-Adensável M. Y. M. OMAR a mohaomar@gmail.com R. B. GOMES b rbggomes@gmail.com A. P. A. REIS c andrea.liserre@gmail.com M. Y. M. OMAR a mohaomar@gmail.com R. B. GOMES b rbggomes@gmail.com A. P. A. REIS c andrea.liserre@gmail.com A. P. A. REIS c andrea.liserre@gmail.com Abstract This paper presents the results of reinforced concrete columns strengthened by addition of a self-compacting concrete overlay at the compressed and at the tensioned face of the member, with and without addition of longitudinal steel bars. Eight columns were submit- ted to loading with an initial eccentricity of 60 mm. These columns had 120 mm x 250 mm of rectangular cross section, 2000 mm in length and four longitudinal reinforcement steel bars with 10 mm in diameter. Reference columns P1 and P2 were tested to failure without any type of rehabilitation. Columns P3 to P8 were loaded to a predefined load (close to the initial yield point of tension reinforce- ment), then unloaded and strengthened for a subsequent test until failure. Results showed that the method of rehabilitation used was effective, increasing the loading capacity of the strengthened pieces by 2 to 5 times the ultimate load of the reference column. Keywords: column; reinforced concrete; strengthening; eccentricity; self-compacting concrete. Received: 14 Sep 2009 • Accepted: 16 Apr 2010 • Available Online: 10 Sep 2010 1. Introduction If the adherence between contact surfaces fails to ensure the monolithicity of the rehabilitation, it is possible to increase the joint shear strength using steel connectors crossing the interface. Such transverse reinforcement is normally used in composite precast parts, in which tangential stresses show high values because of a small contact surface between concretes cast at different ages (El Debs [3]). The guarantee of a satisfactory bond and a monolithic strengthen- ing depends on the correct treatment of the old concrete surface and substrate cleaning and saturation before the subsequent plac- ing of the new concrete layer and steel bars. Besides that, differ- ences in properties of the new concrete and original concrete must be considered, especially concerning elastic modulus, shrinkage and creep strains. To match properties of the new repair material as closely as possible to the original concrete of the structure, the use of Portland cement concrete or similar cementitious composi- tions are frequently the best choices (Clímaco [1]). Abu-Tair et al. [4] evaluated the surface roughness of concrete of three different repair materials: epoxy resin, a modified cementi- tious material, and Portland cement concrete. It also was analyzed four types of substrate surface preparation: manual removal of the superficial concrete layer with hammer and chisel, electric ham- mering, wire brushing and no intervention. The most successful re- sults of adherence strength were obtained by the Portland cement concrete repair, with a surface treated with hammer and chisel and no adhesive agent. Adorno [5] and Araújo [6] have carried out tests with reinforced concrete columns submitted to combined bending and axial com- pression. Their experiments have shown the influence of load ec- centricity variation on a column’s load-bearing capacity. The selec- tion of a 60 mm load eccentricity in this study was supported by the results of such experiments. This eccentricity allowed the tested columns’ reinforcements to reach their yield. This paper highlights the study of reinforced concrete columns strengthened with self-compacting concrete (SCC) at the com- pressed face or at the tensioned face and at the compressed and tensioned faces simultaneously; with all columns submitted to combined bending and axial compression at an initial eccentricity of 60 mm. Depending on the strengthened face this eccentricity may increase or decrease in the post-strengthening test. Resumo Este artigo apresenta os resultados de pilares de concreto armado reforçados por meio da adição de camadas de concreto auto- adensável, tanto na face tracionada quanto na face comprimida, com ou sem adição de barras de aço longitudinais. Oito pilares foram submetidos a um carregamento com excentricidade inicial de 60mm. Estes pilares possuíam seção transversal retangular de 120 mm x 250 mm, comprimento de 2000 mm, e armadura longitudinal constituída por quatro barras de 10 mm de diâmetro. Os pilares de refer- ência, P1 e P2, foram ensaiados até a ruptura sem nenhum tipo de reabilitação. Os pilares P3 à P8 foram inicialmente submetidos a um pré-carregamento que provocava na armadura longitudinal, tensões próximas a do escoamento do aço. Em seguida, estas peças foram descarregadas e reforçadas. Após o concreto do reforço atingir resistência adequada, estes pilares foram ensaiados até a ruptura. Os resultados mostraram que o método de reforço estudado foi eficiente, pois dependendo da face do pilar em que se aplicava a camada de reforço, as peças reabilitadas apresentaram uma capacidade de carga de 2 a 5 vezes maior que a dos pilares não reforçados. Palavras-chave: pilar; concreto armado; reforço; excentricidade; concreto auto-adensável. © 2010 IBRACON Experimental Analysis of Reinforced Concrete Columns Strengthened with Self-Compacting Concrete Experimental Analysis of Reinforced Concrete Columns Strengthened with Self-Compacting Concrete Experimental Analysis of Reinforced Concrete Columns Strengthened with Self-Compacting Concrete 1. Introduction In order to obtain a material with mechanical properties similar to original concrete, to facilitate the application of the strengthening overlay and to avoid execution defects, self-compacting concrete (SCC) may be employed as a strengthening material. This type of concrete may be cast on formwork, thus filling all empty spaces without the aid of external compaction or vibration. It provides fluid- ity, cohesion, and segregation resistance. The repair, rehabilitation and upgrading of reinforced con- crete structures is a major challenge to civil engineers. Thus, the knowledge of upgrading or strengthening techniques of re- inforced concrete structures is very interesting for engineering purposes. Despite the considerable development in this area in the last years, many professionals still use methods based only on empirical experience. Probably this happens because each structure has individual characteristics which demand a specific rehabilitation process. Although numerous structures have been rehabilitated adding new material to the concrete section, limited data are available on their performance under applied loads until failure. Silica fume is used as an admixture to concrete to improve the engineering properties. According to Aïtcin [2], the silica fume add- ed to the concrete improves the paste-aggregate interface zone, adherence, impermeability, axial compression strength, and cohe- sion of fresh concrete, avoiding exudation. Thus, additions of silica fume may improve the bond conditions between the new concrete and the substrate. It is desired that strengthened elements acts like monolithic structures guarantying the complete load transfer from the base concrete to the new material in the vertical joint region, to avoid the debonding between these materials. Aiming to increase the knowledge about the structural behavior of rehabilitated constructions, this paper analyzes the behavior of columns strengthened by an increase of the cross section with or without the addition of longitudinal steel bars embedded by self- compacting concrete (SCC). The re-casting of the cross section does not necessarily have to be applied all around the column, thus it was studied the rehabilitation adding new concrete restrict- ed to some of its faces. The shear resistance of the joint is the sum of the contribution of the cohesion between the two concretes of the joint, the concrete- to-concrete friction resistance mobilized when the joint is simulta- neously subject to shear and normal compression, and the dowel action of the reinforcing bars crossing the interface. 1. Introduction Our spe- cific aims were: 1) to analyze the effect of load (pre-loading) on the column prior to strengthening; 2) to check experimentally whether strengthened columns may be regarded as monolithically cast col- umns, in view of a perfect adherence between strengthening mate- rial and the substrate; 3) to verify the influence of strengthening on columns as regards faces (tension or compression), thickness, and consequent eccentricity variation. IBRACON Structures and Materials Journal • 2010 • vol. 3 • nº 3 272 M. Y. M. OMAR | R. B. GOMES | A. P. A. REIS 2. Materials and experimental program The load was applied by a hydraulic actua- tor which was activated by a hydraulic hand pump. Load cells of 500 kN were placed on the upper and lower ends of the columns whereas a single 1000 kN load cell was placed on the upper end of columns PC45T10, PC45T12, and PC55. A frame anchored to the strong floor was used for the reaction of specimens. Columns P5 and P6 were strengthened on the tension faces add- ing longitudinal bars measuring 2 φ 10.0 mm and 2 φ 12.5 mm re- spectively, and with 45 mm thick SCC layers on both compressed and tension faces. They were respectively named PC45T10 and PC45T12, and their initial eccentricity of 60 mm was maintained. The values of yield strength of the steel bars were 613 MPa and 619 MPa for 10 mm and 12.5 mm diameter bars, respectively. The strain corresponding to yield strength was 2.87 mm/m for 10 mm diameter bar and 2.53 mm/m for 12.5mm diameter bar. A digital dial gauge of 0.01 mm precision (R1) measured the hori- zontal displacement at the middle section of specimens. Figure [4] shows the test scheme with the positions of load cells and of the comparing watch, as well as a photograph with details of the equip- ment used for the measurements, load application, and bonding of an original column within the testing system. Columns P7 and P8 were strengthened on the compressed faces with 35 mm (PC35) and 55 mm (PC55) thick SCC layers without using any steel bars. Consequently their initial eccentricities were reduced to 42.5 and 32.5 mm, respectively. Table [1] presents the main characteristics of the tested columns and Figure [3] reveals the detail of the cross sections of original and strengthened columns. Table [2] shows the proportion of ma- terials per cubic meter of concrete used in the substrate and the strengthening. Electric strain gauges connected to the data acquisition system, were used to monitor steel and concrete strains on the middle section of specimens. Figure [5] presents the position of the extensometers on the steel and concrete of all the columns. Throughout the analyses, reinforcement strains on tension (T) and compressed (C) faces were the averages of readings sup- plied by the extensometers. The entire substrate surface submitted to strengthening was manually treated with hammer and chisel to expose the aggregate for proper adhesion of the rehabilitation concrete. 2. Materials and experimental program 60 mm). Full details of columns dimensions and bar reinforcement are shown in Figures [1] and [2], respectively. i Two testing stages were performed. In the first stage, two refer- ence columns were tested to failure (P1 and P2) and the remaining six (P3 to P8) were submitted to loading which corresponded to a strain close to the initial yield point of the tension reinforcement. The second stage consisted of testing columns to failure after strengthening. Eight columns (named Original Columns) of 120 mm x 250 mm rectangular cross section, and 2000 mm in length were tested (Omar [7]). The concrete mix design aimed a 28 days compres- sive strength of 30 MPa and an elastic modulus of 28 GPa. Dur- ing each concrete casting cylindrical specimens (150 mm x 300 mm) were moulded to determine the compressive strength of the concrete. Strengthening was performed with unloaded columns positioned outside the test apparatus. Columns P3 and P4 were strengthened on the tension face adding longitudinal steel bars measuring 2 φ 10.0 mm and 2 φ 12.5 mm. These new bars were encased by a 45 The longitudinal reinforcement of the original columns was com- posed of four 10 mm diameter bars. These elements were designed to be submitted to eccentric compression (initial eccentricity of IBRACON Structures and Materials Journal • 2010 • vol. 3 • nº 3 273 Experimental Analysis of Reinforced Concrete Columns Strengthened with Self-Compacting Concrete Experimental Analysis of Reinforced Concrete Columns Strengthened with Self-Compacting Concrete The original columns were submitted to eccentric compression at an initial eccentricity of 60 mm. The initial eccentricity varied among the strengthened columns in terms of thickness and face strengthened. The load was applied by a hydraulic actua- tor which was activated by a hydraulic hand pump. Load cells of 500 kN were placed on the upper and lower ends of the columns whereas a single 1000 kN load cell was placed on the upper end of columns PC45T10, PC45T12, and PC55. A frame anchored to the strong floor was used for the reaction of specimens. mm thick SCC layer. These columns were thus called PT10 and PT12, respectively. Their initial eccentricity increased to 82.5 mm due to strengthening. The original columns were submitted to eccentric compression at an initial eccentricity of 60 mm. The initial eccentricity varied among the strengthened columns in terms of thickness and face strengthened. 2. Materials and experimental program The substrate surface was cleaned and saturated prior to strengthening. Steel U-bolts of 5 mm in diameter, spaced out at 50 mm intervals, and glued with epoxy resin were placed within holes made by 8 mm thick and 50 mm deep drill bits. In all columns, the concrete used had cylinder compressive strength of the substrate be­tween 25.1 (PC35 and PC55) MPa and 30.8 MPa (PC45T12) and the compression strength of the strengthen- ing concrete varied from 43.0 MPa to 46.8 MPa (Table 3). IBRACON Structures and Materials Journal • 2010 • vol. 3 • nº 3 274 M. Y. M. OMAR | R. B. GOMES | A. P. A. REIS 3. Results and discussions This load gain was a result of the increase of the cross section and of the reinforcement rate, despite the increase of the initial eccen- tricity of the force applied. The failure load of column PT12 was 12 kN higher than that of column PT10, even though a higher longitu- dinal reinforcement rate was added to it. The failure of PT10 and PT12 were ductile and occurred by yielding of the longitudinal steel bars at the tension face, followed by the crushing of compressed IBRACON Structures and Materials Journal • 2010 • vol. 3 • nº 3 3.2 Horizontal displacement of the column The reference columns (P1 and P2) registered maximum horizontal displacements at mid-heights of 20.35 mm and 37.63 mm, respec- tively. The columns strengthened on the tension face, PT10 and PT12, had maximum displacements of 32.58 mm and 20.37 mm, respectively, whereas columns PC35 and PC55 – strengthened on the compressed face – reached maximum displacements of 22.05 mm and 13.62 mm, respectively. Column PC45T12 had the lowest horizontal displacement among all the columns (11.67 mm). Col- umn PC45T10 reached a horizontal displacement of 19.46 mm. Figure [7] shows the load-horizontal displacement curves for all the columns. Columns PT10 and PT12 had similar stiffnesses which were higher than that of the reference columns. This probably oc- curred because they had larger cross sections and higher rein- forcement rates. However, these specimens had lower stiffness values than all the other columns strengthened on the compressed face. As regards the failure load of column P1, columns PC45T10, PC45T12, PC35, and PC55 had similar stiffnesses with displace- ments of approximately 14.0 times lower than that of column P1. Column PC35, which had a lower cross section than that of col- umns PT10 and PT12, had higher stiffness. The initial eccentricity The reference columns (P1 and P2) registered maximum horizontal displacements at mid-heights of 20.35 mm and 37.63 mm, respec- tively. The columns strengthened on the tension face, PT10 and PT12, had maximum displacements of 32.58 mm and 20.37 mm, respectively, whereas columns PC35 and PC55 – strengthened on the compressed face – reached maximum displacements of 22.05 mm and 13.62 mm, respectively. Column PC45T12 had the lowest horizontal displacement among all the columns (11.67 mm). Col- umn PC45T10 reached a horizontal displacement of 19.46 mm. Columns PC35 and PC55 failed prematurely due to the debonding of the strengthening concrete and lack of yield of the tension rein- forcement. The debonding of the strengthening concrete of column PC35 occurred close to the lower end of the column, whereas the debonding of the strengthening concrete of column PC55 occurred close to its upper end. The load difference between columns PC35 and PC55 was 126 kN, representing 33% more for the latter. This highlights the influence of initial load eccentricity on the columns and the increase in cross section. Figure [6] presents photographs of columns PC35 and PC55 after failure. Figure [7] shows the load-horizontal displacement curves for all the columns. 3.1Ultimate load and mode of failure 3.1Ultimate load and mode of failure Columns strengthened on the tension face, PT10 and PT12, had ultimate loads of 268 kN and 280 kN respectively, that are 2.1 and 2.2 times greater than the failure load of the reference column (P1). IBRACON Structures and Materials Journal • 2010 • vol. 3 • nº 3 275 Experimental Analysis of Reinforced Concrete Columns Strengthened with Self-Compacting Concrete Experimental Analysis of Reinforced Concrete Columns Strengthened with Self-Compacting Concrete concrete in the mid-span region, at the compressed face. Table [3] shows ultimate loads and modes of failure of all tested columns. Columns PC35 and PC55, which were strengthened on the com- pressed face even with the debonding of the strengthening con- crete, recorded greater load gain than the columns strengthened on the tension face. Such an increase was a result of the increase in cross section and of the reduction of the initial load eccentricity. If the debonding of the strengthening concrete had not occurred, col- umns PC35 and PC55 might have revealed higher failure loads. concrete in the mid-span region, at the compressed face. Table [3] shows ultimate loads and modes of failure of all tested columns. Columns PC45T10 and PC45T12 revealed ultimate loads almost 5 times greater than that of the reference column P1 (645 kN and 630 kN, respectively). This load gain was a result of the increase of the cross section and of the reinforcement rate. The failure was sudden by the crushing of compressed concrete at the mid-span region of the column without the yield of the tension reinforcement. There were no traces of debonding of the strengthening concrete. Columns PC35 and PC55 – strengthened on the compressed face with SCC layers of 35 mm and 55 mm, respectively – showed ulti- mate loads 2.9 and 3.9 times greater than that of the reference col- umn (P1), respectively. This load gain occurred due to the increase of the cross section and to the reduction of the initial eccentricity of the force applied. concrete in the mid-span region, at the compressed face. Table [3] shows ultimate loads and modes of failure of all tested columns. Columns PC45T10 and PC45T12 revealed ultimate loads almost 5 times greater than that of the reference column P1 (645 kN and 630 kN, respectively). This load gain was a result of the increase of the cross section and of the reinforcement rate. 3.1Ultimate load and mode of failure The failure was sudden by the crushing of compressed concrete at the mid-span region of the column without the yield of the tension reinforcement. There were no traces of debonding of the strengthening concrete. Columns PC35 and PC55 – strengthened on the compressed face with SCC layers of 35 mm and 55 mm, respectively – showed ulti- mate loads 2.9 and 3.9 times greater than that of the reference col- umn (P1), respectively. This load gain occurred due to the increase of the cross section and to the reduction of the initial eccentricity of the force applied. 3.2 Horizontal displacement of the column Columns PT10 and PT12 had similar stiffnesses which were higher than that of the reference columns. This probably oc- curred because they had larger cross sections and higher rein- forcement rates. However, these specimens had lower stiffness values than all the other columns strengthened on the compressed face. As regards the failure load of column P1, columns PC45T10, PC45T12, PC35, and PC55 had similar stiffnesses with displace- ments of approximately 14.0 times lower than that of column P1. Column PC35, which had a lower cross section than that of col- umns PT10 and PT12, had higher stiffness. The initial eccentricity In addition, even though columns PT10 and PT12 were strength- ened with greater thickness than column PC35, the failure load was lower because the strengthening was placed on the tension face. This reveals that the reduction of initial eccentricity may ben- efit the load-bearing capacity of the strengthened specimens. IBRACON Structures and Materials Journal • 2010 • vol. 3 • nº 3 276 M. Y. M. OMAR | R. B. GOMES | A. P. A. REIS IBRACON Structures and Materials Journal • 2010 • vol. 3 • nº 3 277 Experimental Analysis of Reinforced Concrete Columns Strengthened with Self-Compacting Concrete Experimental Analysis of Reinforced Concrete Columns Strengthened with Self-Compacting Concrete PC45T12 (strengthened on both compressed and tension faces). These values indicate that the tension steel of all columns had not yet reached the yield point before failure. of column PC35, lower by 17.5 mm, generates a lower bending moment and consequently smaller displacements. The horizontal displacement measured at the failure of column PC35 was ap- proximately 5 times greater than the displacements measured on columns PC45T10 and PC45T12. Figure [8] shows that column PC35 – with a smaller cross section – had smaller steel strains than columns PT10 and PT12. As regards the failure load of column PC35, columns PC45T10 and PC45T12 had smaller strains than one third of the strains of column PC35. This was probably due to the fact that columns PC45T10 and PC45T12 had a larger cross section and a higher reinforcement rate than PC35. 3.3 Steel strain – Tension face reinforcement 3.3 Steel strain – Tension face reinforcement The reference columns (P1 and P2) showed tension steel yield and registered maximum strains of 2.89 mm/m and 3.71 mm/m, respectively. The columns strengthened on the tension face, PT10 and PT12, had maximum strains of 3.35 mm/m and 2.63 mm/m re- spectively, thus indicating the yield of the tension steel. The graph in Figure [8] shows the load-strain curve of the tension steel of all the columns tested. The tension reinforcement strain of column PT10 was approximate- ly 30% higher before failure than the maximum strain of column PT12. The strains of columns PC45T10 and PC45T12 (strength- ened on compressed and tension faces) were similar, despite a 2.3% difference in failure load and a higher reinforcement rate (1.56 times higher) of PC45T12. Even though columns PC35 and PC55 (strengthened by compression) had lower failure loads than columns PC45T10 and PC45T12 (strengthened by compression and tension), the tension strains measured were lower for PC35 up Maximum strains of 1.47 mm/m and 1.20 mm/m were respective- ly registered for columns PC35 and PC55 (strengthened on the compressed face), whereas maximum strains of 1.70 mm/m and 1.61 mm/m were respectively registered for columns PC45T10 and 278 IBRACON Structures and Materials Journal • 2010 • vol. 3 • nº 3 IBRACON Structures and Materials Journal • 2010 • vol. 3 • nº 3 278 M. Y. M. OMAR | R. B. GOMES | A. P. A. REIS to the 340 kN load and for PC55 up to the 460 kN load. This was probably a result of lower initial eccentricities for columns PC35 and PC55 (42.5 mm and 32.5 mm, respectively). to the 340 kN load and for PC55 up to the 460 kN load. This was probably a result of lower initial eccentricities for columns PC35 and PC55 (42.5 mm and 32.5 mm, respectively). show smaller strains. Strains on the compressed reinforcement of column P2 are not shown in the graph due to reading problems. However, it seems reasonably to believe that this column behaves similarly to column P1, as was observed in all the other graphs. 3.4 Steel strain – Compressed face reinforcement 3.5 Concrete strains The maximum compression strain of column PC55 (2.17 mm/m) corresponded to 72.3% of the crushing strain adopted by ACI 318M-02 [8] (3.0 mm/m) for specimens subject to combined bending and axial compression. 4. Conclusions The static short-term loading behavior of the reinforced concrete columns strengthened by recasting with or without addition of tensile steel bars has been confirmed to be very satisfactory in Columns PC35 and PC55 had the lowest strains, probably because of pre- mature failure caused by the debonding of the strengthening concrete. The maximum compression strain of column PC55 (2.17 mm/m) corresponded to 72.3% of the crushing strain adopted by ACI 318M-02 [8] (3.0 mm/m) for specimens subject to combined bending and axial compression. Columns PC35 and PC55 had the lowest strains, probably because of pre- mature failure caused by the debonding of the strengthening concrete. The maximum compression strain of column PC55 (2.17 mm/m) corresponded to 72.3% of the crushing strain adopted by ACI 318M-02 [8] (3.0 mm/m) for specimens subject to combined bending and axial compression. 3.5 Concrete strains The reference columns (P1 and P2) and columns PC35 and PC55 (strengthened on the compressed face) had maximum com- pressed steel strains lower than 1.0 mm/m. Columns PC45T10 and PC45T12, strengthened on both compressed and tension faces, had maximum strains of 1.78 mm/m and 1.71 mm/m respectively, whereas columns PT10 and PT12, strengthened on the tension face, reached maximum strains of up to 3.89 mm/m. The graph in Figure [10] shows the load-strain curve of the com- pressed concrete of all the columns tested. Columns P1, P2, and PT12 had similar strains up to the 100 kN load; as for higher loads, the strains of column PT12 decrease in relation to the other two, which remain similar up to the failure load of column P1. Columns PC35 and PC55, strengthened on the compressed face, had the lowest maximum strains of compressed concrete, measur- ing 1.63 mm/m and 2.17 mm/m, respectively. The reference col- umns (P1 and P2) had a maximum strain of compressed concrete of 2.22 mm/m and 4.69 mm/m, respectively. The graph in Figure [9] shows the load-strain curve of the com- pressed steel of all the columns tested. As regards columns P1, PT10, and PT12, the compressed reinforcement revealed, since the start of testing, higher strains than those observed in columns PC45T10, PC45T12, PC35, and PC55. Figure [9] also shows that columns PT10 and PT12 had similar compressed steel strains up to the failure load of reference column P1. Probably due to dam- ages caused by pre-loading, these strengthened columns failed to Columns PT10 and PT12, strengthened on the tension face, and PC45T10 and PC45T12, strengthened on both compressed and tension faces, reached maximum strains from 2.84 mm/m to 3.89 mm/m, respectively. IBRACON Structures and Materials Journal • 2010 • vol. 3 • nº 3 279 Experimental Analysis of Reinforced Concrete Columns Strengthened with Self-Compacting Concrete Experimental Analysis of Reinforced Concrete Columns Strengthened with Self-Compacting Concrete Experimental Analysis of Reinforced Concrete Columns Strengthened with Self-Compacting Concrete Analysis of Reinforced Concrete Columns Strengthened with Self-Compacting Concrete 280 IBRACON Structures and Materials Journal • 2010 • vol. 3 • nº 3 Columns PC35 and PC55 had the lowest strains, probably because of pre- mature failure caused by the debonding of the strengthening concrete. 4. Conclusions The static short-term loading behavior of the reinforced concrete columns strengthened by recasting with or without addition of tensile steel bars has been confirmed to be very satisfactory in 280 IBRACON Structures and Materials Journal • 2010 • vol 3 • nº 3 IBRACON Structures and Materials Journal • 2010 • vol. 3 • nº 3 280 M. Y. M. OMAR | R. B. GOMES | A. P. A. REIS the present experimental program. However, the problem of joint cracking was a point of concern and it was understood to be of in- terest for the research to explore further the capability of the repair method carrying out more tests. Since the start of testing, columns PC35, PC55, PC45T10, and PC45T12 were stiffer than column P1, thus proving the efficiency of strengthening on the reduction of horizontal displacements. The columns strengthened on compressed and tension faces showed greater stiffness (PC45T10 and PC45T12). As regards reference columns (P1 and P2) and columns strength- the present experimental program. However, the problem of joint cracking was a point of concern and it was understood to be of in- terest for the research to explore further the capability of the repair method carrying out more tests. PC45T12 were stiffer than column P1, thus proving the efficiency of strengthening on the reduction of horizontal displacements. The columns strengthened on compressed and tension faces showed greater stiffness (PC45T10 and PC45T12). As regards reference columns (P1 and P2) and columns strength- Since the start of testing, columns PC35, PC55, PC45T10, and IBRACON Structures and Materials Journal • 2010 • vol. 3 • nº 3 281 Experimental Analysis of Reinforced Concrete Columns Strengthened with Self-Compacting Concrete Experimental Analysis of Reinforced Concrete Columns Strengthened with Self-Compacting Concrete Experimental Analysis of Reinforced Concrete Columns Strengthened with Self-Compacting Concrete 5. Acknowledgements ened only on the tension face (PT10 and PT12), the steel is sub- jected to tension from the start of testing. However, the reinforce- ment only begins to be effectively tensioned at 19% of the failure load of columns PC45T10 and PC45T12 and at 63% and 73% of the respective failure loads of columns PC35 and PC55. One of the contributing factors is the fact that columns PC35 and PC55 had reduced initial eccentricities. ened only on the tension face (PT10 and PT12), the steel is sub- jected to tension from the start of testing. However, the reinforce- ment only begins to be effectively tensioned at 19% of the failure load of columns PC45T10 and PC45T12 and at 63% and 73% of the respective failure loads of columns PC35 and PC55. One of the contributing factors is the fact that columns PC35 and PC55 had reduced initial eccentricities. The authors gratefully acknowledge to CNPq and Procad/ Capes for their financial support. They also thanks to Carlos Campos Consultoria Limitada, Impercia, Perfinasa, and Real- mix for providing materials and for performing material charac- terization tests. Columns strengthened on the tension face (PT10 and PT12) and on both compressed and tension faces simultaneously (PC45T10 and PC45T12) did not reveal signs of debonding of the strengthening concrete. Even though, the difference of the reinforcement rate placed on the strengthening of these col- umns’ tension faces reached 36%, the difference in failure load was less than 5%. Therefore, an increase of the reinforcement ratio on the tension face may not produce a considerable load gain because of the possibility of concrete crushing or of an eventual debonding. 6. References [01] CLIMACO, J. C. T.. Repair of structural concrete involving the addition of new concrete. Ph.D. Thesis, Polythecnic of Central London, London, UK. 285p, 1990. [02] AÏTCIN, P. C., High-Performance Concrete. London, U. K., Editora E & FN SPON, 1st Edition, 1998, 591 p. Despite concrete debonding, columns strengthened on the com- pressed face had a higher strength gain than those strengthened on the tension face. Considering the tension reinforcement ratio used in these specimens, these results suggest a significant in- crease in load-bearing capacity when the strengthening occurs on the column’s compressed face, thus leading to a reduction of initial load eccentricity. [03] EL Debs, M. K. Precast Concret: Fundamentals and Applications. Department of Structural Engineering, São Carlos Engineering School, São Paulo University, EESC-USP. 456p, 2000, São Paulo, Brazil. [04] ABU-TAIR, A. I.; RIGDEN, S. R.; LAVERY, D.; NADJAI, A. e AHMED, T. M. A. A new method for evaluating the surface roughness of concrete cut for repair or strengthening. Construction and Building Materials, 14, p. 171-176, 2000. Self-compacting concrete as a strengthening material proved to be satisfactory, shaping the section of specimens without segre- gation or faults. As to adherence, this material worked together with the substrate, thus increasing the load-bearing capacity of the strengthened specimens. Debonding only occurred on col- umns PC35 and PC55, which were strengthened solely on the compressed face. [05] ADORNO, A. L. C. Theoretical and experimental analysis of concrete columns and reinforced concrete columns under eccentric load. Ph.D. thesis, Department of Civil and Environmental Engineering, [05] ADORNO, A. L. C. Theoretical and experimental analysis of concrete columns and reinforced concrete columns under eccentric load. Ph.D. thesis, Department of Civil and Environmental Engineering, 282 IBRACON Structures and Materials Journal • 2010 • vol. 3 • nº 3 IBRACON Structures and Materials Journal • 2010 • vol. 3 • nº 3 282 M. Y. M. OMAR | R. B. GOMES | A. P. A. REIS University of Brasilia, Brasilia, DF, Brazil, 399p, 2004. (In Portuguese). [06] ARAÚJO, L. M. B. Theoretical and experimental analysis of reinforced concrete columns subjected to axial load and flexure. M.Sc. thesis, Federal University of Goias, Goiânia, Brazil. 196p, 2004. (In Portuguese). [07] OMAR, M. Y. M. Experimental analysis of reinforced concrete columns strengthened with self-compacting concrete. M.Sc. thesis, Federal University of Goias, Goiânia, Brazil. 266p, 2006. (In Portuguese). [08] ACI COMMITTEE 318. IBRACON Structures and Materials Journal • 2010 • vol. 3 • nº 3 6. References Building Code Requirements for Reinforced Concrete and Commentary - ACI 318M/02. Detroit, American Concrete Institute, 2001. IBRACON Structures and Materials Journal • 2010 • vol. 3 • nº 3 283
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Murine Anti-vaccinia Virus D8 Antibodies Target Different Epitopes and Differ in Their Ability to Block D8 Binding to CS-E
PLOS pathogens
2,014
cc-by
12,388
Abstract The IMV envelope protein D8 is an adhesion molecule and a major immunodominant antigen of vaccinia virus (VACV). Here we identified the optimal D8 ligand to be chondroitin sulfate E (CS-E). CS-E is characterized by a disaccharide moiety with two sulfated hydroxyl groups at positions 49 and 69 of GalNAc. To study the role of antibodies in preventing D8 adhesion to CS-E, we have used a panel of murine monoclonal antibodies, and tested their ability to compete with CS-E for D8 binding. Among four antibody specificity groups, MAbs of one group (group IV) fully abrogated CS-E binding, while MAbs of a second group (group III) displayed widely varying levels of CS-E blocking. Using EM, we identified the binding site for each antibody specificity group on D8. Recombinant D8 forms a hexameric arrangement, mediated by self-association of a small C-terminal domain of D8. We propose a model in which D8 oligomerization on the IMV would allow VACV to adhere to heterogeneous population of CS, including CS-C and potentially CS-A, while overall increasing binding efficiency to CS-E. Citation: Matho MH, de Val N, Miller GM, Brown J, Schlossman A, et al. (2014) Murine Anti-vaccinia Virus D8 Antibodies Target Diffe Their Ability to Block D8 Binding to CS-E. PLoS Pathog 10(12): e1004495. doi:10.1371/journal.ppat.1004495 N, Miller GM, Brown J, Schlossman A, et al. (2014) Murine Anti-vaccinia Virus D8 Antibodies Target Different Epitopes and Differ in ing to CS-E. PLoS Pathog 10(12): e1004495. doi:10.1371/journal.ppat.1004495 Editor: Daved H. Fremont, Washington University, United States of America Received April 4, 2014; Accepted September 29, 2014; Published December 4, 2014 Copyright:  2014 Matho et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. uthors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its es. Funding: This project has been funded in whole or in part with Federal funds from the National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. Abstract HHSN272200900048C (www.niaid.nih.gov) and National Institute of General Medical Sciences grant GM093627 to LCHW (www.nigms.nih.gov). ABW was supported from start up funds from the Scripps Research Institute. EM data were collected at the National Resource for Automated Molecular Microscopy at the Scripps Research Institute, which is supported by the Biomedical Technology Research Center program (GM103310) of the National Institute of General Medical Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: dzajonc@lji.org * Email: dzajonc@lji.org Murine Anti-vaccinia Virus D8 Antibodies Target Different Epitopes and Differ in Their Ability to Block D8 Binding to CS-E Michael H. Matho1, Natalia de Val2, Gregory M. Miller3, Joshua Brown3, Andrew Schlossman1, Xiangzhi Meng4, Shane Crotty5, Bjoern Peters5, Yan Xiang4, Linda C. Hsieh-Wilson3, Andrew B. Ward2, Dirk M. Zajonc1* 1 Division of Cell Biology, La Jolla Institute for Allergy and Imunology (LIAI), La Jolla, California, United States of America, 2 Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, United States of America, 3 Howard Hughes Medical Institute, Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, United States of America, 4 Department of Microbiology and Immunology, University of Texas Health Science Center, San Antonio, Texas, United States of America, 5 Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology (LIAI), La Jolla, California, United States of America PLOS Pathogens | www.plospathogens.org December 2014 | Volume 10 | Issue 12 | e1004495 Identification of CS-E as an optimal ligand for D8 and role of MAbs in blocking CS-E adhesion Group II and III MAbs were previously identified to bind peptide 58 (residue 91–110). Residues 91–110 are located on one side of the positively charged crevice of D8 that we previously proposed to be the CS binding site, using computational docking [8]. While group II antibodies did not interfere with CS-E binding, group III antibodies BH7 and EE11 blocked CS-E binding to varying degrees. These results illustrated diversity in this group, which constitutes the majority of the anti-D8 antibodies (eight out of twelve). Despite both group II and III antibodies binding to peptide 58, they surprisingly have different effects on CS-E binding, indicating that they have an overlapping epitope but differ in their binding footprint at the CS-E binding site on D8. Because group II and III MAbs target overlapping epitopes juxtaposing the sugar-binding site, we found such a wide variation in CS-E blocking (BH7 40%, EE11 80%) quite surprising within a single specificity group. As EE11 and BH7 are the most distant representatives of group III antibodies, based on their light chain (LC) sequences (Fig. S1), we next asked whether the other group III antibodies have intermediate CS-E blocking abilities. We tested 6 out of all 8 Group III MAbs. We also used monomeric D8, instead of the oligomer, to avoid potential steric hindrance of antibody binding to D8, which itself could result in CS-E blocking differences (Fig. 1C). Nevertheless, BH7 and EE11 blocked binding to monomeric D8 similarly when compared to oligomeric D8 (30 vs. 40% for BH7 and 60 vs. 80% for EE11). All other analyzed Group III MAbs fall within the same CS-E blocking range (35–60%), indicating subtle differences in their binding footprint at the CS-E binding site of D8 (Fig. 1C, D). Since group II MAbs did not cross-block CS-E, in contrast to group III MAbs, we proposed that the shared epitope residues of peptide 58 could not be responsible for the group III MAbs cross-blocking of CS-E In this study, we used glycosaminoglycan microarrays printed with natural polysaccharides enriched in specific sulfated struc- tures to identify the molecular species of CS that optimally binds to D8 [10,11]. We further asked whether any of the antibodies that target different binding sites on D8 block D8 adhesion to CS. Lastly, using single particle electron microscopy (EM), we mapped the binding site of a representative antibody of each antibody specificity group on D8. Identification of CS-E as an optimal ligand for D8 and role of MAbs in blocking CS-E adhesion Cross-blocking power may be explained in terms of intersecting buried surface areas (BSAs), or in terms of competing intermolecular electrostatic interactions. We have recently identified four different antibody specificity groups among an ensemble of twelve murine monoclonal antibodies specific to D8 [9]. D8 MAbs were first sorted by competitive ELISA according to their targeted epitopes on the basis of which other MAbs they cross-block. Four main specificity groups resulted from this experiment (group I: JE11; II: AB12 and CC7.1; III: BG9.1, BH7.2, EB2.1, EE11, JA11.2, JE10, and JF11; IV: FH4.1 and LA5) [9]. Partial epitope definitions for each of the antibody groups were previously determined. For group I MAbs, the epitope definition was mapped by Deuterium Exchange Mass Spectrometry (DXMS) to residues 10–14 and 80–90 [9]. Peptide ELISA, using 20-mer peptides that overlap by 10 amino acids and cover the entire D8 protein sequence, suggested that group II antibodies target a linear epitope called peptide 58 (residues 91– 110), while groups I, III and IV target a conformational epitope [9]. Alanine scanning of peptide 58, and D8 point mutation analysis (PMA) refined the group II epitope to 10 residues (H95, W96, N97, K99, Y101, S102, S103, E106, H110 and D112). While the group III MAb epitope is conformational, group III MAbs can also bind peptide 58, albeit much weaker than group II antibodies. Interestingly, group II and III MAbs cross-block each other. Alanine scanning of peptide 58 gave a partial definition of the group III epitope; it includes, but is not limited to H95, W96, N97, Y101, S103, Y104, E105, E106, and K108. Finally, X-ray crystallography identified the D8 epitope of one MAb of group IV, LA5 [8]. Group I and II MAb representatives JE11 and CC7.1 did not affect D8 binding to CS-E significantly, indicating that their epitopes do not overlap with the CS-E binding site on D8 (Fig. 1B, D). LA5, a member of group IV, fully abrogates CS-E binding to D8, as we had previously suggested [8] (Fig. 1B, D). Group I and II MAb representatives JE11 and CC7.1 did not affect D8 binding to CS-E significantly, indicating that their epitopes do not overlap with the CS-E binding site on D8 (Fig. 1B, D). LA5, a member of group IV, fully abrogates CS-E binding to D8, as we had previously suggested [8] (Fig. 1B, D). Introduction attachment to host cells. GAGs are linear polysaccharides with repeating disaccharide units predominantly found on cell surfaces and as constituents of the extracellular matrix [3]. While A27 and H3 interact with heparan sulfate (HS) or heparin (HP) [4,5], D8 binds to chondroitin sulfate (CS) [6]. Viral adhesion to GAGs represents a major route of entry for a range of pathogens [7]. As a result, GAG adhesion is an early and important step that initiates viral infection. Vaccinia virus (VACV) is a low virulence orthopoxvirus that was used to eradicate smallpox [1]. Immunization with VACV leads to the production of potent protective antibodies that target the VACV envelope proteins A27, A33, B5, D8, H3 and L1, among others [1]. VACV has two forms of infectious virions. The intracellular mature virion (IMV) is the most abundant form of VACV and mainly responsible for viral spread between hosts. A27, D8, H3 and L1 are expressed on the outer membrane of IMV. A33 and B5 are embedded in the more fragile extracellular enveloped virion (EEV), which has an additional host cell derived envelope. EEV is thought to be involved in cell-to-cell spread within the host and is critical for virulence. Antibody responses against VACV potently target both infectious forms of the virus, likely contributing to the efficacy of the smallpox vaccine [2]. Among the VACV envelope proteins, A27, H3 and D8 are viral adhesion molecules that bind to glycosaminoglycans (GAG) for We have recently determined the crystal structure of the CS adhesion protein D8 [8]. The N-terminal ectodomain contains a carbonic anhydrase fold (CAH, residues 1–234), followed by a smaller domain of unknown function (residues 235–273). A single transmembrane domain (TM, 274–294) and a small intra-virion tail (295–304) constitute the rest of the protein. The CAH domain was suggested to be responsible for CS binding, as it has a central positively charged crevice that complements the negative charge of CS [8]. December 2014 | Volume 10 | Issue 12 | e1004495 1 PLOS Pathogens | www.plospathogens.org Natural Antibody Responses Interfere with D8/CS-E Adhesion Identification of CS-E as an optimal ligand for D8 and role of MAbs in blocking CS-E adhesion Vaccinia virus (VACV) is an orthopox virus and considered the gold standard of vaccines as it was used to eradicate smallpox from the human population. Inoculation with VACV leads to a strong B cell immune response and the production of potent antibodies that simultaneously target several envelope proteins of the virus. Among those viral proteins, D8 is an adhesion molecule that binds chondroitin sulfate, a glycosaminoglycan, on the host cell surface. Here, we identified chondroitin sulfate E (CS-E), as the preferred ligand for D8 and assessed the role of a panel of anti-D8 antibodies in preventing D8 binding to CS-E. We further mapped the binding site of each antibody on the D8 surface to reveal the targeted epitopes. Finally, using several truncated D8 constructs, we identified that the C- terminal domain of D8 that is not involved in CS-E binding is in fact involved in oligomerization of native D8 in vitro and likely, also on the virion, as a means of increasing binding affinity to increase viral adhesion to CS on the host cell. To broadly assess D8-GAG interactions, we employed micro- arrays containing immobilized chondroitin sulfate polysaccharides enriched in specific sulfation motifs (CS-A, -C, -D, and –E), dermatan sulfate (DS), hyaluronic acid (HA), heparin and heparan sulfate (HS). We observed weak binding of monomeric D8 to chondroitin sulfate, a CS preparation containing a mix of sulfation motifs (CS-A, -C, -D, and –E). In contrast, D8 displayed strong concentration-dependent binding to CS-E. Binding to heparan sulfate or chondroitin sulfate with different sulfation patterns was not observed (Fig. 1A). Having identified the optimal ligand for D8, we then determined the role of anti-D8 antibodies in preventing D8 binding to CS-E using a competition-binding assay. A represen- tative of each of the four antibody specificity groups was first pre- incubated at a saturating concentration with D8 and then tested for binding to CS-E on the GAG microarrays. If the antibody bound at the CS-E binding site on D8, no (or greatly reduced) binding of CS-E would be observed. In contrast, full binding to CS-E would occur for antibodies bound at a site separate from the CS-E binding site. The extent of the CS-E blocking ability (%) of the MAb indicated the degree of overlap of the two binding sites (MAb epitope and CS-E binding site). PLOS Pathogens | www.plospathogens.org Identification of CS-E as an optimal ligand for D8 and role of MAbs in blocking CS-E adhesion Identification of different binding sites on D8 illuminates the molecular details of the murine antibody response against viral D8. Finally, we discovered two opposing sides of the D8 protein surface that are not targeted by antibodies, likely due to their inaccessibility in the viral membrane, and we propose a model in which D8 forms a hexamer. The hexameriza- tion is mediated by self-association of the previously uncharacter- ized C-terminal ectodomain (residues 235–273) downstream of the CAH domain. December 2014 | Volume 10 | Issue 12 | e1004495 PLOS Pathogens | www.plospathogens.org PLOS Pathogens | www.plospathogens.org 2 Natural Antibody Responses Interfere with D8/CS-E Adhesion ( i id H95 W96 N97 Y101 S103 d E106) [9] K108 h i l d CS A [8] I h d k d d l h lf Figure 1. D8 binds to CS-E and anti-D8 MAbs display different levels of competition with CS-E. A. GAG microarray performed with monomeric D8 antigen. B. MAb/CS-E cross-blocking experiments using representatives of all four antibody specificity groups and oligomeric D8. C. MAb/CS-E cross-blocking of group III MAbs D. Summary of CS-E cross-blocking abilities of various MAbs. Group III MAbs are characterized by large variations in cross-blocking ability. Microarray binding experiments were performed in triplicate, and the data represent the average of 10 spots per concentration averaged from the three experiments (6SEM, error bars). doi:10.1371/journal.ppat.1004495.g001 y p Figure 1. D8 binds to CS-E and anti-D8 MAbs display different levels of competition with CS-E. A. GAG microarray performed with monomeric D8 antigen. B. MAb/CS-E cross-blocking experiments using representatives of all four antibody specificity groups and oligomeric D8. C. MAb/CS-E cross-blocking of group III MAbs D. Summary of CS-E cross-blocking abilities of various MAbs. Group III MAbs are characterized by large variations in cross-blocking ability. Microarray binding experiments were performed in triplicate, and the data represent the average of 10 spots per concentration averaged from the three experiments (6SEM, error bars). doi:10.1371/journal.ppat.1004495.g001 the previously used CS-A [8]. In the docked model, each sulfate group is found in the vicinity of the charged D8 residue pairs K48/K98, R44/K108, and K41/R220, delineating the crevice, which corroborates the high specificity of CS-E over CS-A that was used for docking prior to our knowledge of the exact ligand (Fig. 2A). In effect, CS-A bears only one sulfate group, on 49- hydroxyl of GalNAc, while CS-E has an additional sulfate on the 69-hydroxyl group. PLOS Pathogens | www.plospathogens.org Natural Antibody Responses Interfere with D8/CS-E Adhesion illustrating that our experimental epitope definitions agreed with the associated EM maps. necessary to form salt bridges with both sulfate moieties. Alanine substitutions of residues lining the crevice led to moderate (R220A) or severe (R220A/R44A, and R220A/K48A) reductions in CS-E binding (Fig. 2B). This suggests that D8 binding to CS-E is likely mediated by a network of electrostatic interactions that form pairs on opposing sides of the entire D8 crevice, involving residues K41, R44, K48, K98 and K108 (Fig. 2A). In the D8/LA5-Fab complex [MAb of group IV, pdb code 4ETQ], we observed that only two of these residue pairs (K41/R220 and R44/K108) are part of the epitope. However, LA5 fully blocks CS-E binding of D8, even without K48/K98 coverage. Total BSA for group I (JE11) D8:MAb interface is 1238 A˚ 2. EM data suggests the following additional residues for group I epitope, adding to the strict definition previously obtained by DXMS: N9, D75, Y76, Q122, L124, D126, K163, T187, P188, and N190 (Fig. 3B, C). Looking back at the DXMS data [9], we saw that most of the aforementioned residues are in D8 regions where deuterium (H2) exchange decreased upon complex formation, but was weak or inconsistent. Most of these additional residues interact with the JE11 light chain (Fig. 3B, C). The group II (CC7.1) MAb:D8 interface has a total BSA of only 851 A˚ 2, which correlates with the linear nature of group II epitope. Residues 91–110 correspond to a protruding region at the surface of D8. Only minor differences were observed for the group II epitope when comparing EM data to group II alanine scanning definition: EM defined additional residues K98 and K100 as part of the epitope (Fig. 4). However, K98A and K100A showed no reduced binding to MAb CC7.1 [9], suggesting that both residues do not contribute greatly to antibody binding. Identification of anti-D8 MAb epitopes by EM p p y Determining the full epitope for group II and III MAbs is necessary to explain the wide spread in CS-E blocking ability (10– 80%) observed between group II and group III MAbs. To address this question, we used negative stain single particle D8:Fab EM reconstructions. To map the relative positions of the different antibodies on D8, we used Fabs from two different groups, simultaneously bound to D8. JE11-Fab (group I) was included as a reference in all subsequent ternary complexes because it does not cross-block binding of any other MAb groups [9]. We then reconstructed the three-dimensional arrangements by embedding individual atomic models within the low-resolution maps of the multivalent complexes obtained by EM. Docking was guided using previously determined experimental constraints: X-ray crystallog- raphy definition of group IV epitope, DXMS definition of group I epitope, and alanine-scan definitions of groups II and III epitopes [9]. Since we had previously determined the high-resolution crystal structure of D8/LA5 (group IV), we first reconstructed the D8/JE11/LA5 complex (Fig. 3). We then reconstructed the D8/ JE11/CC7.1 complex, since a definition of the group II epitope was available (Fig. 4). Fitting of those reconstructed atomic models gave best-fit correlation values of 0.9016 and 0.9207, respectively. These two first processes served as an internal proof of concept, Identification of CS-E as an optimal ligand for D8 and role of MAbs in blocking CS-E adhesion Hence, docking data converged with the experimental definition of the ligand, since it pointed to the aforementioned positively charged residual pairs that are probably (amino acids H95, W96, N97, Y101, S103 and E106) [9]. K108, however, is unique to the group III epitope and is involved in CS- E binding, based on our docking result (see below). Therefore, we hypothesized that residue K108 is responsible for cross-blocking differences between group II and group III. Other intersecting D8 residues are necessarily involved in the case of group III MAbs that induce high levels of CS-E blocking, such as EE11 (60–80% cross- blocking; Fig. 1B, C). These CS-E binding data led us to refine our previous docking results by using a dodecasaccharide fragment of CS-E, instead of December 2014 | Volume 10 | Issue 12 | e1004495 PLOS Pathogens | www.plospathogens.org 3 Natural Antibody Responses Interfere with D8/CS-E Adhesion Group III (EE11) epitope definition by EM We used EE11-Fab to build a group III ternary complex for which we did not have a full definition (Fig. 5). Model-to-map correlation for this complex was 0.9115. The total EE11:D8 BSA was 1710 A˚ 2, which is larger than group I and II epitopes but smaller than group IV (2434 A˚ 2). A total of twenty-six D8 residues interact with EE11 MAb. Six D8 residues interact with the light chain (LC) and twenty-four with the heavy chain (HC) (Fig. 5B, C). Novel D8 contacts are E30, T34, T35, R44, N46, F47, K48, G49, G50, Y51, N59, E60, V62, L63, S64 and additional peptide 58 residues K98, K99, K100 and S102, with residues involved in CS-E binding indicated in bold. Group IV (LA5) footprint (in orange) intersects with the group III (EE11) epitope at residues R44 and K108 (Fig. 5C). Figure 2. Mapping of the CS-E binding site on vaccinia D8 ectodomain. A. Docking of CS-E dodecasaccharide to D8. Framed regions highlight regularly spaced, positively charged residue pairs K41/R220, R44/K108, and K48/K98, which are predicted to interact with negatively charged sulfate moieties of CS-E. B. Mapping of CS-E binding site. Mutation R220A led to a ,50% decrease in CS-E binding compared to wt, while CS- E binding to the double mutants R220/R44 and R220/K48 was almost fully abrogated, corroborating the CS-E docking model. Data were averaged from three experiments. doi:10.1371/journal.ppat.1004495.g002 Figure 2. Mapping of the CS-E binding site on vaccinia D8 ectodomain. A. Docking of CS-E dodecasaccharide to D8. Framed regions highlight regularly spaced, positively charged residue pairs K41/R220, R44/K108, and K48/K98, which are predicted to interact with negatively charged sulfate moieties of CS-E. B. Mapping of CS-E binding site. Mutation R220A led to a ,50% decrease in CS-E binding compared to wt, while CS- E binding to the double mutants R220/R44 and R220/K48 was almost fully abrogated, corroborating the CS-E docking model. Data were averaged from three experiments. doi:10.1371/journal.ppat.1004495.g002 December 2014 | Volume 10 | Issue 12 | e1004495 PLOS Pathogens | www.plospathogens.org 4 Natural Antibody Responses Interfere with D8/CS-E Adhesion Figure 3. Group I (JE11) footprint. A. EM reconstruction of the D8 monomer in complex with Fab’s JE11 (group I) and LA5 (group IV) at 24 A˚ resolution. Projection Matching and Fourier Shell Correlation (FSC) are shown in figure S5. Top left inset shows one of the class-averages used for building the map. Group III (EE11) epitope definition by EM EM density is shown in gray mesh. D8 monomer crystal structure is represented as a grey surface except for epitope footprints that follow the same color code as [9]: group I (JE11): red; group IV (LA5): orange. Actual Fab chains also follow this color code. B. Summary of JE11 (group I) contacts. D8 residues in red belong to the initial definition of group I epitope, assessed by DXMS. Salmon-colored residues complete the definition of group I conformational epitope. Black bold-contours highlight residues previously picked for mutation analysis [9]. C. Footprint of completed JE11 epitope. Red and salmon footprints evidence initial and additional epitope residues. Despite being juxtaposed to each other, group IV (LA5) and group I (JE11) footprints do not intersect. Black labels inform on residues resulting in a loss of MAb/Ag affinity upon mutation to alanine, while mutated residues in white did not lead to any relevant change in binding. doi:10.1371/journal.ppat.1004495.g003 Figure 3. Group I (JE11) footprint. A. EM reconstruction of the D8 monomer in complex with Fab’s JE11 (group I) and LA5 (group IV) at 24 A˚ resolution. Projection Matching and Fourier Shell Correlation (FSC) are shown in figure S5. Top left inset shows one of the class-averages used for building the map. EM density is shown in gray mesh. D8 monomer crystal structure is represented as a grey surface except for epitope footprints that follow the same color code as [9]: group I (JE11): red; group IV (LA5): orange. Actual Fab chains also follow this color code. B. Summary of JE11 (group I) contacts. D8 residues in red belong to the initial definition of group I epitope, assessed by DXMS. Salmon-colored residues complete the definition of group I conformational epitope. Black bold-contours highlight residues previously picked for mutation analysis [9]. C. Footprint of completed JE11 epitope. Red and salmon footprints evidence initial and additional epitope residues. Despite being juxtaposed to each other, group IV (LA5) and group I (JE11) footprints do not intersect. Black labels inform on residues resulting in a loss of MAb/Ag affinity upon mutation to alanine, while mutated residues in white did not lead to any relevant change in binding. doi:10.1371/journal.ppat.1004495.g003 Figure 4. Group II (CC7.1) footprint. A. EM reconstruction of the D8 monomer in complex with Fabs JE11 (group I) and CC7.1 (group II) at 21 A˚ resolution. See figure legend 3 for general description. Group III (EE11) epitope definition by EM Projection Matching and Fourier Shell Correlation (FSC) are shown in figure S6. Epitope footprints follow the same color code as [9]: group I (JE11): red; group II (CC7.1): green. B. Summary of CC7.1 (group II) contacts. D8 residues colored in forest green belong to the initial definition of group I epitope, assessed by alanine scanning. Lighter green residues complete the definition of group II epitope. C. Footprint of completed CC7.1 epitope. Forest green and light green footprints evidence initial and current epitope definitions. Group IV (LA5) footprint in orange does not intersect with group II (CC7.1) epitope. doi:10.1371/journal.ppat.1004495.g004 PLOS Pathogens | www plospathogens org 5 December 2014 | Volume 10 | Issue 12 | e1004495 Figure 4. Group II (CC7.1) footprint. A. EM reconstruction of the D8 monomer in complex with Fabs JE11 (group I) and CC7.1 (group II) at 21 A˚ resolution. See figure legend 3 for general description. Projection Matching and Fourier Shell Correlation (FSC) are shown in figure S6. Epitope footprints follow the same color code as [9]: group I (JE11): red; group II (CC7.1): green. B. Summary of CC7.1 (group II) contacts. D8 residues colored in forest green belong to the initial definition of group I epitope, assessed by alanine scanning. Lighter green residues complete the definition of group II epitope. C. Footprint of completed CC7.1 epitope. Forest green and light green footprints evidence initial and current epitope definitions. Group IV (LA5) footprint in orange does not intersect with group II (CC7.1) epitope. doi:10.1371/journal.ppat.1004495.g004 December 2014 | Volume 10 | Issue 12 | e1004495 5 Natural Antibody Responses Interfere with D8/CS-E Adhesion Figure 5. Group III (EE11) footprint. A. EM reconstruction of the D8 monomer in complex with Fabs JE11 (group I) and EE11 (group III) at 22 A˚ resolution. See figure legend 3 for general description. Projection Matching and Fourier Shell Correlation (FSC) is shown in figure S7. Epitope footprints follow the same color code as [9]: group I (JE11): red; group III (EE11): blue. Actual Fab chains follow the same color code. B. Summary of EE11 (group III) contacts. D8 residues colored in blue belong to the initial definition of group III epitope, assessed by alanine scanning. Cyan residues complete the definition of group III epitope. C. Footprint of completed EE11 epitope. Group III (EE11) epitope definition by EM When subjected to SEC both D8 oligomers, either lacking (1–261) or containing (1–262) the C-terminal cysteine, eluted at the identical volume (Fig. 7). When the D8 oligomers were subjected to non- reducing SDS PAGE, no D8 monomer, but only disulfide-linked D8 dimers were observed [8]. Therefore, only an even numbered D8 oligomer, such as a hexamer appears possible, as an octamer would not be supported by the SEC-MALS data. We speculate that the 7th and central drupelet is not a result of heptameric D8 but rather formed by the convergence of the C-terminal extremities (235–262) of all six D8 subunits (SUs). We confirmed the monodispersity of the sample by size SEC-MALS (Fig. S3), The resolution of the EM maps obtained with negative staining provides an accurate epitope definition of group III but not at atomic resolution. In order to validate this newly defined interface, we picked three seemingly critical residues for alanine scanning mutagenesis, two of which are involved in CS-E binding (E30, R44, K48). A 3-fold decrease in affinity was observed for D8 E30A (Fig. S2). However, this was likely due to suboptimal folding of D8 E30A, as the control antibody (JE11) also showed reduced binding (9-fold). In both cases, only the association phase was affected. This suggested that E30 does not contribute greatly to EE11 binding. We observed an almost 10-fold decrease in EE11 affinity with D8 R220A/R44A and R220A/K48A mutants compared to either wild-type (wt) D8 or R220A D8, suggesting both R44 and K48 are important residues for EE11 binding (Fig. S2). Both mutants bound normally to control antibodies. Figure 6 summarizes the details of and the techniques used to identify the complete murine D8 epitome. Most of the CS-E binding residues of D8 are targeted by the HC of EE11 (K41, R44, K98, and K108), while the EE11 LC has a single contact (K98; Fig. 5B). Hence, our model is compatible with the general assumption that the HC frequently drives most Ag/MAb interactions [12]. However, the EM model does not corroborate our hypothesis that LC differences were responsible for the CS-E blocking differences within group III. Group III (EE11) epitope definition by EM The initial definition obtained by alanine scanning and PMA is depicted in blue and the current definition deduced from the EM particle reconstruction is in cyan. Group IV (LA5) footprint in orange does intersect with group III (EE11) epitope at residues R44 and K108 (orange/cyan or orange/blue stripes). doi:10.1371/journal.ppat.1004495.g005 Figure 5. Group III (EE11) footprint. A. EM reconstruction of the D8 monomer in complex with Fabs JE11 (group I) and EE11 (group III) at 22 A˚ resolution. See figure legend 3 for general description. Projection Matching and Fourier Shell Correlation (FSC) is shown in figure S7. Epitope footprints follow the same color code as [9]: group I (JE11): red; group III (EE11): blue. Actual Fab chains follow the same color code. B. Summary of EE11 (group III) contacts. D8 residues colored in blue belong to the initial definition of group III epitope, assessed by alanine scanning. Cyan residues complete the definition of group III epitope. C. Footprint of completed EE11 epitope. The initial definition obtained by alanine scanning and PMA is depicted in blue and the current definition deduced from the EM particle reconstruction is in cyan. Group IV (LA5) footprint in orange does intersect with group III (EE11) epitope at residues R44 and K108 (orange/cyan or orange/blue stripes). doi:10.1371/journal.ppat.1004495.g005 C-terminal cysteine (C262, residues 1–261). However, we have previously shown that the full D8 ectodomain forms a disulfide- linked dimer (through C262) that further associates non-covalently to form an oligomer [8]. Despite oligomeric D8 having an molecular weight that was considerably larger than the 158 kDa MW marker on SEC, we had previously described the D8 oligomeric state as tetrameric, based on D8 migration during native gel electrophoresis [8]. However, size exclusion chroma- tography with inline multi-angle light scattering (SEC-MALS), assigned an average MW of 228 kDa for the D8 oligomer compared to 42 kDa obtained for the D8 monomer, using two different SEC resins (Fig. S3). The SEC-MALS data indicate a hexameric D8 arrangement. EM class averages of the D8 oligomer revealed no more than 7 drupelets, with 6 drupelets surrounding a central drupelet that appears of lesser intensity (Figs. 7A, S3 and S4). While a D8 heptamer remained a possibility, our SEC data coupled to non-reducing SDS-PAGE suggested that the D8 oligomer is formed by en even number of D8 monomers. D8 oligomerization is mediated through the C-terminal domain For structural studies, we prepared monomeric D8 that either only contains the CAH domain (residues 1–234) or lacks the distal December 2014 | Volume 10 | Issue 12 | e1004495 PLOS Pathogens | www.plospathogens.org PLOS Pathogens | www.plospathogens.org 6 Natural Antibody Responses Interfere with D8/CS-E Adhesion Figure 6. Summary of D8 murine epitome and CS-E binding site. A. Updated footprint of groups I (red), II (green), III (blue) and IV (orange) are represented. The yellow line reminds the CS-E path between positively charged residue pairs (black frames). B. Summary of D8 epitope residues f ll VACV ti D8 i MAb f th f it R l ti d d th th d d f ifi t N l d fi d Figure 6. Summary of D8 murine epitome and CS-E binding site. A. Updated footprint of groups I (red), II (green), III (blue) and IV (orange) are represented. The yellow line reminds the CS-E path between positively charged residue pairs (black frames). B. Summary of D8 epitope residues for all VACV anti-D8 murine MAbs of the four epitope groups. Resolution depends on the method used for a specific assessment. Newly-defined epitope residues are highlighted in bold italic. Residues in red are important for both CS-E, and group III and IV MAb binding. doi:10.1371/journal.ppat.1004495.g006 Figure 6. Summary of D8 murine epitome and CS-E binding site. A. Updated footprint of groups I (red), II (green), III (blue) and IV (orange) are represented. The yellow line reminds the CS-E path between positively charged residue pairs (black frames). B. Summary of D8 epitope residues for all VACV anti-D8 murine MAbs of the four epitope groups. Resolution depends on the method used for a specific assessment. Newly-defined epitope residues are highlighted in bold italic. Residues in red are important for both CS-E, and group III and IV MAb binding. doi:10.1371/journal.ppat.1004495.g006 Figure 6. Summary of D8 murine epitome and CS-E binding site. A. Updated footprint of groups I (red), II (green), III (blue) and IV (orange) are represented. The yellow line reminds the CS-E path between positively charged residue pairs (black frames). B. Summary of D8 epitope residues for all VACV anti-D8 murine MAbs of the four epitope groups. Resolution depends on the method used for a specific assessment. Newly-defined epitope residues are highlighted in bold italic. Residues in red are important for both CS-E, and group III and IV MAb binding. Natural Antibody Responses Interfere with D8/CS-E Adhesion Natural Antibody Responses Interfere with D8/CS-E Adhesion which suggested that 2D class averages are of a single species, and most likely represent different orientations of the hexamer (Fig. S4A). However, obtaining three-dimensional maps of the D8 hexamer was problematic due to apparent conformational flexibility of D8. In fact, the CAH fold domains do not adopt a uniform arrangement in the different hexameric D8 class averages of figure 7A, but are instead placed haphazardly around the central drupelet, likely due to flexibility of the connecting linker to the C-terminal domain. The C-terminal domain was also disordered in the crystal structure of D8 [8]. Hence, multiple conformations of the D8 hexamer prevent the reconstruction of a three-dimensional model via EM. This flexibility also suggests that CAH fold domains do not participate in the oligomeric interface and correlates with an apparent higher MW for the D8 hexamer (MW of 228 kDa compared to theoretical 192 kDa). 262 (D8D263 construct), we observed a higher ratio of oligomer vs. monomer (Fig. 7B). The third construct, D8D235, contains only the CAH fold domain. D8D235 was solely monomeric, which confirmed the mapping of the D8 oligomeric interface to the C- terminal region. These findings now assign a function to this domain. A model of the D8 hexameric arrangement is shown in figure 7C. In this model, both sides of a D8 monomer are in close proximity to the two neighboring D8 monomers. Those are also the D8 surfaces that are not targeted by any of the murine antibodies, likely due to inaccessibility, further validating the hexameric ‘‘ring-like’’ model. Finally we have assessed the role of D8 oligomerization in CS binding, using the GAG microarray binding assay (Fig. 7D). A 40% increase in CS-E binding to oligomeric D8 compared to the monomer was observed, correcting for the six times molar excess of D8 monomer. Therefore, oligomerization increases binding avidity. In addition, and maybe more importantly, slight binding to other CS species could also be detected. This was especially true for CS-C, and was also observed for CS-A to a low degree. Together, with more optimal CS binding to host cells, we speculate that D8 oligomerization increases viral avidity to CS-E but also to other CS species. This may improve viral adhesion to cells expressing low levels of surface CS-E, or heterogeneous populations of CS. Until now, no function was assigned to the C-terminal domain of D8. Natural Antibody Responses Interfere with D8/CS-E Adhesion To test our hypothesis that this domain mediates oligomerization, we compared SEC elution profiles of three D8 constructs of different lengths. D8 D262 is mostly monomeric when cells are grown at 37uC, and has a surprisingly higher ratio of hexamer when grown at lower temperature (30uC). This construct was used to solve the structure of the D8 monomer/ LA5-Fab complex. In order to favor crystallization, we intention- ally did not include unique cysteine 262. When including cysteine Figure 7. D8 oligomeric interface is secluded to the C-terminal region 235–262. A. From top to bottom: selected class averages of (i) unliganded D8 oligomer, (ii) oligomeric D8+JE11-Fab, and (iii) oligomeric D8+JE11-Fab+LA5-Fab. Despite the monodispersity of unliganded D8 oligomeric sample, particles showed a varying number of drupelets, because of their orientation on the EM grid. The class averages with the highest number of drupelets always show six drupelets surrounding a central one (red arrows). B. SEC profiles of recombinant D8 D235, D262, and D263 suggest that D8 oligomerises through the C-terminal domain. SEC markers as grey curve with MW given in kDa. C. Putative D8 hexameric model based on EM data and SEC-MALS using biochemical constraints relative to the dimer and oligomer interfaces. The black circle highlights the putative seventh and central drupelet, arising from all six SU C-terminal extremities, converging toward the IMV envelope. D. CS-E microarray indicating that D8 hexamer (0.33 mM) binds more effectively to CS-E compared to 6-times molar excess of D8 monomer (2 mM) E. GAG microarray obtained with D8 oligomer (0.1 mM). D8 oligomer binds CS-E with higher affinity than the monomer, and also weakly binds to other CS species but not to DS, HA, heparin and HS. Micro array binding experiment was performed in triplicate, and the data represent the average of 10 spots per concentration averaged from the three experiments (6SEM, error bars). doi:10.1371/journal.ppat.1004495.g007 Figure 7. D8 oligomeric interface is secluded to the C-terminal region 235–262. A. From top to bottom: selected class averages of (i) unliganded D8 oligomer, (ii) oligomeric D8+JE11-Fab, and (iii) oligomeric D8+JE11-Fab+LA5-Fab. Despite the monodispersity of unliganded D8 oligomeric sample, particles showed a varying number of drupelets, because of their orientation on the EM grid. The class averages with the highest number of drupelets always show six drupelets surrounding a central one (red arrows). B. D8 oligomerization is mediated through the C-terminal domain doi:10.1371/journal.ppat.1004495.g006 December 2014 | Volume 10 | Issue 12 | e1004495 PLOS Pathogens | www.plospathogens.org PLOS Pathogens | www.plospathogens.org 7 Discussion Interestingly, a VACV D8 knockout was unable to infect the rat brain, suggesting that D8 function is connected to neural tissues [17]. An ex or in vivo experiment with cells displaying the CS-E+/ HP-/HS- phenotype is essential to test the hypothesis that orthopoxviruses may use CS-E as a selective infection route. In effect, an infectious route relying on a non-ubiquitous GAG such as CS-E may be strategically effective, since the virus would avoid binding to most cells and therefore more efficiently target the desired cell types for infection. In addition, perhaps binding CS-E is a strategy for orthopoxviruses to build dormant pools of virus, or to travel long distances within axons of neural cells, for example. However, such subtle mechanisms may be hidden under the main infection routes that involve binding to heparin sulfate by the viral attachment proteins A27 and H3 [4,5]. In this study, we have completed the determination of the murine D8 antibody epitome, proposing a novel definition of the group III epitope. Involvement of K108 explains the competition between group III and IV MAbs. These data also explain why group III MAbs block CS-E binding when group II does not. Sequence analysis, together with the observed overlap of MAb group II and III epitopes lead us to consider group II as a sub- group of group III. The group II epitope is a linear, minimal version of a larger epitope space that includes group III conformational epitopes. Group III MAbs have increased CS-E cross-blocking abilities as their epitopes diverge from linear to conformational, as well as increase in size. As a result, CS-E cross- blocking ability of these MAbs increased. Because EE11 includes the maximal number of CS-E binding site residues that group III MAbs may target (R44, K48, K98, K108), we believe that the EE11 cross-blocking ability represents group III’s CS-E blocking maximum. In contrast, glycan microarray data place BH7 at the bottom end of group III MAb cross-blocking, and therefore the BH7 epitope may (i) be narrower than EE11, and (ii) include at least CS-E contacting residue K108. Group III MAbs display cross-blocking levels between those of group II and IV. We conclude that group III MAbs nuance their CS-E blocking abilities by including additional CS-E binding residues to their epitope (R44, K48, and/or K98). One can use CS-E binding data to predict the size of their respective epitopes. D8 cloning D8 D262 construct (amino acids 1–261, lacking Cys 262) was engineered and prepared as reported previously [8]. D8D263 (containing Cys 262) protein expression vector was designed by modification of the pET-22b(+):: D8D262 expression vector through site-directed insertion of the C262 mutation using the QuikChange II Site-Directed Mutagenesis Kit (Agilent) with primers 59-TCCGATTTGAGAGAGACATGCCTCGAGCAC- CACCACCAC-39 and 59-GTGGTGGTGGTGCTCGAGG- CATGTCTCTCTCAAATCGGA-39. The D8D235 construct contains only the carbonic anhydrase domain of D8 and was obtained by overlapping PCR using the following primers to amplify the D8D235 sequence from VACV ACAM2000 genomic DNA with the Accuprime Pfx PCR Kit (Invitrogen). Primer 1: 59-CTTTAAGAAGGAGATATACA- TATG CAACAACTATCTCCTATT-39, Primer 2: 59-GT- GGTGGTGCTCGAGAGAATAATATACTTCTGTGTCAT- C-39 [18]. A 134:1 molar ratio of gel-purified PCR product to pET22b(+) was mixed to 1 uL KOD HiFi Polymerase, 1 mL 106 KOD Buffer, 1 mL 8 mM dNTPs, 1 mL 10 mM MgCl2 in a final 10 mL reaction volume (Toyobo). The following thermocycler protocol was then used: 98uC for 2 min, 20 cycles of 98uC for 30 s, 55uC for 30 s and 72uC for 8 min, followed by final extension at 72uC for 20 min. Template DNA was digested with DpnI for 1 hr at 37uC and used to transform DH5a cells for plating on LB Agar with ampicillin. DNA was isolated from 5 mL cultures of single DH5a colonies by Miniprep (Fermentas) and successful cloning was confirmed by sequencing (Retrogen). g , , , The importance of D8/CS-E adhesion for subsequent VACV infection remains unclear. It is conceivable that the CS-E ligand may be restricted to small pools of target cells in certain organs [13,14]. In vivo infection models have not been discriminative of cells based on their surface sugar profiles. In addition, it is possible that there are species differences in glycosylation between mouse and man that alter poxvirus pathogenesis. Alternatively, the sugar binding properties of D8, H3, and A27 may have evolved to be redundant or combinatorial with each other. The highly sulfated CS-E type has been shown to bind to heparin binding growth factors midkine (MK), pleiotropin (PTN), heparin-binding epider- mal growth factor-like growth factor (HB-EGF), FGF-16, and FGF-18. As many of these growth factors are expressed in the mammalian brain, it was proposed that CS-E and CS proteogly- cans (CSPGs) are critical to the development of the brain and central nervous system [15]. Natural Antibody Responses Interfere with D8/CS-E Adhesion Natural Antibody Responses Interfere with D8/CS-E Adhesion Discussion CS-E blocking abilities of all anti-D8 MAbs is hierarchized as follows: LA5.EE11$ JF11,JE10.BG9.JA11.BH7.CC7.JE11. Total BSAs of the different groups’ epitopes can be ranked as follows: IV (LA5: 2434 A˚ 2) [8] .III (EE11: 1710 A˚ 2).I (JE11: 1238 A˚ 2).II (CC7: 851 A˚ 2). Consequently, we predict epitopes of group III JF11 and JE10 to target a region similar to EE11 on D8’s surface that most likely includes all four possible CS-E binding residues within this epitope, since all three MAbs have maximal CS-E blocking abilities within group III. JA11 is at the other end of the CS-E blocking range, and therefore its epitope might be close to the minimal definition for group III. Lastly, BG9 has an intermediate blocking ability of 50%, suggesting it targets an intermediate-sized epitope that includes K108 and no more than two other positively charged residues, such as R44, K48, and/or K98. D8 cloning Subsequent work using an antibody specific for the CS-E disaccharide revealed its presence in the developing mouse brain. The associated strong expression of the gene for GalNAc4S-6ST transferase confirmed that CS-E chains are critical in brain development, with the implication that CS-E chains participate in neurogenesis, axon guidance, and/or neuronal survival [16]. For mapping of the CS-E binding site, D8 D262 R220A, R44/ R220, and K48/R220 were expressed and purified as reported previously [9] with addition of a final size exclusion chromatog- raphy (SEC) step to isolate monomeric fractions. Natural Antibody Responses Interfere with D8/CS-E Adhesion SEC profiles of recombinant D8 D235, D262, and D263 suggest that D8 oligomerises through the C-terminal domain. SEC markers as grey curve with MW given in kDa. C. Putative D8 hexameric model based on EM data and SEC-MALS using biochemical constraints relative to the dimer and oligomer interfaces. The black circle highlights the putative seventh and central drupelet, arising from all six SU C-terminal extremities, converging toward the IMV envelope. D. CS-E microarray indicating that D8 hexamer (0.33 mM) binds more effectively to CS-E compared to 6-times molar excess of D8 monomer (2 mM) E. GAG microarray obtained with D8 oligomer (0.1 mM). D8 oligomer binds CS-E with higher affinity than the monomer, and also weakly binds to other CS species but not to DS, HA, heparin and HS. Micro array binding experiment was performed in triplicate, and the data represent the average of 10 spots per concentration averaged from the three experiments (6SEM, error bars). doi:10.1371/journal.ppat.1004495.g007 December 2014 | Volume 10 | Issue 12 | e1004495 PLOS Pathogens | www.plospathogens.org 8 PLOS Pathogens | www.plospathogens.org Protein expression and purification BL21-CodonPlus(DE3)-RIL competent cells (Agilent) were transformed with one of the D8 expression vectors and grown in LB media with 1 mM Ampicillin at 37uC until OD600 ,0.6. Protein expression was then induced with 1 mM IPTG for 4 hrs at 37uC, while shaking at 230 rpm. Cells were pelleted and resuspended in lysis buffer containing 100 mM Tris pH 8.0, 300 mM NaCl, 0.5 mM EDTA, 20 mM Imidazole, 0.2 mM PMSF and lysed under 20000 psi pressure using a microfluidizer (Microfluidics). Cell lysate was clarified at 50,000 g for 20 min. Supernatant was loaded onto 5 mL Ni-NTA column (His-Trap, GE). Bound D8 protein was eluted with 20 mM Tris pH 8.0, 300 mM NaCl, 200 mM Imidazole. After overnight dialysis against 20 mM Tris pH 8.0, 200 mM NaCl, the sample was PLOS Pathogens | www.plospathogens.org December 2014 | Volume 10 | Issue 12 | e1004495 9 Natural Antibody Responses Interfere with D8/CS-E Adhesion concentrated and subjected to SEC using a Superdex 200 10/ 300GL column (GE) in the same buffer. The monomeric peak with VE>16.5 mL and oligomeric peak with VE>11.7 mL were collected in separate fractions. with 2% w/w activated papain in 50 mM NaOAc pH 5.5 reaction buffer at 37uC over 4 hrs; CC7.1 (IgG1) was digested with 2% w/w activated papain in 100 mM Tris pH 7.0 reaction buffer with 10 mM cysteine at 37uC over 2 hrs. Papain digestion was terminated by addition of 20 mM iodoacetamide. EE11, JE11, and LA5-Fab containing samples were then dialyzed overnight in 5 L PBS pH 8.0. Samples were then passed through 1 mL FF Protein A column (GE) in PBS pH 8.0 binding buffer and Fab was collected in the flow-through. Fab was further concentrated for subsequent purification by SEC using a Superdex 200 10/300GL column (GE). Purified monomeric Fab peaks (VE>16 mL) were collected for complex preparation. The CC7.1 Fab containing sample was buffer exchanged against 3 M NaCl, 1.5 M Glycine pH 8.9 for Protein A affinity purification. Flow-through was dialyzed overnight against 5 L 20 mM Tris pH 8.0, 200 mM NaCl and subjected to SEC using a Superdex 200 16/60HR column (GE). Fractions containing monomeric Fab were collected for complex preparation (VE>86.4 mL). Complex preparation Monomeric D8 protein was used to prepare the D8 ternary and quaternary complexes with two different specificity groups Fab molecules (D8/JE11/LA5, D8/JE11/LA5/CC7.1, and D8/ JE11/EE11). The D8/LA5 crystal structure was used to position LA5-Fab in the EM map, while subsequently JE11 was used as a position marker for single particle EM reconstruction, since it does not cross-block binding of any of the other specificity group MAbs. The D8 D263 oligomeric protein was used to assess the physiological D8 oligomerization state with or without Fab decoration. D8/JE11/LA5. D8D262 monomer+JE11-Fab complex was prepared by mixing 20% molar excess of D8D262 monomer to purified JE11-Fab at low concentration (,0.2 mg/mL). Sample was then incubated on ice for 5 minutes, concentrated, and loaded onto a Superdex 200 10/300 SEC (GE). The D8D262 monomer+ JE11-Fab complex (,82 kDa) was pooled and mixed with LA5- Fab at equimolar ratio. The ternary complex sample was concentrated and purified by SEC (Superdex 200 10/300) as a ,152 kDa protein complex. D8/JE11/LA5. D8D262 monomer+JE11-Fab complex was prepared by mixing 20% molar excess of D8D262 monomer to purified JE11-Fab at low concentration (,0.2 mg/mL). Sample was then incubated on ice for 5 minutes, concentrated, and loaded onto a Superdex 200 10/300 SEC (GE). The D8D262 monomer+ JE11-Fab complex (,82 kDa) was pooled and mixed with LA5- Fab at equimolar ratio. The ternary complex sample was concentrated and purified by SEC (Superdex 200 10/300) as a ,152 kDa protein complex. D8/JE11/LA5/CC7.1. This complex was prepared as de- scribed above for D8D262 monomer+LA5-Fab+JE11-Fab, with the final addition of CC7.1-Fab at equimolar amounts for a final SEC step, where fractions corresponding to D8D262 monomer+ LA5-Fab+JE11-Fab+CC7.1-Fab quaternary complex were ob- tained upon SEC as a ,200 kDa complex. Only few class- averages pertained to the quaternary complex, and, therefore, this sample lead only to 3D-reconstruction of the ternary complex D8/ JE11/CC7.1. D8/CS-E docking g CS-E ligand was built by fusing two CS-A hexasaccharides extracted from CS/cathepsin K complex structure [pdb code 3C9E] [21]. Additional sulfate groups were added to the ligand at position 69 of N-acetyl-beta-D-galactosamine-4-sulfate, and ligand geometry was regularized and energy-minimized with PRODRG [22]. Docking was performed with Autodock Vina using a D8 pdbqt-formatted structure file [23] along with chondroitin 4,6- sulfate (CS-E) dodecasaccharide as ligand. In the original D8 coordinate file [pdb code 4E9O] [8], K48 Nj interacts with water 334, and water 326 of adjacent symmetry mate, and is pointing outward, suggesting it is not constrained in solution. In order to allow optimal docking of CS-E, side chain of residue K48 was moved away from K98 side chain (Coot, rotamer 5, 4% likelihood, Chi1 = 2177u). Grid box dimensions (x, y, z = 120, 48, 54) and center (x, y, z = 18.169, 20.779, 210.045) were defined based on our early routines [8] in order to accommodate the new, larger ligand. D8/JE11/EE11. The first step for preparing this complex is similar to the ones mentioned above: JE11-Fab was added to 20% molar excess of D8D235 monomer initially, followed by the addition of a 20% molar excess of EE11-Fab to the purified secondary complex pool for final SEC (Superdex 200 10/300). All complexes were freshly SEC-purified prior to EM analysis, and existence of the complex was validated by observation of a proper shift of elution volume upon complex formation, and SDS-PAGE analysis (Fig. S8). D8/JE11/EE11. The first step for preparing this complex is similar to the ones mentioned above: JE11-Fab was added to 20% molar excess of D8D235 monomer initially, followed by the addition of a 20% molar excess of EE11-Fab to the purified secondary complex pool for final SEC (Superdex 200 10/300). All complexes were freshly SEC-purified prior to EM analysis, and existence of the complex was validated by observation of a proper shift of elution volume upon complex formation, and SDS-PAGE analysis (Fig. S8). Glycosaminoglycan microarray assay y g y y y Microarrays containing natural GAGs enriched in CS-A, CS-C, CS-D, and CS-E (Seikagaku Corp., Tokyo, Japan), dermatan sulfate (DS; Sigma-Aldrich, St. Louis, MO), hyaluronic acid (HA; Sigma-Aldrich, St. Louis, MO), heparin (Hep; Neoparin, Alame- da, CA), heparan sulfate (HS; Sigma-Aldrich, St. Louis, MO), or chondroitin sulfate (CS; Sigma-Aldrich, St. Louis, MO) were printed on poly-DL-lysine-coated glass surfaces as described previously [19,20]. Arrays were blocked with 10% FBS in 16 PBS with gentle rocking at room temperature for 1 h, followed by a brief rinse with 16PBS. For binding and mapping experiments, monomeric D8, monomeric D8 mutants R220A, R44A/R220A, K48A/R220A, and oligomeric D8 were diluted to 2 mM (monomeric) and 0.33 mM (oligomeric) in 16 PBS containing 1% BSA; 100 ml was spotted on the microarrays and incubated at room temperature for 3 h. For antibody blocking experiments, 0.1 mM oligomeric D8 was incubated with 1 mM MAb (group I: JE11; II: CC7; III: BH7; BG9; EE11; JA11; JE10; IV: LA5) or alone for 1 h at room temperature; 100 ml was spotted on the microarrays and incubated at room temperature for 3 h. Microarrays were rinsed briefly three times with 16 PBS and incubated with 1:200 rabbit anti-6-His (Bethyl Laboratories, Montgomery, TX) for 1 h with gentle rocking, rinsed briefly three times with 16 PBS, followed by 1:5,000 Cy3-conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch, West Grove, PA) for 1 h in the dark with gentle rocking. The microarrays were then washed (3 times of 16 PBS and 2 times of de-ionized water), dried under a stream of air, and scanned at 532 nm using a GenePix 5000a scanner. Fluorescence quantification was performed using GenePix 6.0 software (Molecular Devices, Sunnyvale, CA). CS-E cross-blocking abilities of the four specificity group MAb representatives, and of group III MAbs were calculated for a CS-E concentra- tion of 5 mM. Data processing and image reconstruction Particles were picked automatically using DoG Picker and put into a particle stack using the Appion software package [25,26]. Initial, reference-free, two-dimensional (2D) class averages were calculated using particles binned by two via the Xmipp Clustering 2D Alignment [27] and sorted into classes. Particles corresponding to complexes were selected into a substack and binned by two before another round of reference-free alignment was carried out using the Xmipp Clustering and 2D alignment and IMAGIC software systems [28]. To analyze the interactions of the Fabs (LA5, JE11 and CC7) with D8 monomer, the reference free 2D class averages were examined. An ab initio common lines model was calculated from reference-free 2D class averages in EMAN2 [29]. Fab densities were visible after 10 iterations. This model was then refined against raw particles for an additional 89 cycles. EMAN [30] was used for all 3D reconstructions. The resolutions of the final models were determined using a FSC cut-off of 0.5. For the 3D reconstruction of D8 monomer with JE11 plus LA5, a number of 5291 particles were used. For the 3D volume of D8 monomer in complex with JE11 and CC7, a number of 8044 particles were used. Fab modeling Full Fab sequences were reconstructed from our in-house Fv sequenced data. V-, D-, and J-alleles were identified using V-quest of the international immunogenetics system [31]. For example, V- and J-genes IGKV4-57*01 and IGKJ5*01 for k chain and V-, D-, and J-genes IGHV14-3*02, IGHJ3*01, and IGHD2-3*01 for c2a chain were assigned for JE11. JE11, CC7.1, and EE11 Fv were modeled using Web Antibody Modeling (WAM) based on the AbM package [32]. Isotype-specific constant region sequences were determined using IMGT database and respective atomic coordinates were appended to the Fv domain after segment fitting. Model fitting into the EM densities First, a guided docking approach was used to obtain putative models of D8/JE11-Fv, D8/CC7-Fv and D8/EE11-Fv: experi- mental epitope definitions were used as input for Z-DOCK docking ([33]; DXMS for JE11, and alanine scan for CC7 and EE11) while assigning CDR residues as potential contacts in the Ag/MAb interface (Kabat definition) while blocking D8 residues that are not part of the epitope. The first ternary complex to be reconstructed was D8/JE11/LA5; D8/JE11/LA5 EM density was segmented into 5 regions corresponding to every domain (C1, Fv1, D8, Fv2, C2), using the UCSF Chimera ‘Segment map’ function [34]. Coordinates of D8/LA5-Fv were extracted from D8/LA5- Fab structure [pdb code 4ETQ] and fitted into the D8/JE11/LA5 EM density. Best-score fit was selected, after testing possible orientations. Putative ternary complexes were obtained by superimposing D8/JE11-Fv docking models onto the EM Natural Antibody Responses Interfere with D8/CS-E Adhesion Natural Antibody Responses Interfere with D8/CS-E Adhesion EM. A 3 mL aliquot containing ,0.05 mg/mL of complex was applied for 15 s onto a carbon-coated 400 Cu mesh grid that had been glow discharged at 20 mA for 30 s, then negatively stained with uranyl formate for 30 s. Data were collected using a FEI Tecnai F20 or T12 electron microscope operating at 120 keV, with an electron dose of ,27 e2/A˚ 2 and a magnification of 52,0006 that resulted in a pixel size of 2.05 A˚ at the specimen plane. Images were acquired with a Gatan US4000 CCD or Tietz TemCam-F416 CMOS camera using a nominal defocus of 1000 nm and the Leginon package [24] at 10u tilt increments and up to 50u. The tilts provided additional particle orientations to improve the image reconstructions. map-fitted D8/LA5-Fv complex, using Coot least square fit (LSQ) algorithm and setting D8 as the reference molecule [35]. The resulting ternary complex with the highest map fitting score was selected as the final solution, and conserved domains of LA5 and JE11 were fitted at last in the two remaining segments of the map, located the farthest away from the D8 molecule. In the two consecutive ternary complexes (JE11/D8/CC7 and JE11/D8/ EE11), a similar routine was applied by fitting the D8/JE11-Fab model deduced formerly first. For the last model (JE11/D8/ EE11), D8 residues outside of the known epitope definition were not blocked. The novel definition of group III epitope was defined by submitting JE11/D8/EE11 model to the Contact Map Analysis server [36]. Buried surface areas (BSA) were calculated using PISA [37]. Size-Exclusion Chromatography Coupled with Multi- angle Light Scattering (SEC-MALS) The D8 D262 wt monomer and the D8 D262 oligomer were loaded onto either a Superose 6 10/30 or Superdex S200 10/30 SEC column (GE Healthcare), which were coupled to an AKTA Avant FPLC system (GE Healthcare) with the following calibrated detection systems: (1) HP1 1050 Hewlett-Packard UV detector; (2) MiniDawn Treos multiangle light scattering (MALS) detector (Wyatt); (3) quasielastic light scattering (QELS) detector (Wyatt); (4) Optilab T-reX refractive index (RI) detector (Wyatt). Analysis of the light scattering data coupled to UV280 and refractive index protein concentration measurements allowed determination of the molar mass of the eluting proteins by using the protein conjugate template in Astra 6 software. Point mutation kinetics analysis by BioLayer Interferometry (BLI) BLI affinity measurements were determined using the Octet Red 96 (Forte´Bio Inc., Menlo Park, CA). Anti-mouse Fc Capture (AMC) biosensors were pre-soaked in 16 kinetics buffer contain- ing PBS pH 7.4, 0.01% BSA, 0.002% Tween 20 for 1 hour. Antibodies were immobilized by dipping the AMC biosensors in the antibody solution containing 16 kinetics buffer to a concentration of 3 ug/mL for 300 s. For the baseline step, the tips were soaked in 16 kinetics buffer for 300 s. Association was measured by dipping the biosensors in monomeric D8D262 diluted in 16 kinetics buffer to concentrations of 20 nM, 10 nM, 5 nM, 2.5 nM, 1.25 nM, 625 pM, 312.5 pM, and 156.2 pM for 900 s. Dissociation was measured in 16 kinetics buffer for 1200s. All steps are processed at 30uC/1000 rpm. Identical assays were performed for wild-type D8D262, and D8D262 R220A, D8D262 E30A, D8D262 R44A/R220A, and D8D262 K48A/R220A mutants against antibodies BH7, EE11 and JE11. A negative control antibody A2C7 that targets the VACV antigen A33 was run in parallel to all assays for subtraction of background binding signal. Data was analyzed with the ForteBio Data Analysis Software 7.1 (Forte´Bio Inc., Menlo Park, CA). Y-axis alignment to baseline step and interstep correction to dissociation step were selected to align curves. Aligned and subtracted curves were processed using Savitzky-Golay Filtering. Curve fittings were derived from application of the 1:1 binding model with full global fitting. Fab preparation Various complexes were prepared with either monomeric D8, monomeric D8 together with the Fabs from either JE11 and CC7.1, or JE11 and EE11, or JE11 and LA5, or oligomeric D8 either unliganded, or in complex with LA5-Fab, or with both JE11- and LA5-Fabs. Samples were analyzed by negative stain IgG’s were subjected to papain digestion to produce Fabs. Conditions for digestion were as follows: EE11-IgG2a was digested with 2% w/w activated papain in 50 mM NaOAc pH 5.5 reaction buffer at 37uC over 3 hrs; JE11 and LA5-IgG2a were digested December 2014 | Volume 10 | Issue 12 | e1004495 PLOS Pathogens | www.plospathogens.org 10 PLOS Pathogens | www.plospathogens.org Supporting Information Figure S1 Phylogenic analysis of light and heavy Fv sequences of murine anti-D8 MAbs. Multiple alignment files of anti-D8 HC and LC CDR sequences were obtained with ClustalW2 [38] and used in Seaview 4.2 to generate the December 2014 | Volume 10 | Issue 12 | e1004495 11 PLOS Pathogens | www.plospathogens.org Natural Antibody Responses Interfere with D8/CS-E Adhesion dendrograms [39]. Specificity groups are overlapped onto the dendrograms, using the same color-code as for our previous study [8]. Alignment highlights hypervariable regions H1, H2, H3 and L1, L2, L3 and residues are color coded (orange: GPST; red: HKR; blue: FWY, green: ILMV). Phylogenic analysis of HC-Fv sequences closely correlates with the specificity grouping obtained by cross-blocking ELISA [9]. However, group III LC CDR sequences overlap with those of group I, II and IV MAbs. (TIF) Figure S6 Negative stain of D8 monomer in complex with Fabs CC7 (group II) and JE11 (group I). A. Projection matching. B. Fourier Shell Correlation graph. (EPS) dendrograms [39]. Specificity groups are overlapped onto the dendrograms, using the same color-code as for our previous study [8]. Alignment highlights hypervariable regions H1, H2, H3 and L1, L2, L3 and residues are color coded (orange: GPST; red: HKR; blue: FWY, green: ILMV). Phylogenic analysis of HC-Fv sequences closely correlates with the specificity grouping obtained by cross-blocking ELISA [9]. However, group III LC CDR sequences overlap with those of group I, II and IV MAbs. (TIF) Figure S7 Negative stain of D8 monomer in complex with Fabs EE11 (group III) and JE11 (group I). A. Projection matching. B. Fourier Shell Correlation graph. (EPS) Figure S8 Preparation of monomeric and oligomeric D8/Fab complexes. A, B, and C. D8-monomer complexes. Associated class averages can be seen in figures S5, S6, and S7 D. D8-hexamer complexes. Associated class averages of unliganded and Fab-bound D8 hexamers can be seen in figure S4. All complexes are prepared by performing recursive SEC runs, starting by purifying the D8 monomer or D8 hexamer, and performing subsequent SEC runs for each additional Fab added to the complex being prepared. Curves are colored according to their order in the sequential process (SEC #1: orange, SEC#2: green, SEC #3: red, SEC#4: cyan). MWapp markers in kDa are shown for reference (grey curve). Based on the cross-blocking data, we also built the quaternary complex D8/JE11/CC4.1/LA5 (panel a, SEC#4). Acknowledgments We thank Enrico Girardi for help with Autodock Vina and Jonathan Torres (TSRI) for technical help with SEC-MALS experiments. We thank Enrico Girardi for help with Autodock Vina and Jonathan Torres (TSRI) for technical help with SEC-MALS experiments. Supporting Information Existence of this complex is evidenced by the class average in figure S9. (TIF) Figure S2 Point mutation kinetics analysis by BioLayer Interferometry. Real-time binding curves of BH7-, EE11- (both group III), and JE11- (positive control) MAbs to wild-type D8D262 and D8D262 R220A (positive controls) and indicated mutants to assess the validity of our complementary group III epitope definition. Association (900 s) and Dissociation (1200 s) steps are represented. Curves are colored according to their specific antigen concentration (80, 40, 20, 10, 5, 2.5, 1.25 nM and 625, 312.5, and 156.2 pM). Association rate (kon), dissociation rate (koff), affinity (KD) constant, and fit quality scores are deduced from each set of curves and reported in the bottom table. BLI experiment was performed once (TIF) Figure S3 SEC-MALS of D8 D262. Elution volume and molar mass (MM) for D8 D262 oligomer (A) and monomer (B) obtained using Superose 6. Elution volume and molar mass (MM) for D8 D262 oligomer (C) and monomer (D) obtained using Superdex S200. The horizontal dark line under each peak corresponds to the MM of the eluting sample as determined by SEC-UV/MALS. E. Reported SEC-UV/MALS MMs. (TIF) Figure S9 Quaternary complex D8/JE11/CC7.1/LA5. A. Superimposed maps of D8/JE11/CC7.1 and D8/JE11/EE11 ternary complexes, showing a ,90u rotation in the way group II and III Fab molecules anchor onto the D8 antigen. B. Reconstruction of D8/JE11/CC7.1/LA5 quaternary complex, obtained by overlapping ternary complexes of figures 3 and 4. (EPS) Figure S4 Negative stain EM data of unliganded and Fab-bound D8 hexamer. 2D class averages of (A) unliganded D8 hexamer, (B) D8 hexamer bound to JE11-Fab, and (C) D8 hexamer bound to JE11- and LA5-Fabs. (EPS) Author Contributions Figure S5 Negative stain of D8 monomer in complex with Fabs LA5 (group IV) and JE11 (group I). A. Projection matching. B. Fourier Shell Correlation graph. (EPS) Conceived and designed the experiments: MHM NdV GMM JB AS LCHW ABW DMZ. Performed the experiments: MHM NdV GMM JB AS. Analyzed the data: MHM NdV GMM JB AS SC BP LCHW ABW DMZ. Contributed reagents/materials/analysis tools: XM SC BP YX. Wrote the paper: MHM NdV GMM AS SC BP LCHW ABW DMZ. References protein D8 and its recognition by the antibody LA5. Journal of virology 86: 8050–8058. 1. Moss B (2011) Smallpox vaccines: targets of protective immunity. Immuno- logical reviews 239: 8–26. 9. Sela-Culang I, Benhnia MR, Matho MH, Kaever T, Maybeno M, et al. (2014) Using a Combined Computational-Experimental Approach to Predict Anti- body-Specific B Cell Epitopes. Structure 22. 646–657. 2. McCausland MM, Benhnia MR, Crickard L, Laudenslager J, Granger SW, et al. 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Journal of structural biology 166: 95–102. December 2014 | Volume 10 | Issue 12 | e1004495 PLOS Pathogens | www.plospathogens.org PLOS Pathogens | www.plospathogens.org 13
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The role of prenatal food insecurity on breastfeeding behaviors: findings from the United States pregnancy risk assessment monitoring system
International breastfeeding journal - Electronic Edition -
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RESEARCH Open Access The role of prenatal food insecurity on breastfeeding behaviors: findings from the United States pregnancy risk assessment monitoring system Lauren M. Dinour1*, Elizabeth I. Rivera Rodas1, Ndidiamaka N. Amutah-Onukagha2 and Laurén A. Doamekpor3 uren M. Dinour1*, Elizabeth I. Rivera Rodas1, Ndidiamaka N. Amutah-Onukagha2 and Laurén A. D © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. (2020) 15:30 (2020) 15:30 Dinour et al. International Breastfeeding Journal (20 https://doi.org/10.1186/s13006-020-00276-x Abstract Background: In addition to its health and nutritional benefits, breastfeeding can save low-income, food insecure mothers the cost of infant formula so that money can be spent on food and other necessities. Yet breastfeeding may exacerbate food insecurity by negatively affecting maternal employment. The relationship between food insecurity and breastfeeding has been explored previously, with varying results. The purpose of this study was to determine the relationship between prenatal food insecurity and breastfeeding initiation and early cessation (< 10 weeks) among U.S. mothers. Methods: Data were pooled from 2012 to 2013 (Phase 7) of the Pregnancy Risk Assessment Monitoring System, a population-based cross-sectional survey of postpartum women administered 2–4 months after delivery. The analytic sample was drawn from Colorado, Maine, New Mexico, Oregon, Pennsylvania, and Vermont, and limited to mothers aged 20 years and older whose infants were alive and living with them at the time of the survey (n = 10,159). We used binomial and multinomial logistic models to assess the predictive association between food insecurity and breastfeeding initiation and early cessation, respectively, while controlling for confounders. Results: Most women reported prenatal food security (90.5%) and breastfeeding initiation (91.0%). Of those who initiated breastfeeding, 72.7% breastfed for > 10 weeks. A larger proportion of food secure women compared to food insecure women, initiated breastfeeding (91.4% vs. 87.6%, P < 0.01), and patterns of early breastfeeding cessation differed significantly between the two groups (P < 0.01). In the final models, prenatal food insecurity was not associated with breastfeeding initiation or early cessation, with one exception. Compared to food secure mothers, mothers reporting food insecurity had a lower risk of breastfeeding for 4–6 weeks than for > 10 weeks, independent of covariates (relative risk ratio 0.65; 95% CI 0.50, 0.85; P < 0.01). Women who were married, had a college degree, and did not smoke were more likely to initiate breastfeeding and breastfeed for a longer time, regardless of food security status (P < 0.01). (Continued on next page) (Continued on next page) * Correspondence: dinourl@montclair.edu 1College of Education and Human Services, Montclair State University, 1 Normal Avenue, Montclair, NJ 07043, USA Full list of author information is available at the end of the article Background recall of exclusive breastfeeding or any breastfeeding at several time points after birth. Yet, greater household food insecurity is associated with a reduced volume of breast milk intake among infants [18], and mothers reporting moderate to severe household hunger are more likely than mothers experiencing little to no household hunger to cease exclusive breastfeeding be- tween 4 and 6 months [19]. Additionally, food insecure mothers are significantly more likely than food secure mothers to believe exclusive breastfeeding for 6 months would be an insufficient mode of infant feeding, and that they would be unable to exclusively breastfeed for 6 months if recommended to do so by a healthcare worker [20]. Qualitative findings from Haiti paint a more nu- anced relationship, whereby food insecurity among some mothers led to breastfeeding cessation due to perceived breast milk insufficiency and maternal weakness from undernutrition [21]. Conversely, some Haitian mothers reported breastfeeding continuation as a last resort in the absence of other foods or liquids [21]. Breast milk is superior to infant formula, protecting in- fants from a host of infectious and chronic conditions [1– 4]. Consequently, most national and international public health authorities recommend that infants be breastfed ex- clusively for the first 6 months of life, with continued breastfeeding alongside complementary foods through at least the first year or more [2, 4–6]. Even any breastfeed- ing confers health benefits compared to never breastfeed- ing [1, 3]. In the U.S., breastfeeding rates have improved over time [7] due to multi-pronged efforts aimed at educa- tion and policy changes. Yet many mothers, particularly low-income women, are not offered paid maternity leave and thus must return to work soon after birth [8, 9], mak- ing it difficult to establish a successful breastfeeding rela- tionship. Likewise, employers may not fully support women who wish to express breast milk at work, and women may terminate breastfeeding as a result [10–13]. y g Food insecurity, the lack of enough money to purchase adequate amounts, variety, and quality of food, can act as a potential double burden for low-income women. When food insecurity is severe, there may be prolonged periods of disrupted eating patterns and reduced food intake [14]. Although the prevalence of food insecurity in the U.S. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Dinour et al. International Breastfeeding Journal (2020) 15:30 Page 2 of 16 (Continued from previous page) Conclusions: Socioeconomic, psychosocial, and physiological factors explain the association between prenatal food insecurity and breastfeeding outcomes among this U.S. sample. More targeted and effective interventions and policies are needed to encourage the initiation and duration of breastfeeding, regardless of food security status. Keywords: Breastfeeding initiation, Breastfeeding cessation, Food insecurity, PRAMS Study variables h d d The independent variable was prenatal maternal food in- security measured retrospectively at the time of the sur- vey using the single standard question, “During the past 12 months before your new baby was born, did you ever eat less than you felt you should because there wasn’t enough money to buy food?” (yes/no). This standard question is one of the items included in the U.S. Depart- ment of Agriculture’s (USDA) abbreviated 6-item food security module [32] and has been used elsewhere [33, 34]. Responses were dichotomized to food insecure (yes) and food secure (no). Human participant protection The Montclair State University Institutional Review Board determined that no protocol approval was re- quired because the study used secondary, de-identified data. Background International Breastfeeding Journal (2020) 15:30 Dinour et al. International Breastfeeding Journal (2020) 15:30 The lack of consistent findings is largely due to the variety of geographies, confounding circumstances (e.g., HIV status), breastfeeding outcome variables, food se- curity measures, and research methods (e.g., qualitative and quantitative) used. Additionally, there is a need for representative studies from the U.S. that assess the rela- tionship between food security and breastfeeding behav- iors. The U.S. is unique in that it is the only developed country without a federally-mandated paid maternity leave [28], and it offers the Special Supplemental Nutri- tion Program for Women, Infants, and Children (WIC) [29] to provide low-income, at-risk participants with supplemental food, nutrition education, breastfeeding support, and infant formula. The purpose of the current study is to utilize the national Pregnancy Risk Assess- ment Monitoring System (PRAMS) dataset to determine if food insecurity is associated with breastfeeding initi- ation and early cessation (< 10 weeks) among U.S. mothers. PRAMS data is ideal for this research, as it sur- veys a U.S.-based representative sample of women dur- ing the first few months after birth and asks questions regarding both breastfeeding and household food security. random sample of resident women who delivered a live infant. PRAMS uses birth certificates as the sampling frame and identifies women who gave birth to a live in- fant within the previous 2–4 months. It then uses mailed questionnaires and telephone follow-ups to obtain infor- mation from a stratified representative sample of these women, with members of high-risk groups oversampled, and links questionnaire answers to birth certificate data. Additional details regarding the general methodology of PRAMS are available elsewhere [31]. For this study, the sample was limited to mothers aged 20 years and older whose infants were alive and living with them at the time of the survey (n = 11,830; Fig. 1). Background has declined in recent years (11.8% in 2017, down from 14.9% in 2011), risk remains substantially higher among households with incomes near or below the poverty line (30.8%) and among households with children headed by single women (30.3%) [14]. In theory, breastfeeding could save food insecure mothers the cost of formula so that money can be spent on food and other necessities. However, in countries like the U.S. without guaranteed paid maternity leave, adhering to breastfeeding recommendations may exacerbate food in- security by preventing or negatively affecting maternal employment and earnings [15–17]. It is therefore im- portant to understand the relationship between food in- security and breastfeeding practices to provide every woman, regardless of income, with the true choice to breastfeed. Results vary in more developed countries, as well. Among a sample of Brazilian children under 2 years old, an association between breastfeeding and food insecurity was found for children between 12 and 24 months (but not younger than 12 months), whereby breastfeeding prevalence was higher among children living in food in- secure households compared to food secure households [22]. In Canada, while household food insecurity is not related to breastfeeding initiation, the odds of exclusive breastfeeding at 4 months were significantly lower among women living in food insecure, compared to food secure, households [23]. Qualitative findings from Canada, however, suggest that food insecurity is a major contributor to breastfeeding initiation due to the worry over the cost of infant formula [24]. Additionally, food insecurity may be a root cause of breastfeeding cessation due to maternal fears of producing milk that is inad- equate in quantity or quality [24]. In considering the re- verse temporal pathway, Wong et al. found that breastfeeding duration does not predict household food insecurity [25]. Finally, among two samples of low- income, Hispanic or primarily Hispanic mother-infant pairs in New York City, neither partial nor exclusive breastfeeding was significantly associated with food inse- curity, whether experienced prenatally, postnatally, or both [26, 27]. The relationship between food insecurity and breast- feeding behaviors has been assessed in several different geo-cultural contexts and appears complex. Among some less developed countries, such as Kenya [18] and Uganda [19], where HIV rates are relatively high, house- hold food insecurity is not associated with maternal Page 3 of 16 Page 3 of 16 Dinour et al. International Breastfeeding Journal (2020) 15:30 Dinour et al. Data source and study population This study pools cross-sectional PRAMS data from 2012 to 2013 (Phase 7) for Colorado, Maine, New Mexico, Oregon, Pennsylvania, and Vermont (N = 13,284). These states were chosen because they were the only ones with data on food insecurity, which is an optional question that states can choose to ask of their PRAMS respon- dents. However, it should be noted that the majority of these states exhibited higher than national averages for both breastfeeding initiation [7] and food security [30] (Table 1). Two dependent variables were used: breastfeeding ini- tiation and early breastfeeding cessation. To assess breastfeeding initiation, PRAMS asks the following ques- tion: “Did you ever breastfeed or pump breast milk to feed your new baby, even for a short period of time?” (yes/no). Responses were dichotomized as mothers who did initiate breastfeeding (yes) and those who did not (no). To determine early cessation among respondents who initiated breastfeeding, the number of weeks of breastfeeding was measured using the following survey questions: “Are you currently breastfeeding or feeding pumped milk to your new baby?” (yes/no) and if no, “How many weeks or months did you breastfeed or pump milk to feed your baby?” For mothers who were still breastfeeding at the time of the survey, we deter- mined the age of the infant in days and removed respon- dents whose infants were less than 10 weeks (70 days) old (n = 7). We chose 10 weeks as the cut-point to maximize our sample size, since all but seven respon- dents who were still breastfeeding at the time of the sur- vey completed the survey when the infant was 10 weeks old or older. Later cut-points, such as 4 months, would PRAMS is a joint effort of the U.S. Center for Disease Control and Prevention (CDC) and state health depart- ments and is a public health survey that uses standard- ized collection techniques to gather information from a Table 1 Breastfeeding initiation and household food security prevalence nationally and among states included in this study State Breastfeeding Initiation Rate, 2013 (7) Household Food Security Prevalence, Average 2011–2013 (21) U.S. National 81.1% 85.4% Colorado 88.6% 86.1% Maine 86.6% 84.9% New Mexico 85.5% 86.8% Oregon 92.5% 84.8% Pennsylvania 73.3% 88.1% Vermont 84.5% 86.8% Table 1 Breastfeeding initiation and household food security prevalence nationally and among states included in this study Dinour et al. Statistical analysis All l All analyses were conducted with survey procedures in STATA MP version 16 and the standard errors adjusted for the complex sampling survey design. PRAMS’ weighted variables were used to account for the PRAMS survey design and the statistical weighting of the data. The analytic sample was limited to women with data for both food security status and breastfeeding outcomes (n = 10,159, Fig. 1). We assessed the sample by food se- curity status and conducted chi-square tests and t-tests to examine the distribution of each variable. To assess the best predictive association between prenatal food in- security and breastfeeding initiation, we used binomial logistic models and report the odds ratios (OR). Model 1 included prenatal food security status to assess the un- adjusted relationship between the independent and dependent variables. Model 2 added socioeconomic sta- tus variables: maternal age, income, marital status, ma- ternal education, maternal race/ethnicity, insurance type at the time of survey, breastfeeding information provided by a healthcare provider, and WIC status during preg- nancy. Model 3 added psychosocial variables: postpar- tum depression, number of stresses during pregnancy, and pregnancy intention. Model 4 included physiological variables: delivery method, Kotelchuck index, length of hospital stay, and current smoking status. Psychosocial variables included postpartum depression (yes/no), total number of stresses in the 12 months be- fore birth (a continuous variable between 0 and 14 based on a sum of positive responses to 14 possible stressful events that have happened during the 12 months before the new baby was born such as moving, divorce, loss of a job, or a death), and whether the mother wanted to be pregnant (later; sooner; then; never; unsure). Physio- logical variables included delivery method (vaginal deliv- ery; non-vaginal delivery), the Kotelchuck index for prenatal care (inadequate; intermediate; adequate; ad- equate plus), infant’s length of hospital stay (0–2 days; 3–5 days; > 6 days), and mother’s smoking status at the time of the survey (smoker; non-smoker). Multinomial logistic models were used to assess the best predictive association between prenatal food inse- curity and early breastfeeding cessation among respon- dents who initiated breastfeeding (n = 9239). From these models, we present the relative risk ratios (RRR) com- paring breastfeeding durations of < 1 week, 1–3 weeks, 4–6 weeks, and 7–9 weeks to the reference of breastfeed- ing for > 10 weeks. Data source and study population International Breastfeeding Journal (2020) 15:30 Page 4 of 16 Dinour et al. International Breastfeeding Journal Fig. 1 Flow diagram of sample selection for this study: PRAMS, United States, 2012–2013 initiate breastfeeding excluded from analysis. Exclusive breastfeeding could not be determined as PRAMS does not ask about the feeding of anything other than breastmilk. have removed over half of the sample. Additionally, much of the prior breastfeeding research based on PRAMS data utilizes the 10-week cut-point in an effort to give all respondents an equal opportunity for inclu- sion in the analysis [35–39]. The early breastfeeding ces- sation variable was classified into five categories (< 1 week, 1–3 weeks, 4–6 weeks, 7–9 weeks, and > 10 weeks), with those still breastfeeding at the time of the survey classified as > 10 weeks and those who did not Potentially confounding variables included in our mul- tivariable analysis were identified from previous studies of food insecurity and breastfeeding. Of particular note, Webb-Girard et al. [20] posited multiple mechanisms by which food insecurity may adversely affect exclusive Page 5 of 16 Dinour et al. International Breastfeeding Journal (2020) 15:30 Dinour et al. International Breastfeeding Journal (2020) 15:30 Page 5 of 16 breastfeeding. These include socio-economic factors, psychosocial factors, and physiological factors. For ex- ample, with regards to socio-economic factors, food in- security may lead women to seek employment outside of the home, which can negatively impact breastfeeding be- haviors. Psychosocially, the distress and depression caused by food insecurity may undermine women’s con- fidence and self-efficacy to breastfeed. The stress associ- ated with food insecurity may also physiologically impair milk output, and thus reduce the likelihood of exclusive breastfeeding [20]. For our study, we grouped potentially confounding variables using similar categories presented by Webb-Girard et al.’s [20] conceptual framework: sociodemographic, psychosocial, and physiological variables. Sociodemographic variables included maternal age in years (20–24; 25–29; 30–34; > 35), annual income (< $22,000; $22,001–$37,000; $37,001–$52,000; $52, 001–$67,000; > $67,001), self-reported marital status (married; not married), years of maternal education (< 12 years; 12 years; 13–15 years, > 16 years), race/ethnicity (Hispanic of any race; non-Hispanic White; non- Hispanic Asian/Pacific Islander; non-Hispanic Black; non-Hispanic American Indian/Alaska Native; non- Hispanic other or mixed race; unknown race and/or eth- nicity), educational information regarding breastfeeding provided by a healthcare worker (received breastfeeding information; did not receive breastfeeding information), and receipt of WIC during pregnancy (received WIC; did not receive WIC). Data source and study population Medical insurance at the time of the survey was categorized as Government (Medicaid; Medicare; Children’s Health Insurance Program (CHIP)/ State CHIP; or other governmental insurance), Private (insurance paid by an employer; purchased from a com- pany; or TRICARE/military insurance), Other (insurance from some other source), and None (no insurance or in- surance through the Indian Health Service). These insur- ance categories were based on prior research analyzing PRAMS data [40]. for maternal age, marital status, maternal education, ma- ternal race/ethnicity, delivery method, and Kotelchuck Index, which originated from the birth certificate. Add- itional potentially confounding variables were considered but not included in the final analysis, either because there were values for only a small percentage of the sam- ple (i.e., trying to get pregnant, or already pregnant, at the time of the survey), issues with multicollinearity (i.e., maternal pre-pregnancy body mass index), or non- significant relationships in binomial logistic regression models that predicted breastfeeding initiation or early cessation (i.e., plurality, birth order, gestational age, birthweight, birth defect, infant in intensive care at birth, and maternal medical risk factors). for maternal age, marital status, maternal education, ma- ternal race/ethnicity, delivery method, and Kotelchuck Index, which originated from the birth certificate. Add- itional potentially confounding variables were considered but not included in the final analysis, either because there were values for only a small percentage of the sam- ple (i.e., trying to get pregnant, or already pregnant, at the time of the survey), issues with multicollinearity (i.e., maternal pre-pregnancy body mass index), or non- significant relationships in binomial logistic regression models that predicted breastfeeding initiation or early cessation (i.e., plurality, birth order, gestational age, birthweight, birth defect, infant in intensive care at birth, and maternal medical risk factors). Results Compared to non-Hispanic White women, those iden- tifying as Hispanic, non-Hispanic Native American, non- Hispanic Asian, or non-Hispanic of a race not otherwise classified were more than twice as likely to initiate breastfeeding. Women with insurance other than private or government-sponsored were less likely to have ever breastfed compared to women with private insurance (OR 0.38; 95% CI 0.24, 0.60). Women who reported that a healthcare worker discussed breastfeeding before birth were over 1.5 times more likely to initiate breastfeeding than those who did not experience such conversations (OR 1.68; 95% CI 1.39, 2.03). Notably, prenatal WIC re- cipients were 27% less likely to have ever breastfed (OR 0.73; 95% CI 0.60, 0.88) compared to those who did not receive WIC. Table 2 describes the sample. A total of 10,159 women were included in the final analysis, most of whom were 25–29 years old (31.1%), non-Hispanic White (69.9%), married (66.8%), and had at least some college education (68.2%). Thirty-three percent of women had an annual income of $22,000 or below and 29.6% made more than $67,000 in the 12 months before giving birth. A little over half of the sample had private insurance (54.9%) and 39.9% of women were receiving WIC benefits during pregnancy. Nearly 80% of the sample received adequate or adequate plus prenatal care, 85.1% had a prebirth conversation with a healthcare worker about breastfeed- ing, 10.9% reported experiencing postpartum depression, and 15.2% smoked at the time of the survey. Differences were found between food security groups for most so- cioeconomic, psychosocial, and physiological characteris- tics (Table 2). With the exception of postpartum depression and pregnancy intention, all psychosocial and physiological variables under study were significantly associated with breastfeeding initiation in the final model. For example, with each additional stress a woman experienced in the 12 months prior to birth, the odds of breastfeeding initi- ation increased by 5% (OR 1.05; 95% CI 1.01, 1.09). The adequacy of prenatal care during pregnancy was also as- sociated with breastfeeding initiation. Women who re- ceived intermediate and inadequate prenatal care were 22 -23% less likely to start breastfeeding compared to women who received adequate plus care. Breastfeeding initiation In the unadjusted multivariate model, women who were food insecure in the 12 months prior to birth were less likely to have ever breastfed compared to women who were food secure (OR 0.67; 95% Confidence Interval [CI] 0.54, 0.82; Table 3). However, this effect disappeared when the potentially confounding variables were in- cluded. Among the sociodemographic variables, all but Results Further, a lon- ger than typical newborn hospital stays (6 + days) was as- sociated with an increased odd of breastfeeding initiation (OR 1.45; 95% CI 1.15, 1.82), whereas a non- vaginal delivery decreased the odds of breastfeeding ini- tiation (OR 0.79; 95% CI 0.67, 0.94). Finally, smokers were more than 50% less likely to initiate breastfeeding (OR 0.43; 95% CI 0.36, 0.52) compared to non-smokers. The majority of the sample reported they were food secure during the 12 months prior to birth (90.5%) and initiated breastfeeding (91.0%). Among those who initi- ated breastfeeding, 72.7% of the sample breastfed for > 10 weeks. A larger proportion of food secure women compared to food insecure women, initiated breastfeed- ing (91.4% vs. 87.6%, P < 0.01). Patterns of early breast- feeding cessation were also significantly different between the two groups (P < 0.01; Table 2). Notably, among food secure women, the highest percentage of early breastfeeding cessation occurred during 4–6 weeks (9.1%), whereas the largest percentage of food insecure women stopped breastfeeding between 1 and 3 weeks (12.2%), followed by 7–9 weeks (11.7%). Statistical analysis All l Four models were also conducted for the multinomial logistic models where Model 1 assessed the relationship between prenatal food insecurity and early breastfeeding cessation without controlling for any potential confounders. Model 2 added socioeconomic Potentially confounding variables were collected by self-report from mothers via the PRAMS survey, except Dinour et al. International Breastfeeding Journal (2020) 15:30 Page 6 of 16 Page 6 of 16 income were significantly associated with breastfeeding initiation in the final model (Model 4). Specifically, com- pared to older women (> 35 years), those between the ages of 20–24 were about 1.5 times more likely to initi- ate breastfeeding (OR 1.45; 95% CI 1.12, 1.88). Married women had a greater odds of initiating breastfeeding compared to their unmarried counterparts (OR 1.29; 95% CI 1.09, 1.54). Less educated women were less likely to initiate breastfeeding compared to those with 16+ years of education. Of note, women with some college (13–15 years of education) were nearly half as likely (OR 0.53; 95% CI 0.41, 0.67), and those with < 12 years about 70% less likely, to initiate breastfeeding compared to their more educated counterparts. status variables, Model 3 added psychosocial variables, and Model 4 added physiological variables. The traditional P < 0.05 criterion of statistical signifi- cance was employed for all tests within each model. However, in order to avoid inflated likelihood of error in the multinomial logistic models, we use an adjusted p - value to test for significance between models. To calcu- late this using Bonferroni’s method, we divided our ori- ginal p - value of 0.05 by the number of models (4), giving us a new threshold of significance (P < 0.0125). For simplicity, we use P < 0.01 which maintains our 95% confidence in our set of analyses as a whole. Early breastfeeding cessation Prenatal food insecurity was significantly related to early breastfeeding cessation in the unadjusted model (Table 4). Women who were food insecure in the 12 months prior to birth were more likely to breastfeed for < 1 week than Dinour et al. International Breastfeeding Journal (2020) 15:30 Page 7 of 16 Table 2 Maternal characteristics, overall and by prenatal food security status Characteristics Overall (N = 10, 159) Food Secure (n = 9, 190) Food Insecure (n = 969) T-test or χ2 P- Breastfeeding initiation, No. (%) 9246 (91.01) 8397 (91.37) 849 (87.62) 0.00 Early breastfeeding cessation, No. (%)a < 1 week 241 (2.61) 203 (2.42) 38 (4.48) 0.00 1–3 weeks 747 (8.09) 644 (7.68) 103 (12.13) 4–6 weeks 848 (9.18) 761 (9.07) 87 (10.25) 7–9 weeks 687 (7.44) 588 (7.01) 99 (11.66) ≥10 weeks 6716 (72.69) 6194 (73.83) 522 (61.48) Grouped maternal age, No. (%) 20–24 years old 2279 (22.43) 1884 (20.50) 395 (40.76) 0.00 25–29 years old 3158 (31.09) 2882 (31.36) 276 (28.48) 30–34 years old 3021 (29.74) 2830 (30.79) 191 (19.71) 35+ years old 1701 (16.74) 1594 (17.34) 107 (11.04) Income 12 months before, No. (%) $0–$22,000 3390 (33.37) 2736 (29.77) 654 (67.49) 0.00 $22,001-37,000 1685 (16.59) 1487 (16.18) 198 (20.43) $37,001-52,000 1178 (11.60) 1096 (11.93) 82 (8.46) $52,001-67,000 900 (8.86) 878 (9.55) 22 (2.27) $67,001+ 3006 (29.59) 2993 (32.57) 13 (1.34) Marital status, No. (%) Not married 3372 (33.19) 2815 (30.63) 557 (57.48) 0.00 Married 6787 (66.81) 6375 (69.37) 412 (42.52) Years of maternal education, No. (%) 0–11 years 971 (9.56) 823 (8.96) 148 (15.27) 0.00 12 years 2261 (22.26) 1929 (20.99) 332 (34.26) 13–15 years 3105 (30.56) 2724 (29.64) 381 (39.32) 16+ years 3822 (37.62) 3714 (40.41) 108 (11.15) Race/ethnicity, No. (%) Hispanic 1283 (12.63) 1151 (12.52) 132 (13.62) 0.33 NH White 7098 (69.87) 6483 (70.54) 615 (63.47) 0.00 NH Black 402 (3.96) 338 (3.68) 64 (6.60) 0.00 NH Asian 416 (4.09) 399 (4.34) 17 (1.75) 0.00 NH Native American 374 (3.68) 310 (3.37) 64 (6.60) 0.00 NH Other 422 (4.15) 358 (3.90) 64 (6.60) 0.00 Unknown race/ethnicity 164 (1.61) 151 (1.64) 13 (1.34) 0.48 Insurance type, No. (%) Government insurance 3088 (30.40) 2561 (27.87) 527 (54.39) 0.00 Private insurance 5578 (54.91) 5357 (58.29) 221 (22.81) 0.00 Other insurance 149 (1.47) 132 (1.44) 17 (1.75) 0.43 No insurance 1344 (13.23) 1140 (12.40) 204 (21.05) 0.00 Healthcare worker talked about breastfeeding before birth, No. Early breastfeeding cessation NH non-Hispanic, SD standard deviation, WIC Special Supplemental Nutrition Program for Women, Infants, and Children; aEarly breastfeeding cessation analysis excludes respondents who did not initiate breastfeeding (n = 913) and those who were still breastfeeding at the time of the survey but completed the survey before 10 weeks post birth (n = 7). Thus, the early breastfeeding cessation sample size is N = 9239 for overall, n = 8390 for food secure, and n = 849 for food insecure. Percentages within this category are calculated based on these sample sizes to breastfeed for > 10 weeks compared to women who were food secure (RRR 2.22; 95% CI 1.55, 3.18). In other words, the risk of breastfeeding for < 1 week compared to breastfeeding for > 10 weeks more than doubled when prenatal food insecurity was reported. Similarly, women who were food insecure were more likely to stop breast- feeding at 1–3 weeks and 7–9 weeks compared to breast- feeding for > 10 weeks (Table 4). Although women who were food insecure also showed a higher risk of breast- feeding cessation at 4–6 weeks compared to > 10 weeks, this finding was not significant at the P < 0.01 level (to account for familywise error). compared to women 35 years and older, though no other age ranges or cessation periods showed significance. Among married women, the risk of breastfeeding cessa- tion at 1–3 weeks and 7–9 weeks versus > 10 weeks was between 22 and 23% lower compared to unmarried women. Additionally, women with less years of education were more likely to discontinue breastfeeding earlier. In fact, women with < 16 years of education had more than a 3–5 fold greater risk of breastfeeding for < 1 week versus > 10 weeks compared to women with > 16 years of education. Racial/ethnic identity was not associated with early breastfeeding cessation, except among non-Hispanic women of a race not otherwise classified, who exhibited a 46% lower risk of breastfeeding cessation at 4–6 weeks versus > 10 weeks compared to non-Hispanic White women. No other sociodemographic variables (i.e., income, insurance type at the time of survey, breastfeeding information provided by healthcare provider, and WIC status during pregnancy) were significantly related to early breastfeeding cessation at the P < 0.01 level. Early breastfeeding cessation (%) 8641 (85.06) 7808 (84.96) 833 (85.96) 0.40 On WIC during pregnancy, No. (%) 4053 (39.90) 3369 (36.66) 684 (70.59) 0.00 Experienced postpartum depression, No. (%) 1111 (10.94) 856 (9.31) 255 (26.32) 0.00 Number of stresses in 12 months before birth, mean (SD) 1.99 (2.02) 1.74 (1.81) 4.32 (2.42) 0.00 Dinour et al. International Breastfeeding Journal (2020) 15:30 Page 8 of 16 Table 2 Maternal characteristics, overall and by prenatal food security status (Continued) Characteristics Overall (N = 10, 159) Food Secure (n = 9, 190) Food Insecure (n = 969) T-test or χ2 P-value Wanted to be pregnant, No. (%) Later 2061 (20.29) 1779 (19.36) 282 (29.10) 0.00 Sooner 1513 (14.89) 1416 (15.41) 97 (10.01) 0.00 Then 4665 (45.92) 4398 (47.86) 267 (27.55) 0.00 Never 591 (5.82) 479 (5.21) 112 (11.56) 0.00 Unsure 1329 (13.08) 1118 (12.17) 211 (21.78) 0.00 Vaginal delivery, No. (%) 6774 (66.68) 6119 (66.58) 655 (67.60) 0.52 Kotelchuck Index, No. (%) Inadequate 1028 (10.12) 857 (9.33) 171 (17.65) 0.00 Intermediate 1175 (11.57) 1067 (11.61) 108 (11.15) Adequate 4441 (43.71) 4070 (44.29) 371 (38.29) Adequate Plus 3515 (34.60) 3196 (34.78) 319 (32.92) Hospital baby stay length grouped, No. (%) 0–2 days 5967 (58.74) 5439 (59.18) 528 (54.49) 0.02 3–5 days 2698 (26.56) 2419 (26.32) 279 (28.79) 6+ days 1494 (14.71) 1332 (14.49) 162 (16.72) Smoking at time of survey, No. (%) 1526 (15.20) 1176 (12.80) 350 (36.12) 0.00 Note. NH non-Hispanic, SD standard deviation, WIC Special Supplemental Nutrition Program for Women, Infants, and Children; aEarly breastfeeding cessation analysis excludes respondents who did not initiate breastfeeding (n = 913) and those who were still breastfeeding at the time of the survey but completed the survey before 10 weeks post birth (n = 7). Thus, the early breastfeeding cessation sample size is N = 9239 for overall, n = 8390 for food secure, and n = 849 for food insecure. Percentages within this category are calculated based on these sample sizes Table 2 Maternal characteristics, overall and by prenatal food security status (Continued) Characteristics Overall (N = 10, 159) Food Secure (n = 9, 190) Food Insecure (n = 969) T-test or χ2 P-value Note. Early breastfeeding cessation When the sociodemographic potentially confounding variables were introduced in Model 2, the relationship between prenatal food insecurity and early breastfeeding cessation was no longer significant (Additional file 1). Likewise, when adding psychosocial variables into Model 3, no significant relationships between prenatal food in- security and early breastfeeding cessation were noted at the P < 0.01 level (Additional file 2). However, as Table 5 indicates, when the physiological variables are included, the risk of breastfeeding for 4–6 weeks compared to breastfeeding for > 10 weeks was about 35% less for food insecure women relative to food secure women (RRR 0.65; 95% CI 0.50, 0.85; P < 0.01), though no other early cessation time frames achieved significance. Psychosocially, women who experienced postpartum depression had a greater risk of breastfeeding cessation at 1–3 weeks (RRR 1.50; 95% CI 1.19, 1.89) than to breastfeed for > 10 weeks. Compared to those who wanted to be pregnant at the time of their pregnancy, women who never wanted to be pregnant had a 2-fold higher risk of breastfeeding for < 1 week than > 10 weeks Women aged 20–24 years old had an increased risk of breastfeeding cessation at 1–3 weeks versus > 10 weeks Dinour et al. Discussion However, this sample was drawn from the federally funded community-based projects of the Canada Prenatal Nutrition Programs (CPNP), which work to able 4 Multinomial logistic regression using “breastfeeding for > 10 weeks” as reference category for Model 1 Breastfeeding < 1 week, RRR (95% CI) Breastfeeding 1–3 weeks, RRR (95% CI) Breastfeeding 4–6 weeks, RRR (95% CI) Breastfeeding 7–9 weeks, RRR (95% CI) od secure (Ref.) 1.00 1.00 1.00 1.00 od insecure 2.22** (1.55, 3.18) 1.90** (1.51, 2.38) 1.36* (1.07, 1.72) 2.00** (1.59, 2.52) te. CI confidence interval, RRR relative risk ratio; *P < 0.05, **P < 0.01 Ninety percent of women in this study were food secure prior to the birth of their infants and/or initiated breast- feeding, and more than half continued breastfeeding for > 10 weeks regardless of their food security status. Among this sample, food insecurity was not associated with breastfeeding initiation after adjusting for socioeco- nomic, psychosocial, and physiological factors. This find- ing is congruent with results from a similar study by Orr et al. [23], who found that among 10,450 Canadian women, breastfeeding initiation was unrelated to house- hold food security once sociodemographic characteris- tics were taken into account. Conversely, in a qualitative study of 20 food insecure Nova Scotian mothers by Frank [24], 95% initiated breastfeeding due to the health benefits and high cost of formula. Frank states that “worry over the cost of formula was a driving factor in initiation, indicating that household food insecurity could be a predictor of breastfeeding initiation” [24]. However, this sample was drawn from the federally funded community-based projects of the Canada Prenatal Nutrition Programs (CPNP), which work to Among the physiological variables, women who did not deliver vaginally had significantly higher risk of breastfeeding cessation at 1–3 weeks (RRR 1.60; 95% CI 1.34, 1.92) and 4–6 weeks (RRR 1.27; 95% CI 1.07, 1.51) than to breastfeed for > 10 weeks. In addition, women whose infants remained in the hospital for > 6 days were 84 -112% more likely to stop breastfeeding at 4–6 weeks or 7–9 weeks than to breastfeed for > 10 weeks when compared to women whose infants stayed in the hospital for 0–2 days. Early breastfeeding cessation International Breastfeeding Journal (2020) 15:30 Page 10 of 16 Dinour et al. International Breastfeeding Journal (2020) 15:30 Page 10 of 16 Table 3 Results of binomial logistic regression predicting breastfeeding initiation (Continued) Model 1, OR (95% CI) Model 2, OR (95% CI) Model 3, OR (95% CI) Model 4, OR (95% CI) Later 1.12 (0.92, 1.37) 1.15 (0.94, 1.41) Sooner 1.04 (0.82, 1.31) 1.03 (0.81, 1.30) Never 0.75* (0.57, 0.98) 0.77 (0.58, 1.01) Unsure 0.90 (0.73, 1.11) 0.96 (0.78, 1.19) Vaginal delivery (Ref.) 1.00 No 0.79** (0.67, 0.94) Kotelchuck Index Inadequate 0.78* (0.61, 0.99) Intermediate 0.77* (0.61, 0.98) Adequate 1.04 (0.88, 1.24) Adequate Plus (Ref.) 1.00 Hospital baby stay length 0–2 days (Ref.) 1.00 3–5 days 1.08 (0.91, 1.30) 6+ days 1.45** (1.15, 1.82) Smoking at time of survey 0.43** (0.36, 0.52) No (Ref.) 1.00 Note. CI confidence interval, HCW healthcare worker, NH non-Hispanic, OR odds ratio, WIC Special Supplemental Nutrition Program for Women, Infants, and Children; *P < 0.05, **P < 0.01. The sample size is N = 10,159 Table 3 Results of binomial logistic regression predicting breastfeeding initiation (Continued) Model 1, OR (95% CI) Model 2, OR (95% CI) Model 3, OR (95% CI) Model 4, OR (95% CI) Later 1.12 (0.92, 1.37) 1.15 (0.94, 1.41) Sooner 1.04 (0.82, 1.31) 1.03 (0.81, 1.30) Never 0.75* (0.57, 0.98) 0.77 (0.58, 1.01) Unsure 0.90 (0.73, 1.11) 0.96 (0.78, 1.19) Vaginal delivery (Ref.) 1.00 No 0.79** (0.67, 0.94) Kotelchuck Index Inadequate 0.78* (0.61, 0.99) Intermediate 0.77* (0.61, 0.98) Adequate 1.04 (0.88, 1.24) Adequate Plus (Ref.) 1.00 Hospital baby stay length 0–2 days (Ref.) 1.00 3–5 days 1.08 (0.91, 1.30) 6+ days 1.45** (1.15, 1.82) Smoking at time of survey 0.43** (0.36, 0.52) No (Ref.) 1.00 Note. CI confidence interval, HCW healthcare worker, NH non-Hispanic, OR odds ratio, WIC Special Supplemental Nutrition Program for Women, Infants, and Children; *P < 0.05, **P < 0.01. The sample size is N = 10,159 Smoking at time of survey Note. CI confidence interval, HCW healthcare worker, NH non-Hispanic, OR odds ratio, WIC Special Supplemental Nutrition Program for Women, Infants, and Children; *P < 0.05, **P < 0.01. The sample size is N = 10,159 Table 4 Multinomial logistic regression using “breastfeeding for > 10 weeks” as reference category for Model 1 Breastfeeding < 1 week, RRR (95% CI) Breastfeeding 1–3 weeks, RRR (95% CI) Breastfeeding 4–6 weeks, RRR (95% CI) Breastfeeding 7–9 weeks, RRR (95% CI) Food secure (Ref.) 1.00 1.00 1.00 1.00 Food insecure 2.22** (1.55, 3.18) 1.90** (1.51, 2.38) 1.36* (1.07, 1.72) 2.00** (1.59, 2.52) Note. CI confidence interval, RRR relative risk ratio; *P < 0.05, **P < 0.01 Early breastfeeding cessation International Breastfeeding Journal (2020) 15:30 Page 9 of 16 Table 3 Results of binomial logistic regression predicting breastfeeding initiation Model 1, OR (95% CI) Model 2, OR (95% CI) Model 3, OR (95% CI) Model 4, OR (95% CI) Food insecurity Food secure (Ref.) 1.00 1.00 1.00 1.00 Food insecure 0.67** (0.54, 0.82) 1.09 (0.88, 1.36) 1.09 (0.87, 1.37) 1.17 (0.92, 1.48) Maternal age 20–24 years old 1.52** (1.18, 1.95) 1.43** (1.11, 1.85) 1.45** (1.12, 1.88) 25–29 years old 1.18 (0.94, 1.49) 1.14 (0.90, 1.44) 1.16 (0.91, 1.47) 30–34 years old 1.03 (0.81, 1.30) 1.00 (0.79, 1.27) 1.02 (0.80, 1.29) 35+ years old (Ref.) 1.00 1.00 1.00 Income $0–$22,000 0.76 (0.55, 1.04) 0.77 (0.56, 1.05) 0.82 (0.59, 1.12) $22,001-37,000 1.07 (0.79, 1.46) 1.08 (0.80, 1.47) 1.08 (0.79, 1.46) $37,001-52,000 1.10 (0.80, 1.50) 1.10 (0.80, 1.51) 1.11 (0.81, 1.52) $52,001-67,000 0.89 (0.64, 1.24) 0.91 (0.65, 1.26) 0.92 (0.66, 1.28) $67,001+ (Ref.) 1.00 1.00 1.00 Marital status Married 1.39** (1.17, 1.64) 1.38** (1.17, 1.64) 1.29** (1.09, 1.54) Not married (Ref.) 1.00 1.00 1.00 Years of maternal education 0–11 years 0.25** (0.18, 0.34) 0.26** (0.19, 0.35) 0.33** (0.24, 0.45) 12 years 0.26** (0.20, 0.33) 0.26** (0.20, 0.33) 0.30** (0.23, 0.39) 13–15 years 0.48** (0.38, 0.61) 0.49** (0.39, 0.62) 0.53** (0.41, 0.67) 16+ years (Ref.) 1.00 1.00 1.00 Race/ethnicity NH White (Ref.) 1.00 1.00 1.00 Hispanic 2.87** (2.21, 3.73) 2.82** (2.17, 3.67) 2.33** (1.79, 3.05) NH Black 1.36 (0.98, 1.89) 1.38 (0.99, 1.92) 1.22 (0.87, 1.71) NH Native American 2.98** (1.97, 4.53) 3.00** (1.97, 4.55) 2.56** (1.68, 3.92) NH Other 3.25** (1.97, 5.36) 3.20** (1.94, 5.27) 3.17** (1.92, 5.25) NH Asian 2.83** (1.60, 5.01) 2.85** (1.61, 5.03) 2.50** (1.41, 4.42) Unknown race/ethnicity 1.70 (0.92, 3.12) 1.74 (0.94, 3.21) 1.51 (0.82, 2.79) Insurance type Private insurance (Ref.) 1.00 1.00 1.00 Government insurance 0.75* (0.60, 0.93) 0.75* (0.60, 0.94) 0.81 (0.65, 1.01) Other insurance 0.36** (0.23, 0.58) 0.37** (0.24, 0.59) 0.38** (0.24, 0.60) No insurance 1.18 (0.90, 1.56) 1.18 (0.90, 1.55) 1.18 (0.89, 1.55) HCW talked about breastfeeding before birth 1.70** (1.41, 2.05) 1.69** (1.40, 2.03) 1.68** (1.39, 2.03) No (Ref.) 1.00 1.00 1.00 On WIC during pregnancy 0.73** (0.61, 0.88) 0.73** (0.61, 0.88) 0.73** (0.60, 0.88) No (Ref.) 1.00 1.00 Experienced postpartum depression 0.81 (0.66, 1.00) 0.82 (0.67, 1.01) No (Ref.) 1.00 1.00 Table 3 Results of binomial logistic regression predicting breastfeeding initiation Model 1, OR (95% CI) Model 2, OR (95% CI) Model 3, OR (95% CI) Model 4, OR (95% CI) Food insecurity Food secure (Ref.) Food insecure Maternal age 20–24 years old 25–29 years old 30–34 years old 35+ years old (Ref.) Income $0–$22,000 $22,001-37,000 $37,001-52,000 $52,001-67,000 $67,001+ (Ref.) Marital status Married Not married (Ref.) Years of maternal education 0–11 years 12 years 13–15 years 16+ years (Ref.) Race/ethnicity NH White (Ref.) Hispanic NH Black NH Native American NH Other NH Asian Unknown race/ethnicity Insurance type Private insurance (Ref.) Government insurance Other insurance No insurance HCW talked about breastfeeding before birth No (Ref.) On WIC during pregnancy No (Ref.) Experienced postpartum depression No (Ref.) Number of stresses in 12 months before birth Wanted to be pregnant Then (Ref.) 1.00 1.00 1.00 1.09 (0.88, 1.36) 1.09 (0.87, 1.37) 1.17 (0.92, 1.48) 1.52** (1.18, 1.95) 1.43** (1.11, 1.85) 1.45** (1.12, 1.88) 1.18 (0.94, 1.49) 1.14 (0.90, 1.44) 1.16 (0.91, 1.47) 1.03 (0.81, 1.30) 1.00 (0.79, 1.27) 1.02 (0.80, 1.29) 1.00 1.00 1.00 0.76 (0.55, 1.04) 0.77 (0.56, 1.05) 0.82 (0.59, 1.12) 1.07 (0.79, 1.46) 1.08 (0.80, 1.47) 1.08 (0.79, 1.46) 1.10 (0.80, 1.50) 1.10 (0.80, 1.51) 1.11 (0.81, 1.52) 0.89 (0.64, 1.24) 0.91 (0.65, 1.26) 0.92 (0.66, 1.28) 1.00 1.00 1.00 1.39** (1.17, 1.64) 1.38** (1.17, 1.64) 1.29** (1.09, 1.54) 1.00 1.00 1.00 0.25** (0.18, 0.34) 0.26** (0.19, 0.35) 0.33** (0.24, 0.45) 0.26** (0.20, 0.33) 0.26** (0.20, 0.33) 0.30** (0.23, 0.39) 0.48** (0.38, 0.61) 0.49** (0.39, 0.62) 0.53** (0.41, 0.67) 1.00 1.00 1.00 1.00 1.00 1.00 2.87** (2.21, 3.73) 2.82** (2.17, 3.67) 2.33** (1.79, 3.05) 1.36 (0.98, 1.89) 1.38 (0.99, 1.92) 1.22 (0.87, 1.71) 2.98** (1.97, 4.53) 3.00** (1.97, 4.55) 2.56** (1.68, 3.92) 3.25** (1.97, 5.36) 3.20** (1.94, 5.27) 3.17** (1.92, 5.25) 2.83** (1.60, 5.01) 2.85** (1.61, 5.03) 2.50** (1.41, 4.42) 1.70 (0.92, 3.12) 1.74 (0.94, 3.21) 1.51 (0.82, 2.79) 1.00 1.00 1.00 0.75* (0.60, 0.93) 0.75* (0.60, 0.94) 0.81 (0.65, 1.01) 0.36** (0.23, 0.58) 0.37** (0.24, 0.59) 0.38** (0.24, 0.60) 1.18 (0.90, 1.56) 1.18 (0.90, 1.55) 1.18 (0.89, 1.55) 1.70** (1.41, 2.05) 1.69** (1.40, 2.03) 1.68** (1.39, 2.03) 1.00 1.00 1.00 0.73** (0.61, 0.88) 0.73** (0.61, 0.88) 0.73** (0.60, 0.88) 1.00 1.00 0.81 (0.66, 1.00) 0.82 (0.67, 1.01) 1.00 1.00 1.02 (0.98, 1.06) 1.05* (1.01, 1.09) No (Ref.) No (Ref.) Number of stresses in 12 months before birth 1.00 1.00 Then (Ref.) Dinour et al. Discussion (RRR 1.99; 95% CI 1.25, 3.16). The number of stresses in the 12 months prior to birth was not significant for any time interval. RRR 1.99; 95% CI 1.25, 3.16). The number of stresses in e 12 months prior to birth was not significant for any me interval. Among the physiological variables, women who did ot deliver vaginally had significantly higher risk of eastfeeding cessation at 1–3 weeks (RRR 1.60; 95% CI 34, 1.92) and 4–6 weeks (RRR 1.27; 95% CI 1.07, 1.51) an to breastfeed for > 10 weeks. In addition, women hose infants remained in the hospital for > 6 days were 4 -112% more likely to stop breastfeeding at 4–6 weeks 7–9 weeks than to breastfeed for > 10 weeks when ompared to women whose infants stayed in the hospital r 0–2 days. With regards to prenatal care, those receiv- g adequate care had a lower risk of breastfeeding ces- tion at 1–3 weeks (RRR 0.73; 95% CI 0.61, 0.88) and –6 weeks (RRR 0.70; 95% CI 0.59, 0.84) versus > 10 eeks, compared to women receiving adequate plus pre- atal care. Finally, women who smoked at the time of e survey had more than a two-fold greater risk of dis- ontinuing breastfeeding at all shorter time intervals an to breastfeed for > 10 weeks, compared to non- mokers (Table 5). Discussion Ninety percent of women in this study were food secure prior to the birth of their infants and/or initiated breast- feeding, and more than half continued breastfeeding for > 10 weeks regardless of their food security status. Among this sample, food insecurity was not associated with breastfeeding initiation after adjusting for socioeco- nomic, psychosocial, and physiological factors. This find- ing is congruent with results from a similar study by Orr et al. [23], who found that among 10,450 Canadian women, breastfeeding initiation was unrelated to house- hold food security once sociodemographic characteris- tics were taken into account. Conversely, in a qualitative study of 20 food insecure Nova Scotian mothers by Frank [24], 95% initiated breastfeeding due to the health benefits and high cost of formula. Frank states that “worry over the cost of formula was a driving factor in initiation, indicating that household food insecurity could be a predictor of breastfeeding initiation” [24]. Discussion With regards to prenatal care, those receiv- ing adequate care had a lower risk of breastfeeding ces- sation at 1–3 weeks (RRR 0.73; 95% CI 0.61, 0.88) and 4–6 weeks (RRR 0.70; 95% CI 0.59, 0.84) versus > 10 weeks, compared to women receiving adequate plus pre- natal care. Finally, women who smoked at the time of the survey had more than a two-fold greater risk of dis- continuing breastfeeding at all shorter time intervals than to breastfeed for > 10 weeks, compared to non- smokers (Table 5). Table 4 Multinomial logistic regression using “breastfeeding for > 10 weeks” as reference category for Model 1 Breastfeeding < 1 week, RRR (95% CI) Breastfeeding 1–3 weeks, RRR (95% CI) Breastfeeding 4–6 weeks, RRR (95% CI) Breastfeeding 7–9 weeks, RRR (95% CI) Food secure (Ref.) 1.00 1.00 1.00 1.00 Food insecure 2.22** (1.55, 3.18) 1.90** (1.51, 2.38) 1.36* (1.07, 1.72) 2.00** (1.59, 2.52) Note. CI confidence interval, RRR relative risk ratio; *P < 0.05, **P < 0.01 Dinour et al. Discussion International Breastfeeding Journal (2020) 15:30 Page 12 of 16 Table 5 Multinomial logistic regression using “breastfeeding for > 10 weeks” as reference category for Model 4 (Continued) Breastfeeding < 1 week, RRR (95% CI) Breastfeeding 1–3 weeks, RRR (95% CI) Breastfeeding 4–6 weeks, RRR (95% CI) Breastfeeding 7–9 weeks, RRR (95% CI) Wanted to be pregnant Then (Ref.) 1.00 1.00 1.00 1.00 Later 1.24 (0.86, 1.78) 1.10 (0.89, 1.35) 0.99 (0.80, 1.22) 1.08 (0.87, 1.34) Sooner 1.09 (0.69, 1.72) 0.99 (0.77, 1.28) 1.16 (0.92, 1.45) 0.92 (0.71, 1.20) Never 1.99** (1.25, 3.16) 0.83 (0.58, 1.19) 1.05 (0.77, 1.45) 0.73 (0.49, 1.06) Unsure 1.10 (0.73, 1.66) 1.04 (0.81, 1.33) 1.15 (0.91, 1.45) 1.02 (0.79, 1.31) Vaginal delivery (Ref.) 1.00 1.00 1.00 1.00 No 1.31 (0.96, 1.77) 1.60** (1.34, 1.92) 1.27** (1.07, 1.51) 1.10 (0.91, 1.33) Kotelchuck Index Inadequate 0.89 (0.56, 1.41) 1.05 (0.81, 1.37) 0.83 (0.64, 1.08) 1.05 (0.79, 1.38) Intermediate 0.94 (0.60, 1.48) 0.93 (0.71, 1.21) 0.90 (0.70, 1.16) 0.98 (0.74, 1.30) Adequate 0.88 (0.65, 1.20) 0.73** (0.61, 0.88) 0.70** (0.59, 0.84) 0.97 (0.80, 1.17) Adequate Plus (Ref.) 1.00 1.00 1.00 1.00 Hospital baby stay length 0–2 days (Ref.) 1.00 1.00 1.00 1.00 3–5 days 1.09 (0.79, 1.50) 1.03 (0.85, 1.26) 1.24* (1.03, 1.50) 1.16 (0.94, 1.44) 6 + days 0.73 (0.46, 1.14) 1.14 (0.89, 1.45) 1.84** (1.49, 2.27) 2.12** (1.69, 2.66) Smoking at time of survey 2.59** (1.88, 3.57) 2.74** (2.22, 3.38) 2.46** (2.01, 3.02) 2.12** (1.69, 2.65) No (Ref.) 1.00 1.00 1.00 1.00 Note. CI confidence interval, HCW healthcare worker, NH non-Hispanic, RRR relative risk ratio, WIC Special Supplemental Nutrition Program for Women, Infants, and Children; *P < 0.05, **P < 0.01. The sample size is N = 9239 Table 5 Multinomial logistic regression using “breastfeeding for > 10 weeks” as reference category for Model 4 (Continued) Breastfeeding < 1 week, RRR (95% CI) Breastfeeding 1–3 weeks, RRR (95% CI) Breastfeeding 4–6 weeks, RRR (95% CI) Breastfeeding 7–9 we RRR (95% CI) improve maternal and infant health though breastfeeding promotion and other types of support [41]. The high breastfeeding initiation rates in this sample of food insecure mothers may be influenced by CPNP participation [24]. Unfortunately, our data do not clearly explain why the risk of breastfeeding cessation at 4–6 weeks compared to breastfeeding for > 10 weeks is significantly lower when food insecurity is experienced prenatally, independent of other known influences on breastfeeding. Discussion International Breastfeeding Journal (2020) 15:30 Page 11 of 16 Table 5 Multinomial logistic regression using “breastfeeding for > 10 weeks” as reference category for Model 4 Breastfeeding < 1 week, RRR (95% CI) Breastfeeding 1–3 weeks, RRR (95% CI) Breastfeeding 4–6 weeks, RRR (95% CI) Breastfeeding 7–9 we RRR (95% CI) Food insecurity Food secure (Ref.) 1.00 1.00 1.00 1.00 Food insecure 0.92 (0.61, 1.37) 0.93 (0.72, 1.20) 0.65** (0.50, 0.85) 1.03 (0.79, 1.34) Maternal age 20–24 years old 0.95 (0.60, 1.48) 1.64** (1.23, 2.20) 1.36* (1.04, 1.77) 1.21 (0.91, 1.62) 25–29 years old 0.69 (0.45, 1.07) 1.25 (0.95, 1.64) 1.04 (0.82, 1.33) 0.97 (0.74, 1.28) 30–34 years old 0.65 (0.41, 1.03) 1.03 (0.78, 1.37) 0.85 (0.66, 1.09) 0.87 (0.66, 1.14) 35+ years old (Ref.) 1.00 1.00 1.00 1.00 Income $0–$22,000 1.40 (0.76, 2.58) 1.21 (0.86, 1.72) 1.14 (0.83, 1.57) 1.53* (1.08, 2.17) $22,001-37,000 0.95 (0.52, 1.73) 1.00 (0.72, 1.40) 0.89 (0.66, 1.20) 1.31 (0.95, 1.82) $37,001-52,000 1.35 (0.75, 2.42) 1.13 (0.81, 1.56) 0.96 (0.71, 1.30) 1.14 (0.81, 1.59) $52,001-67,000 0.95 (0.47, 1.93) 1.17 (0.83, 1.67) 1.01 (0.73, 1.39) 0.90 (0.61, 1.33) $67,001+ (Ref.) 1.00 1.00 1.00 1.00 Marital status Married 0.70* (0.51, 0.96) 0.78** (0.64, 0.94) 0.83* (0.69, 0.99) 0.77** (0.63, 0.94) Not married (Ref.) 1.00 1.00 1.00 1.00 Years of maternal education 0–11 years 3.71** (1.98, 6.96) 2.73** (1.93, 3.85) 2.79** (2.01, 3.88) 2.11** (1.48, 3.02) 12 years 5.18** (3.08, 8.70) 3.03** (2.29, 4.00) 3.36** (2.60, 4.34) 2.53** (1.91, 3.35) 13–15 years 3.14** (1.92, 5.13) 2.14** (1.66, 2.76) 2.28** (1.81, 2.87) 2.13** (1.66, 2.74) 16+ years (Ref.) 1.00 1.00 1.00 1.00 Race/ethnicity NH White (Ref.) 1.00 1.00 1.00 1.00 Hispanic 0.61* (0.39, 0.96) 0.95 (0.74, 1.23) 1.00 (0.78, 1.26) 1.05 (0.81, 1.35) NH Black 0.46 (0.21, 1.00) 0.81 (0.54, 1.21) 1.01 (0.70, 1.44) 1.02 (0.69, 1.50) NH Native American 0.97 (0.56, 1.68) 0.87 (0.59, 1.28) 0.75 (0.50, 1.12) 0.91 (0.61, 1.35) NH Other 0.52 (0.26, 1.04) 0.66* (0.44, 0.98) 0.54** (0.35, 0.82) 0.65* (0.42, 0.99) NH Asian 0.27 (0.07, 1.10) 0.79 (0.48, 1.31) 1.11 (0.75, 1.66) 1.24 (0.81, 1.91) Unknown Race 0.62 (0.19, 2.02) 0.63 (0.31, 1.27) 0.87 (0.49, 1.55) 0.48 (0.21, 1.11) Insurance type Private insurance (Ref.) 1.00 1.00 1.00 1.00 Government insurance 1.13 (0.76, 1.66) 1.00 (0.79, 1.26) 0.86 (0.68, 1.07) 1.03 (0.80, 1.31) Other insurance 1.05 (0.31, 3.53) 1.46 (0.80, 2.69) 0.79 (0.40, 1.60) 1.11 (0.55, 2.22) No insurance 0.79 (0.49, 1.27) 0.84 (0.64, 1.12) 0.72* (0.55, 0.95) 1.09 (0.82, 1.43) HCW talked about breastfeeding before birth 0.75 (0.52, 1.09) 1.39* (1.07, 1.81) 1.00 (0.80, 1.24) 1.42* (1.09, 1.85) No (Ref.) 1.00 1.00 1.00 11.00 On WIC during pregnancy 1.43* (1.01, 2.02) 1.23 (1.00, 1.51) 1.27* (1.04, 1.55) 0.95 (0.77, 1.17) No (Ref.) 1.00 1.00 1.00 11.00 Experienced postpartum depression 0.93 (0.61, 1.41) 1.50** (1.19, 1.89) 1.32* (1.05, 1.65) 1.14 (0.89, 1.47) No (Ref.) 1.00 1.00 1.00 11.00 Number of stresses in 12 months before birth 1.00 (0.93, 1.07) 0.97 (0.93, 1.01) 1.02 (0.98, 1.06) 1.00 (0.96, 1.05) Table 5 Multinomial logistic regression using “breastfeeding for > 10 weeks” as reference category for Model 4 Breastfeeding < 1 week, Breastfeeding 1–3 weeks, Breastfeeding 4–6 weeks, Breastfeeding 7–9 w Dinour et al. Discussion First, a more diverse sample by race, ethni- city, education level, food insecurity, and breastfeeding initiation may show stronger associations between the variables of interest. As the data were taken from a small selection of states that mostly exhibit higher-than- average rates of breastfeeding initiation and household food security, these results may not be generalizable to all food insecure women in other regions of the U.S. or internationally. We recommend that more states include food security questions in their PRAMS questionnaires, or that food security questions become part of the core PRAMS survey. Similarly, it is possible that food inse- cure populations may not participate in national surveys like PRAMS due to high mobility and a possible lack of consistent phone coverage. This represents a potential selection bias that must be considered when interpreting our results. It is notable that participation in WIC during preg- nancy decreased the odds of breastfeeding initiation given the measures WIC has put in place to support breastfeeding. In 1997, WIC launched the Loving Sup- port Makes Breastfeeding Work campaign to increase ini- tiation and duration rates among WIC participants and raise public acceptance and breastfeeding support through mass media campaigns, participant education materials, and technical assistance for WIC staff [47]. Additionally, WIC participants who breastfeed receive a greater quantity and variety of foods than non- breastfeeding participants, along with breast pumps, one-on-one support from WIC Peer Counselors, and longer participation in the program [47]. Still, not all mothers are aware of WIC’s breastfeeding resources, and not all WIC agencies have delivered the campaign con- sistently [48]. Likewise, while the benefits of breastfeed- ing are known, WIC participants lack breastfeeding skills and confidence and find breastfeeding to be more difficult than expected [48]. WIC provides free infant formula (coupled with less food) to mothers who are unable or choose not to breastfeed, and WIC’s budget for breastfeeding promotion is small in comparison to the amount spent on obtaining infant formula [49]. Critics suggest that WIC’s provision of no-cost infant formula is both an implicit endorsement of formula by the U.S. government and an incentive for use by Logistic regressions typically require a large sample size and a general guideline is that a minimum of 10 cases is needed for the least frequent outcome of each independent variable in the model [50]. Discussion Calls to phase out WIC’s large-scale formula distribution [49] have not been answered to date, though WIC launched a new breastfeeding promotion and support campaign (Learn Together. Grow Together.) in 2018 to address the awareness and implementation issues of the prior campaign [48]. Although WIC cannot be expected to address all of the social determinants of breastfeeding behaviors, future research is needed to determine the extent to which WIC’s updated campaign is successful in increasing recipients’ breastfeeding skills, confi- dence, and utilization of resources. WIC participants [49]. Calls to phase out WIC’s large-scale formula distribution [49] have not been answered to date, though WIC launched a new breastfeeding promotion and support campaign (Learn Together. Grow Together.) in 2018 to address the awareness and implementation issues of the prior campaign [48]. Although WIC cannot be expected to address all of the social determinants of breastfeeding behaviors, future research is needed to determine the extent to which WIC’s updated campaign is successful in increasing recipients’ breastfeeding skills, confi- dence, and utilization of resources. method [24]. Further qualitative research is needed in the U.S. to explore the factors driving this association. Our findings regarding other breastfeeding predictors mirror prior work that suggests initiation and longer du- rations are related to a variety of sociodemographic, psy- chosocial, and physiological characteristics [4, 20, 43, 44]. Specifically, we found that married and college- educated women, non-smokers, women not receiving WIC during pregnancy, and women who had vaginal de- liveries were more likely to initiate breastfeeding and/or breastfeed for longer, regardless of food security status. Further public health and policy efforts to improve breastfeeding initiation and duration rates should there- fore concentrate on single mothers, those without a col- lege degree, smokers, prenatal WIC recipients, and those who have non-vaginal births. For example, smokers often choose to formula feed because they fear their breast milk is contaminated by smoking and thus harm- ful to the baby [45]. Yet the CDC states that although mothers should be encouraged to quit tobacco and e- cigarette smoking, breastfeeding is not contraindicated among mothers who smoke but instead remains the rec- ommended food for an infant [46]. While pregnancy and lactation are opportune times for promoting smoking cessation, healthcare professionals should educate mothers that they can continue to breastfeed whether or not they are able to quit. This study is not without limitations that should be mentioned. Discussion It may be a re- sult of a relatively small number (n = 87) of women who reported both food insecurity and breastfeeding for 4–6 weeks, as no other breastfeeding cessation timeframe was significantly different from > 10 weeks when covari- ates were controlled. Additionally, whereas the percent- age of food insecure women who stopped breastfeeding at 4–6 weeks was lower than either 1–3 weeks or 7–9 weeks, the percentage of food secure women who stopped breastfeeding was higher at 4–6 weeks than either 1–3 weeks and 7–9 weeks. These differences in cessation patterns by food security status may have led to the finding that food insecurity appears protective for early breastfeeding cessation at 4–6 weeks. An alterna- tive explanation may be that non-working or under- employed mothers with low socioeconomic status may breastfeed longer due to increased proximity to their in- fants coupled with the high cost of formula feeding, of up to $1500 for a year [42]. Yet reports from food- insecure mothers in Canada indicate that breastfeeding does not save money because there is often little money for food to begin with, regardless of the infant feeding Likewise, prenatal food security status was not related to three of the four early breastfeeding cessation time frames we analyzed. Our findings support the results from Gomes and Gubert’s [22] nationally representative study in Brazil that show no significant relationship between food insecurity and any breastfeeding among children younger than 12 months. It should be noted, though, that the researchers did not account for sociode- mographic variables known to be associated with breast- feeding and food security, such as maternal education level, marital status, employment, maternal health fac- tors, etc. [22]. However, our findings differ from the qualitative results from Haiti [21] and Nova Scotia [24], which suggest that food insecurity leads to breastfeeding cessation due to maternal weakness, perceived or actual breast milk insufficiency, and/or concern that breastmilk is not nutritionally adequate because of poor maternal diet. Other studies that have measured exclusive breast- feeding show either no relationship with hunger [18, 19, 26, 27], or an association between food insecurity and increased likelihood of earlier exclusive breastfeeding cessation [19, 23]. Dinour et al. International Breastfeeding Journal (2020) 15:30 Page 13 of 16 Page 13 of 16 WIC participants [49]. Funding This work was supported by the Montclair State University Separately Budgeted Research Internal Award. The funding body had no role in the design of the study or the collection, analysis, and interpretation of data or in writing the manuscript. Conclusions Although PRAMS is a cross-sectional survey, the results of this study add to the small body of international lit- erature on food insecurity and breastfeeding outcomes. Among this U.S. sample, socioeconomic, psychosocial, and physiological factors explain the association between prenatal food insecurity and breastfeeding outcomes. Our findings indicate the need for more targeted and effective interventions and policies in the U.S. that encourage the initiation and duration of breastfeeding regardless of food security status, particularly among single mothers, those without a college degree, smokers, prenatal WIC recipients, and those recovering from non-vaginal births. Additionally, food security status was determined from a single question, which may not provide the sensitivity needed to identify all women experiencing food insecur- ity nor capture the latent construct as precisely as the standard 18-item or abbreviated 6-item food security modules used by the USDA [32]. More specifically, the food security question asked in PRAMS reflects reduced adult food intake, and while this alone cannot capture the full range of food insecurity, it is asking for a level of severity that goes beyond anxiety about food budgets or food supply. There are no studies, to our knowledge, that examine food insecurity and breastfeeding using a 1-item measure for food insecurity, however other stud- ies examining food insecurity using the PRAMS dataset do use the 1-item measure [33, 34]. Future PRAMS sur- veys would benefit from including additional validated food security questions to better discern between various levels of food security. Discussion As such, we checked for small cells by performing a cross-tabulation between categorical predictors and breastfeeding cessa- tion. There were a few cases where the cell size did fall below the threshold of 10. All but one of these instances occurred in the race categories. For example, among women who breastfed for < 1 week, there were only seven non-Hispanic Black women, nine non-Hispanic women of a race not otherwise classified, two non- Hispanic Asian women, and three women of an un- known race and ethnicity. Likewise, among women of an unknown race and ethnicity, there were nine who breastfed for 1–3 weeks and six who breastfeed for 7–9 weeks. One other small cell occurrence was noted for the three participants with other insurance who breast- fed for < 1 week. Since these small cells have the poten- tial to make our model unstable, we re-ran our models with larger time-based intervals: < 4 weeks, 5–9 weeks, and > 10 weeks. The results from these multinomial lo- gistic regressions were similar to those of our original models included in this study. Unfortunately, these Page 14 of 16 Page 14 of 16 Dinour et al. International Breastfeeding Journal (2020) 15:30 Dinour et al. International Breastfeeding Journal (2020) 15:30 Dinour et al. International Breastfeeding Journal (2020) 15:30 larger time-based intervals are not useful for policy rec- ommendations as they do not represent meaningful postpartum time frames (i.e., 6-week maternity leave). Although the use of weighted variables can potentially reduce accuracy because the sampling variance, standard deviation, and standard error increase, it is important to note that the data were weighted and are representative of the 12-month PRAMS-eligible population. As a result, our weighted sample is representative of the women age 20 years or older who gave birth to live infants in the selected states. Abbreviations CDC: Centers for Disease Control and Prevention; CHIP: Children’s Health Insurance Program; CI: Confidence interval; CPNP: Canada Prenatal Nutrition Programs; OR: Odds ratio; PRAMS: Pregnancy Risk Assessment Monitoring System; RRR: Relative risk ratio; USDA: United States Department of Agriculture; WIC: Special Supplemental Nutrition Program for Women, Infants, and Children To understand how food insecurity, as measured, can impact a distal outcome such as breastfeeding be- yond 10 weeks, it is important to understand the na- ture of food insecurity. Food insecurity is rarely an acute problem, but rather a prolonged and chronic issue that actively occurs over extended periods of time [14]. Although we only capture a snapshot of food security status, we can safely assume that some respondents may be food insecure for periods longer than 12 months. Due to the limitations in this ques- tion we cannot ascertain the exact time period, but this measure may not be as distal as it seems on the surface. Likewise, PRAMS does not include a measure of current food security status at the time of the sur- vey, so it remains unclear if women are food insecure while breastfeeding. Finally, there are variables not in- cluded in PRAMS that are related to food insecurity and/or breastfeeding, such as employment status at the time of the survey, percent of the federal poverty level, and postnatal participation in WIC or other fed- eral food assistance programs. Including these ques- tions in future PRAMS questionnaires would allow for testing alternative explanations for the mainly null associations we found between food security and breastfeeding outcomes. Acknowledgements We are indebted to Jacalyn Szaro and Jacqueline Bavaro for providing research assistance during the conceptualization and writing of this manuscript. We also thank the members of the Pregnancy Risk Assessment Monitoring System (PRAMS) Working Group and the Centers for Disease Control and Prevention (CDC) PRAMS Team, Applied Sciences Branch, Division of Reproductive Health. Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s13006-020-00276-x. Supplementary information accompanies this paper at https://doi.org/10. 1186/s13006-020-00276-x. Supplementary information accompanies this paper at https://doi.org/10. 1186/s13006-020-00276-x. Additional file 1. Table of multinomial logistic regression using “breastfeeding for > 10 weeks” as reference category for Model 2: Pregnancy Risk Assessment Monitoring System, Colorado, Maine, New Mexico, Oregon, Pennsylvania, and Vermont, 2012–2013. Additional file 2. Table of multinomial logistic regression using “breastfeeding for > 10 weeks” as reference category for Model 3: Pregnancy Risk Assessment Monitoring System, Colorado, Maine, New Mexico, Oregon, Pennsylvania, and Vermont, 2012–2013. Additional file 1. Table of multinomial logistic regression using “breastfeeding for > 10 weeks” as reference category for Model 2: Pregnancy Risk Assessment Monitoring System, Colorado, Maine, New Mexico, Oregon, Pennsylvania, and Vermont, 2012–2013. Additional file 1. Table of multinomial logistic regression using “breastfeeding for > 10 weeks” as reference category for Model 2: Pregnancy Risk Assessment Monitoring System, Colorado, Maine, New Mexico, Oregon, Pennsylvania, and Vermont, 2012–2013. Additional file 2. Table of multinomial logistic regression using “breastfeeding for > 10 weeks” as reference category for Model 3: Pregnancy Risk Assessment Monitoring System, Colorado, Maine, New Mexico, Oregon, Pennsylvania, and Vermont, 2012–2013. Authors’ contributions LMD conceived of the study, obtained the data, and supervised all aspects of the study. LMD, NA-O, and LAD contributed to the design of the study, ana- lysis of the data, and interpretation of the findings. ERR conducted the statis- tical analyses. All authors contributed to the writing of the manuscript and approved the final manuscript. References 24. Frank L. Exploring infant feeding practices in food insecure households: what is the real issue? Food Foodways. 2015;23:186–209. 1. Dieterich CM, Felice JP. O’ Sullivan E, Rasmussen KM. Breastfeeding and health outcomes for the mother-infant dyad. Pediatr Clin N Am. 2013; 60:31–48. 1. Dieterich CM, Felice JP. O’ Sullivan E, Rasmussen KM. Breastfeeding and health outcomes for the mother-infant dyad. Pediatr Clin N Am. 2013; 60:31–48. 25. Wong PD, Parkin PC, Moodie RG, Dai DWH, Maguire JL, Birken CS, et al. Total breastfeeding duration and household food insecurity in healthy urban children. Acad Pediatr. 2019;19:884–90. 2. Eidelman AI, Schanler RJ. American Academy of Pediatrics policy statement: breastfeeding and the use of human milk. Pediatrics. 2012;129:e827–41. 2. Eidelman AI, Schanler RJ. American Academy of Pediatrics policy statement: breastfeeding and the use of human milk. Pediatrics. 2012;129:e827–41. 26. Gross RS, Mendelsohn AL, Messito MJ. Additive effects of household food insecurity during pregnancy and infancy on maternal infant feeding styles and practices. Appetite. 2018;130:20–8. 3. Ip S, Chung M, Raman G, Trikalinos TA, Lau J. A summary of the Agency for Healthcare Research and Quality’s evidence report on breastfeeding in developed countries. Breastfeed Med. 2009;4:S17–30. 3. Ip S, Chung M, Raman G, Trikalinos TA, Lau J. A summary of the Agency for Healthcare Research and Quality’s evidence report on breastfeeding in developed countries. Breastfeed Med. 2009;4:S17–30. 27. Gross RS, Mendelsohn AL, Fierman AH, Racine AD, Messito MJ. Food insecurity and obesogenic maternal infant feeding styles and practices in low-income families. Pediatrics. 2012;130:254–61. 4. U.S. Department of Health and Human Services. The Surgeon General’s call to action to support breastfeeding. Washington D.C.: U.S. Department of Health and Human Services, Office of the Surgeon General; 2011. https:// www.ncbi.nlm.nih.gov/books/NBK52682/. Accessed 30 Nov 2017. 4. U.S. Department of Health and Human Services. The Surgeon General’s call to action to support breastfeeding. Washington D.C.: U.S. Department of Health and Human Services, Office of the Surgeon General; 2011. https:// www.ncbi.nlm.nih.gov/books/NBK52682/. Accessed 30 Nov 2017. 28. Organisation for Economic Co-operation and Development. PF2.1 Parental leave systems. OECD family database. 2019. https://www.oecd.org/els/soc/ PF2_1_Parental_leave_systems.pdf. Accessed 5 Aug 2019. 5. Lessen R, Kavanagh K. Position of the academy of nutrition and dietetics: promoting and supporting breastfeeding. J Acad Nutr Diet. 2015;115:444–9. 5. Lessen R, Kavanagh K. Position of the academy of nutrition and dietetics: promoting and supporting breastfeeding. J Acad Nutr Diet. 2015;115:444–9. PF2_1_Parental_leave_systems.pdf. Accessed 5 Aug 2019. 29. Author details 1 Author details 1College of Education and Human Services, Montclair State University, 1 Normal Avenue, Montclair, NJ 07043, USA. 2School of Medicine, Tufts University, 136 Harrison Avenue, Boston, MA 02111, USA. 3Scientific Research, Health Policy Research Consortium, CTIS Inc, 6401 Golden Triangle Drive, Suite #310, Greenbelt, MD 20770, USA. 21. Lesorogol C, Bond C, Dulience SJL, Iannotti L. Economic determinants of breastfeeding in Haiti: the effects of poverty, food insecurity, and employment on exclusive breastfeeding in an urban population. Matern Child Nutr. 2018;14(2):e12524. 22. Gomes GP, Gubert MB. Breastfeeding in children under 2 years old and household food and nutrition security status. J Pediatr. 2012;88:279–82. Consent for publication Not applicable. 19. Young SL, Plenty AHJ, Luwedde FA, Natamba B, Natureeba P, Achan J, et al Household food insecurity, maternal nutritional status, and infant feeding practices among HIV-infected Ugandan women receiving combination antiretroviral therapy. Matern Child Health J. 2014;18:2044–53. References Special Supplemental Nutrition Program for Women, Infants, and Children (WIC). U.S. Department of Agriculture, Food and Nutrition Service website. https://www.fns.usda.gov/wic. Published date unknown. Accessed 5 Aug 2019. 6. World Health Organization. Global strategy for infant and young child feeding. Geneva: World Health Organization; 2003. http://apps.who.int/iris/ bitstream/10665/42590/1/9241562218.pdf. Accessed 30 Nov 2017. 6. World Health Organization. Global strategy for infant and young child feeding. Geneva: World Health Organization; 2003. http://apps.who.int/iris/ bitstream/10665/42590/1/9241562218.pdf. Accessed 30 Nov 2017. 7. Centers for Disease Control and Prevention. Percentage of US children who were ever breastfed, by birth year, national immunization survey, United States. 2016. https://www.cdc.gov/breastfeeding/data/nis_data/results.html. Accessed 4 Mar 2017. 30. Coleman-Jensen A, Rabbitt MP, Gregory C, Singh A. Household food security in the United States in 2013. U.S. Department of Agriculture, Economic Research Service; 2014. https://www.ers.usda.gov/publications/ pub-details/?pubid=45268. Accessed 18 Feb 2017. 8. Shepherd-Banigan M, Bell JF. Paid leave benefits among a national sample of working mothers with infants in the United States. Matern Child Health J. 2014;18:286–95. 8. Shepherd-Banigan M, Bell JF. Paid leave benefits among a national sample of working mothers with infants in the United States. Matern Child Health J. 2014;18:286–95. 31. Shulman HB, Gilbert BC, Lansky A. The pregnancy risk assessment monitoring system (PRAMS): current methods and evaluations of 2001 response rates. Public Health Rep. 2006;121:74–83. 9. U.S. Department of Health and Human Services, Health Resources and Services Administration. Women’s health USA 2011. Rockville, Maryland: U. S Department of Health and Human Services; 2011. https://mchb.hrsa.gov/ whusa11/more/downloads/pdf/w11.pdf. Accessed 30 Nov 2017. 32. Bickel G, Nord M, Price C, Hamilton W, Cook J. Guide to measuring household food security. Alexandria, VA: U. S Department of Agriculture, Food and Nutrition Service; 2000. https://www.fns.usda.gov/guide- measuring-household-food-security-revised-2000. Accessed 8 Nov 2017. 10. Amin RM, Said ZM, Sutan R, Shah SA, Darus A, Shamsuddin K. Work related determinants of breastfeeding discontinuation among employed mothers in Malaysia. Int Breastfeed J. 2011;6:4. 33. Adams EJ, Hoffmann LL, Rosenberg KD, Peters D, Pennise M. Increased food insecurity among mothers of 2 year olds with special health care needs. Matern Child Health J. 2015;19:2206–14. 11. Kozhimannil KB, Jou J, Gjerdingen DK, McGovern PM. Access to workplace accommodations to support breastfeeding after passage of the affordable care act. Womens Health Issues. 2016;26:6–13. 34. Cunningham TJ, Barradas DT, Rosenberg KD, May AL, Kroelinger CD, Ahluwalia IB. Is maternal food security a predictor of food and drink intake among toddlers in Oregon? Matern Child Health J. 2012;16(Suppl 2):339–46. Received: 15 May 2019 Accepted: 13 April 2020 Received: 15 May 2019 Accepted: 13 April 2020 y ; 23. Orr SK, Dachner N, Frank L, Tarasuk V. Relation between household food insecurity and breastfeeding in Canada. Can Med Assoc J. 2018;190:E312–9. 24. Frank L. Exploring infant feeding practices in food insecure households: what is the real issue? Food Foodways. 2015;23:186–209. 23. Orr SK, Dachner N, Frank L, Tarasuk V. Relation between household food insecurity and breastfeeding in Canada. Can Med Assoc J. 2018;190:E312–9 Competing interests The authors declare that they have no competing interests. 20. Webb-Girard A, Cherobon A, Mbugua S, Kamau-Mbuthia E, Amin A, Sellen DW. Food insecurity is associated with attitudes towards exclusive breastfeeding among women in urban Kenya. Matern Child Nutr. 2012;8: 199–214. Availability of data and materials The data that support the findings of this study are available from the United States Centers for Disease Control and Prevention (CDC), but restrictions apply to the availability of these data, which were used under license for the current study, and so the authors are unable to release the data set. Data are however available from the CDC via a proposal submission process outlined here: https://www.cdc.gov/prams/prams-data/researchers.htm. Page 15 of 16 Page 15 of 16 Dinour et al. International Breastfeeding Journal (2020) 15:30 The survey questionnaires, analytic variables, and codebooks are publicly available from the PRAMS website: https://www.cdc.gov/prams/. 15. Venu I, van den Heuvel M, Wong JP, Borkhoff CM, Moodie RG, Ford-Jones EL, et al. The breastfeeding paradox: relevance for household food insecurity. Paediatr Child Health. 2017;22:180–3. 16. Noonan MC, Rippeyoung PLF. The economic costs of breastfeeding for women. Breastfeed Med. 2011;6:325–7. Ethics approval and consent to participate pp p p The Montclair State University Institutional Review Board determined that no protocol approval was required because the study used secondary, de- identified data. 17. Rippeyoung PLF, Noonan MC. Is breastfeeding truly cost free? Income consequences of breastfeeding for women. Am Sociol Rev. 2012;77:244–67. 18. Miller JD, Young SL, Boateng GO, Oiye S, Owino V. Greater household food insecurity is associated with lower breast milk intake among infants in western Kenya. Matern Child Nutr. 2019;15(4):e12862. References Breastfeeding initiation in a rural sample: predictive factors and the role of smoking. J Hum Lact. 2012;27:33–40. 46. Centers for Disease Control and Prevention. Should mothers who smoke breastfeed? Centers for Disease Control and Prevention. 2018. https://www. cdc.gov/breastfeeding/breastfeeding-special-circumstances/vaccinations- medications-drugs/tobacco-and-e-cigarettes.html. Accessed 21 Nov 2018. 47. Institute of Medicine. Updating the USDA national breastfeeding campaign: workshop summary. Washington, DC: The National Academies Press; 2011. https://doi.org/10.17226/13235. Accessed 1 Sep 2019. 48. About WIC breastfeeding support and formative research findings. U.S. Department of Agriculture, Food and Nutrition Service website. https:// wicbreastfeeding.fns.usda.gov/about-wic-breastfeeding-support-and- formative-research-findings. Published date unknown. Accessed 1 Sept 2019. 49. Kent G. WIC’s promotion of infant formula in the United States. Int Breastfeed J. 2006;1:8. 50. Peduzzi P, Concato J, Kemper E, Holford TR, Feinstein AR. A simulation study of the number of events per variable in logistic regression analysis. J Clin Epidemiol. 1996;49:1373–9. References 12. Mirkovic KR, Perrine CG, Scanlon KS, Grummer-Strawn LM. Maternity leave duration and full-time/part-time work status are associated with US mothers’ ability to meet breastfeeding intentions. J Hum Lact. 2014; 30:416–9. 35. Rosenberg KD, Eastham CA, Kasehagen LJ, Sandoval AP. Marketing infant formula through hospitals: the impact of commercial hospital discharge packs on breastfeeding. Am J Public Health. 2008;98:290–5. 13. Tsai SY. Impact of a breastfeeding-friendly workplace on an employed mother’s intention to continue breastfeeding after returning to work. Breastfeed Med. 2013;8:210–6. 36. Colaizy TT, Saftlas AF, Morriss FH Jr. Maternal intention to breast-feed and breast-feeding outcomes in term and preterm infants: pregnancy risk assessment monitoring system (PRAMS), 2000-2003. Public Health Nutr. 2011;15:702–10. 14. Coleman-Jensen A, Rabbitt MP, Gregory CA, Singh A. Household food security in the United States in 2017. U.S. Department of Agriculture, Economic Research Service; 2018. https://www.ers.usda.gov/publications/ pub-details/?pubid=90022. Accessed 17 Feb 2017. 37. Ahluwalia IB, Morrow B, D’Angelo D, Li R. Maternity care practices and breastfeeding experiences of women in different racial and ethnic groups: Page 16 of 16 Dinour et al. International Breastfeeding Journal (2020) 15:30 pregnancy risk assessment and monitoring system (PRAMS). Matern Child Health J. 2012;16:1672–8. pregnancy risk assessment and monitoring system (PRAMS). Matern Child Health J. 2012;16:1672–8. 38. Marshall C, Gavin L, Bish C, Winter A, Williams L, Wesley M, et al. WIC participation and breastfeeding among white and black mothers: data from Mississippi. Matern Child Health J. 2013;17:1784–92. 39. Ma X, Liu J, Smith M. WIC participation and breastfeeding in South Carolina: updates from PRAMS 2009-2010. Matern Child Health J. 2014;18:1271–9. 40. D’Angelo DV, Le B, O’Neil ME, Williams L, Ahluwalia IB, Harrison LL, et al. Patterns of health insurance coverage around the time of pregnancy among women with live-born infants--pregnancy risk assessment monitoring system, 29 states, 2009. Morb Mortal Wkly. 2015;64:1–19. 41. Public Health Agency of Canada. Canada Prenatal Nutrition Program (CPNP). 2019. https://www.canada.ca/en/public-health/services/health-promotion/ childhood-adolescence/programs-initiatives/canada-prenatal-nutrition- program-cpnp.html. Accessed 1 Sep 2019. 42. Association of Women’s Health, Obstetric, and Neonatal Nurses. Breastfeeding position statement. J Obstet Gynecol Neonatal Nurs. 2015:44, 145–150. 43. Mathews ME, Leerkes EM, Lovelady CA, Labban JD. Psychosocial predictors of primiparous breastfeeding initiation and duration. J Hum Lact. 2014;30: 480–7. 44. Rollins NC, Bhandari N, Hajeebhoy N, Horton S, Lutter CK, Martines JC, et al. Why invest, and what it will take to improve breastfeeding practices? Lancet. 2016;387:491–504. 45. Bailey B, Wright H. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Beyond burnout: a four-year survey of osteopathic medical student mental health and the implications for the development of wellness and mental health programs
Journal of osteopathic medicine
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Alyse Folino Ley , DO, Joan J. Han, DO, Emma Hare, DO, Alla Sikorskii, PhD, John R. Taylor, PsyD, Arpon Shahed, DO and Celia Guro, PhD Beyond burnout: a four-year survey of osteopathic medical student mental health and the implications for the development of wellness and mental health programs https://doi.org/10.1515/jom-2022-0179 Received August 24, 2022; accepted February 2, 2023; published online February 27, 2023 Results: A total of 973 students in 2016, 889 students in 2017, 547 students in 2018, and 606 students in 2019 participated in the study. For depression and burnout subscales, an increase in symptom scores were observed every survey year (2016, 2017, 2018, and 2019) by the second or third year of medical school with a clinically significant effect size. Persistently high levels of anxiety were observed throughout medical school, with significant increases after the first year noted in the 2016 and 2017 surveys, but not in the 2018 or 2019 surveys. Similarly, significant changes in sleep disturbance were found in the 2016 and 2017 surveys, but not in 2018 or 2019. Conclusions: Symptoms of burnout, depression, and anxi- ety were observed throughout all four years of medical school, with increases starting after the first year. Early intervention is needed to support students’ mental health and increase access to care and resources. This work is licensed under the Creative Commons Attribution 4.0 International License. *Corresponding author: Alyse Folino Ley, DO, Department of Psychiatry, Michigan State University College of Osteopathic Medicine and College of Human Medicine, A233 East Fee Hall, 965 Wilson Road, East Lansing, MI 48824-9902, USA, E-mail: folinoal@msu.edu Joan J. Han, DO, Department of Psychiatry, University of Maryland Medical Center, Baltimore, MD, USA Emma Hare, DO, Michigan State University College of Osteopathic Medicine, East Lansing, MI, USA Alla Sikorskii, PhD, John R. Taylor, PsyD and Celia Guro, PhD, Department of Psychiatry, Michigan State University College of Osteopathic Medicine, East Lansing, MI, USA Arpon Shahed, DO, Michigan State University Institute for Global Health, East Lansing, MI, USA Original Article Original Article Behavioral Health Behavioral Health J Osteopath Med 2023; 123(5): 225–233 Original Article Open Access. © 2023 the author(s), published by De Gruyter. This w clinically significant disturbance in an individual’s cogni- tion, emotion regulation, or behavior that reflects a dysfunction in the psychological, biological, or developmental processes underlying mental functioning” [5]. Acknowledge- ment of burnout is critical, but it should be differentiated from treatable psychiatric disorders such as depressive and anxiety disorders, which place the individual at risk for sui- cide. If all mental health symptoms are misinterpreted as burnout, we will miss the opportunity to recognize mental health symptoms that may be an indication of a treatable psychiatric condition. help us to intervene, ameliorate, and ultimately prevent devastating outcomes. To gauge the mental health of the student body, a survey guided by the concepts in standard- ized psychiatric inventories was created and distributed to the entire medical student body over the course of 4 years. The aim of our study was to better understand the emer- gence and prevalence of burnout and psychiatric symptoms in osteopathic medical students. Participants All enrolled medical students were eligible to participate in the survey each year. The participants were not compensated. Totals of 1,257 in Fall 2016, 1,254 in Fall 2017, 1,224 in Fall 2018, and 1,228 in Fall 2019 were invited to complete the yearly survey. Study design The Medical Student Mental Health Committee (MSMHC) was created in 2015 at the Michigan State University College of Osteopathic Medi- cine, with the goal of improving access to care and resources available to students, and ultimately increasing awareness and decreasing stigma related to mental health problems. The MSMHC created a sur- vey with the aim of characterizing symptoms and risk factors that students experience in medical school. The study was not funded. The survey was initially considered mandatory, but anonymous. After the initial survey was completed in 2016, MSMHC felt that the result was not only valuable for the development of resources, curricula modi- fication, and improvement, but it also provided insight into the mental health of those in the medical profession as a whole. The survey was then submitted to the Michigan State University Institutional Review Board (IRB) for approval. The IRB required that students had the ability to consent to their information being utilized; therefore, the survey was changed to voluntary for the subsequent years (2017–2019) of distribution. An IRB exemption was granted allowing use of the 2016 survey data. Data on mental health symptoms of first-, second-, third-, and fourth-year medical students in each survey year (2016, 2017, 2018, and 2019) were analyzed in this study. Methods Untreated psychiatric illness is a major risk factor for suicide, and more than 90% of suicide victims have a psychiatric disorder [6]. Overall, suicide is one of the top nine leading causes of death in the United States and sec- ond leading cause of death among individuals 10 to 14 and 25–34 years old and is the third leading cause of death for ages 15–24 years old [7]. Compared to the general popula- tion, physician suicide statistics are staggering. According to the 2012 to 2016 National Violent Death Reporting Sys- tem, physicians have death rates from suicide that are comparable to the general populations; female physicians, however, appear to have higher rates than female non- physicians [8]. A previous meta-analysis indicates that the suicide rate among male physicians is 1.41 times higher than the general male population, and the rate among female physicians is 2.27 times greater than the general female population [9]. Like the general population, the prevalence of physician suicide rates have increased from 2003 to 2017 [10]. Although rates of physician suicide are increasing, the onset of psychiatric symptoms and suicidal ideation during medical training has not yet been delin- eated. Prior studies have revealed that compared to the general population, burnout and depressive symptoms are more prevalent among medical students and may progress through medical school [11, 12]. A 2021 meta- analysis indicated that the main risk factors for suicidal ideation and suicide in the medical student population include depression, burnout and comorbid psychiatric conditions, and stress [3]. A study examining factors that contribute to suicide have shown that job dissatisfaction and/or suffering from a known mental health disorder significantly increases the risk of physician suicide [13]. The high rates of depression and suicide in the medical profession should thrust the conversation to move beyond burnout toward the identification and treatment of psy- chiatric symptoms in the individual. Abstract Context: The mental health crisis in medicine cannot be explained by burnout alone. Physicians are not immune to this crisis and are known to have higher rates of suicide and depression than the general population. A high prevalence of mental health symptoms has been observed in early medical training. Objectives: This study was completed to characterize medical students’ mental well-being and provide guid- ance for timely intervention. Methods: An annual prospective, voluntary, anonymous, cross-sectional survey of medical students was completed over a 4-yearperiod in medical school from 2016 to 2019.The survey was created based on standardized psychiatric screening tools assessing symptoms of depression, anxiety, burnout, and sleep problems. In each of those years, 1,257 (2016), 1,254 (2017), 1,221 (2018), and 1,220 (2019) enrolled students, respectively, were invited to participate. Data on students’ mental health were analyzed in relation to their year of school separately for each survey year utilizing SAS 9.4. Keywords: burnout; medical student anxiety; medical student depression; medical student mental health; medical student wellness. Physicians and medical students who experience loss of meaning in their work accompanied by mental health symptoms are often told that they are experiencing burnout. Burnout is a work-related syndrome characterized by emotional exhaustion, cynicism and depersonalization, and reduction in personal accomplishment and effectiveness [1]. Burnout is associated with lower overall empathy and decreased professionalism in medical students, residents, and faculty [2]. Although many suspect that burnout is a risk factor for psychiatric illness and suicide, the true nature of the relationship remains unclear [3]. A 2019 systematic review and meta-analysis by Koutsimani et al. [4] found that there is “no conclusive overlap between burnout and depression and burnout and anxiety, indicating that they are different and robust constructs.” According to The Diag- nostic and Statistical Manual of Mental Disorders, Fifth Edition, “A mental disorder is a syndrome characterized by Alla Sikorskii, PhD, John R. Taylor, PsyD and Celia Guro, PhD, Department of Psychiatry, Michigan State University College of Osteopathic Medicine, East Lansing, MI, USA Arpon Shahed, DO, Michigan State University Institute for Global Health, East Lansing, MI, USA Ley et al.: Beyond burnout: a four-year survey 226 Results A total of 973 students in 2016 (77.4% out of 1,257 invited), 889 students in 2017 (70.9% out of 1,254 invited), 547 stu- dents in 2018 (44.7% out of 1,224 invited), and 606 students in 2019 (49.7% out of 1,220 invited) participated in the study (Table 2). Correlations among depression, burnout, and anxiety scores were strong (>0.70) in all survey years. Correlations between these three construct scores and sleep were moderate to strong and ranged from 0.57 to 0.60 in 2016, from 0.60 to 0.64 in 2017, from 0.56 to 0.58 in 2018, and from 0.57 to 0.60 in 2019. The mean subscale scores and their SEs for each student class and survey year are pre- sented in Table 3. identification of repeated measures of the same individual across time, which was not possible due to the de-identified nature of the survey. Combining data from all survey years into one, without specification of the random effect for nesting of repeated measures within the indi- vidual, would have resulted in incorrect standard errors (SEs). Therefore, survey data from 2016, 2017, 2018, and 2019 were analyzed separately. The scores for the subscales were summarized utilizing descriptive statistics and Pearson correlations among subscale scores. Analyses of variance (ANOVA) were utilized to relate depression, anxiety, burnout, and sleep problems subscale scores to student class (first, second, third, or fourth year). If significant differences among the four classes were indicated by the ANOVA, pairwise comparisons of classes were performed utilizing Bonferroni adjustment for multiple comparisons (alpha level of 0.008). To facilitate interpretation of the magnitude of differences by class, Cohen’s d effect size was calculated as the difference between means of two classes divided by the pooled standard deviation. All analyses were performed utilizing SAS 9.4 [18]. 2-week period every fall semester on the Michigan State University online educational platform. To standardize response time, students were allowed to complete the survey at their leisure. Most questions asked, “Since the start of medical school, how often have you been bothered by the following?” and then listed various symptoms related to mental health and wellness. The survey included over 40 multiple- choice or short-answer questions focused on symptoms of depression, anxiety, burnout, sleep, and eating habits. Additional questions were added to the Fall 2017 to 2019 surveys to determine the extent of mental health conditions prior to medical school and whether they worsened during medical school. Qualitative short-answer questions asked whether they needed help for mental health concerns and, if so, why they failed to seek help. Students were also asked if they knew where to seek help and what resources were available. Students were notified that their responses would be anonymous and were provided with a link to the Office of Personal Counseling and Health Promotion’s mental health resource page, which listed various resources (24-h crisis numbers, emergency services, the college mental health team, campus resources, and community resources). For 2017 to 2019 sur- veys, students could choose to opt out and have their responses removed from the data pool and analysis. Survey The survey (Appendix A) was created by the authors, including a board-certified psychiatrist and medical student, utilizing standard- ized screening inventories as a guide [14–17]. No specific questions were utilized from the screening tools, but the constructs of depres- sion, anxiety, sleep problems, and burnout were identified a priori, and the items were selected to reflect each question constructed based on the authors’ subject matter expertise. There were no preliminary reliability or validity analyses conducted on the survey, which was revised after 2016 to enable the survey to be voluntary. The survey was not intended to be diagnostic due to the inability to identify study participants if acute safety concerns were revealed. The students were notified via email of the assigned survey, which was available for a In light of the recent pandemic and growing evidence that physicians have a significant risk of developing psy- chiatric symptoms during their professional training, efforts aimed at early identification of psychiatric symptoms may Ley et al.: Beyond burnout: a four-year survey 227 Depression subscale The striking number of medical students in our study endorsed depressive symptoms (37.7–77.6%). The overall differences for the mean depression subscale scores among student classes were statistically significant in 2016 (F[3, 908]=14.87, p<0.0001), in 2017 (F[3, 879]=24.46, p<0.0001), in 2018 (F[3, 542]=4.24, p=0.01), and in 2019 (F[3, 602]=2.74, p=0.04). Utilizing the Bonferroni adjustment, in 2016 and 2017, first-year students had statistically significantly lower depressive symptoms compared to students from the three other classes. In 2018 and 2019, first-year students had significantly lower depressive symptoms compared to third- year students. None of the other pairwise differences were statistically significant when controlling for multiple com- parisons. A common finding from all surveys was that depressive symptoms were higher for second-or third-year Outcome measures The survey was constructed utilizing a Likert scale with “never, rarely, sometimes, often, or always” options, yes/no answers, and multiple response questions (i.e., “check all that apply”). Items were grouped into four constructs (subscales of the survey): depression (7 items), anxiety (7 items), sleep problems (4 items), and burnout (13 items) (Table 1). The depression subscale was comprised of questions focused on mood, anhedonia, hope, excessive crying, guilt, irritability, and decreases in energy. The anxiety subscale included questions on con- centration, control, worrying, trouble relaxing, future orientation, and appetite. The sleep subscale contained questions regarding difficulty with sleep, average hours slept, feeling refreshed in the morning, and the need for medications for sleep. Sleep was quantified into 8+ hours, 6–8 h, 4–6 h, and less than 4 h. The burnout subscale focused on questions relating to failure, giving-up, feeling broken down, exhaus- ted, tired, insensitive, lacking achievement, pressure, having more work than is achievable, being in the wrong profession, unappreciated, disappointment, and others are problem-free. For each subscale separately, items were subjected to an exploratory factor analysis to determine dimensionality. Because there was a single eigenvalue greater than 1 for each subscale, and the remaining eigenvalues were much smaller than the first in magnitude, each subscale was deemed unidimensional, and a single summary score (mean across items, range 0–4) was created for each construct. For each survey year, the internal consistency reliability (Cronbach’s alpha) was excellent (>0.85 across four survey years) for depression, burnout (>0.91), and anxiety (>0.89). For sleep, it was lower (0.59–0.64 across survey years) but acceptable given that the sleep scale had only 4 items. Never, rarely, sometimes, often, or always Table : Mental health questions as subscale constructs. Wake up feeling unrefreshed Since the start of medical school, how often have you done the following? Take anything (e.g., medication, alcohol, etc) to help sleep Burnout construct questions Since the start of medical school, how often have you been bothered by the following? Feeling disappointed in myself, feelings of failure, or I have let myself or my family down’ Never, rarely, sometimes, often, or always Since the start of medical school, how often have you been bothered by the following? Feeling like giving up or think “I can’t take it anymore” Since the start of medical school, how often have you been bothered by the following? Feeling like medical school is breaking me down Since the start of medical school, how often have you been bothered by the following? Feel physically or emotionally exhausted Since the start of medical school, how often have you been bothered by the following? Feel tired when I get up in the morning and have to face another day of school Since the start of medical school, how often have you been bothered by the following? Have become more insensitive to people Since the start of medical school, how often have you been bothered by the following? Feeling down or depressed Never, rarely, sometimes, often, or always Since the start of medical school, how often have you been bothered by the following? Little interest or pleasure in doing things Since the start of medical school, how often have you been bothered by the following? Feeling discouraged or hopeless about the future Since the start of medical school, how often have you been bothered by the following? Crying more than usual Since the start of medical school, how often have you been bothered by the following? Feeling guilty Since the start of medical school, how often have you been bothered by the following? Feeling tired, low in energy, or slowed down Since the start of medical school, how often have you done the following? Become easily annoyed or irritable Anxiety construct questions Since the start of medical school, how often have you been bothered by the following? Trouble concentrating or making decisions Never, rarely, sometimes, often, or always Since the start of medical school, how often have you been bothered by the following? Feeling nervous, anxious, or on edge Since the start of medical school, how often have you been bothered by the following? Table : Mental health questions as subscale constructs. Table : Mental health questions as subscale constructs. Depression construct questions Since the start of medical school, how often have you been bothered by the following? Feeling down or depressed Never, rarely, sometimes, often, or always Since the start of medical school, how often have you been bothered by the following? Little interest or pleasure in doing things Since the start of medical school, how often have you been bothered by the following? Feeling discouraged or hopeless about the future Since the start of medical school, how often have you been bothered by the following? Crying more than usual Since the start of medical school, how often have you been bothered by the following? Feeling guilty Since the start of medical school, how often have you been bothered by the following? Feeling tired, low in energy, or slowed down Since the start of medical school, how often have you done the following? Become easily annoyed or irritable Anxiety construct questions Since the start of medical school, how often have you been bothered by the following? Trouble concentrating or making decisions Never, rarely, sometimes, often, or always Since the start of medical school, how often have you been bothered by the following? Feeling nervous, anxious, or on edge Since the start of medical school, how often have you been bothered by the following? Not being able to control my worrying Since the start of medical school, how often have you been bothered by the following? Worrying too much about multiple things Since the start of medical school, how often have you been bothered by the following? Trouble relaxing Since the start of medical school, how often have you been bothered by the following? Feeling afraid that something awful might happen Since the start of medical school, how often have you been bothered by the following? Worrying to the extent that I lost appetite or overate Sleep construct questions Since the start of medical school, how many hours of sleep do you get per night on average? + hours, –h, –h, or less than h Since the start of medical school, how often have you been bothered by the following? Trouble falling asleep or staying asleep, or sleeping too much Never, rarely, sometimes, often, or always Since the start of medical school, how often have you been bothered by the following? Data management and statistical analysis The data for the 4 survey years included data from some of the same individuals, but due to the deidentified voluntary nature of the survey- logitudinal analysis was not possible. Longitudinal analyses require 228 Ley et al.: Beyond burnout: a four-year survey 228 Table : (continued) Table : (continued) Since the start of medical school, how often have you been bothered by the following? Feel like I am achieving less than I should Since the start of medical school, how often have you been bothered by the following? Feeling an unpleasant level of pressure to succeed Since the start of medical school, how often have you been bothered by the following? Feel that there is more work to do than I can practically handle Since the start of medical school, how often have you been bothered by the following? Feel like I am in the wrong profession Since the start of medical school, how often have you been bothered by the following? Feel unappreciated or misunderstood Since the start of medical school, how often have you been bothered by the following? Feel like I am not getting what I want out of medical school Since the start of medical school, how often have you been bothered by the following? Feel like everyone else is problem-free except for me Table : Mean scores and their SEs for each student class and survey year. Table : Number of responses per survey year and year of medical school. students compared to first-year students. The effect size for the differences between the first- and second-year students in 2016 was d=0.43. In 2017, the difference between the year 1 and year 2 students corresponded to the effect size d=0.73. The difference between year 1 and 3 students corresponded to d=0.36 in 2018 and d=0.25 in 2019. Anxiety subscale The majority of students in our survey acknowledged anxi- ety symptoms (67.0–88.2%). Table : Mental health questions as subscale constructs. Not being able to control my worrying Since the start of medical school, how often have you been bothered by the following? Worrying too much about multiple things Since the start of medical school, how often have you been bothered by the following? Trouble relaxing Since the start of medical school, how often have you been bothered by the following? Feeling afraid that something awful might happen Since the start of medical school, how often have you been bothered by the following? Worrying to the extent that I lost appetite or overate Sleep construct questions Since the start of medical school, how many hours of sleep do you get per night on average? + hours, –h, –h, or less than h Since the start of medical school, how often have you been bothered by the following? Trouble falling asleep or staying asleep, or sleeping too much Never, rarely, sometimes, often, or always Since the start of medical school, how often have you been bothered by the following? Wake up feeling unrefreshed Since the start of medical school, how often have you done the following? Take anything (e.g., medication, alcohol, etc) to help sleep Burnout construct questions Since the start of medical school, how often have you been bothered by the following? Feeling disappointed in myself, feelings of failure, or I have let myself or my family down’ Never, rarely, sometimes, often, or always Since the start of medical school, how often have you been bothered by the following? Feeling like giving up or think “I can’t take it anymore” Since the start of medical school, how often have you been bothered by the following? Feeling like medical school is breaking me down Since the start of medical school, how often have you been bothered by the following? Feel physically or emotionally exhausted Since the start of medical school, how often have you been b h d b h f ll i ? Feel tired when I get up in the morning and have to face h d f h l Never, rarely, sometimes, often, or always Never, rarely, sometimes, often, or always Ley et al.: Beyond burnout: a four-year survey 229 Results for help seeking Most students (43.1–48.0%) who acknowledged experi- encing mental health symptoms sought help. Of those who did not seek help, the vast majority knew where to find mental health resources and recognized that their concerns would remain confidential. Students who chose not to seek Burnout subscale The effect size for the difference between year 1 and year 3 was d=0.33 in 2016. In 2017, the difference between year 1 and year 2 corresponded to d=0.41. 2016, first-year students had significantly lower burnout symptoms compared to other classes, but there was no significant difference between third- and fourth-year stu- dents that existed in 2016. In 2018 and 2019, the only sta- tistically significant pairwise difference was in lower burnout scores among first-year students compared to third-year students. A common finding from all four sur- veys is similar to that for depressive symptoms in that the burnout scores increased by the second or third years of school. The magnitude of difference between year 1 and year 2 corresponded to d=0.54 in 2016 and d=0.72 in 2017. The effect size for the comparisons of first-and third-year students was d=0.13 in 2018 and 2019. Burnout subscale The overall differences among student classes were statistically significant in 2016 (F[3, 906]=6.80, p=0.0002), in 2017 (F[3, 879]=9.67, p<0.0001), but not in 2018 (F[3, 542]=0.43, p=0.73) or 2019 (F[3, 602]=2.51, p=0.058). In 2016, first-year students had statistically significant lower mean scores compared to the other classes, with no differences among second-, third-, and fourth-year students. In 2017, first-year students had significantly lower mean scores compared to second- or third-year students. The difference between first- and fourth-year students in 2017 was not significant after Bonferroni adjustment (p=0.0281). Differences between year one and year two corresponded to d=0.33 in 2016, and d=0.44 in 2017. Many of our surveyed students also experienced burnout symptoms. Burnout symptoms were most elevated in years 2 and 3 in the 2016 and 2018 surveys, years 2–4 in the 2017 survey, and years 3–4 in the 2019 survey. For burnout, the overall differences among student classes were also sta- tistically significant in 2016 (F[3, 906]=24.41, p<0.0001), in 2017 (F[3, 879]=30.30, p<0.0001), in 2018 (F[3, 542]=3.60, p=0.0134), and in 2019 (F[3, 602]=3.23, p=0.0220). In 2016, first-year students had statistically significantly lower burnout symptoms compared to all other classes; in addi- tion, scores were significantly lower among fourth-year students compared to third-year students. In 2017, as in 230 Ley et al.: Beyond burnout: a four-year survey 230 Figure 1: Medical students’ reasons for not seeking help. Figure 1: Medical students’ reasons for not seeking help. were statistically significant in 2016 (F[3, 905]=3.42, p=0.0168) and in 2017 (F[3, 879]=11.53, p<0.0001), but not in 2018 (F[3, 542] =0.80, p=0.497) or 2019 (F[3, 602]=0.19, p=0.90). In the 2016 sur- vey, sleep problems were statistically significantly lower for first-year students compared to third-year students. In the 2017 survey, sleep problems were significantly lower for first-year students compared to all other classes. The effect size for the difference between year 1 and year 3 was d=0.33 in 2016. In 2017, the difference between year 1 and year 2 corresponded to d=0.41. were statistically significant in 2016 (F[3, 905]=3.42, p=0.0168) and in 2017 (F[3, 879]=11.53, p<0.0001), but not in 2018 (F[3, 542] =0.80, p=0.497) or 2019 (F[3, 602]=0.19, p=0.90). In the 2016 sur- vey, sleep problems were statistically significantly lower for first-year students compared to third-year students. In the 2017 survey, sleep problems were significantly lower for first-year students compared to all other classes. Discussion The rates of depression and suicidal ideation during medical training cannot be ignored or simply categorized as burnout. A 2015 meta-analysis indicated that depressive symptoms in resi- dent physicians ranged from 20.9 to 43.2%, and its prevalence increased with each calendar year by 0.5%, with the median absolute increase in depressive symptoms of 15.8% at the onset of residency training [19]. Another 2016 meta-analysis among medical students, including studies from 43 countries, revealed a13.5%increasein depressive symptoms during medicalschool with no significant difference between clinical and preclinical students. Unfortunately, the authors of this study found that only 15.7% of medical students who screened positive for depression sought treatment [20]. With such concerning data, theCOVID-19 pandemic revealed again that physicians are not immune to the exacerbation of psychiatric symptoms under stress [21]. In a 2020 meta-analysis of over 33,000 healthcare workers, a significant proportion of healthcare workers experienced anxiety (23.2%), depression (22.8%), and insomnia (38.9%)duringthe outbreak[22]. Discussion of burnoutwithout further investigation into mental health symptoms may foster missed opportunities to address treatable psychiatric condi- tions. Although acknowledging the existence of burnout in the medical profession has propelled efforts toward much- needed change, categorizing all mental health symptoms into burnout fails to delineate symptoms that may be a sign of a treatable medical condition. g Although there are growing collaborations aimed at enhancing interventions for mental wellness in the med- ical profession, there is no mandate for medical schools to report mental health information [26], making it difficult to evaluate trends over time. Unlike previous literature, reporting that only 15.7% of students who screened posi- tive for depression sought treatment [20], our study showed the unexpected result that 43.1–48.0% of students sought mental health assistance. And more importantly, the majority knew where and how to seek treatment (Figure 1). The MSUCOM MSMHC gathered each medical student class, twice per year, during a mandatory mental health awareness event and discussed mental health symptoms, and resources in the college, larger university, and community. Once available, the MSMHC also shared the survey results with the student body. We suspect the dissemination of information regarding services avail- able, and the normalization of seeking help allowed the students to access care at higher rates. The committee events helped destigmatize mental illness symptoms and establish a culture at MSUCOM that allowed more students who were suffering from mental health symptoms to know how and where to seek assistance. 231 help cited lack of time (35.0–43.3%) as the most common reason for failing to do so (Figure 1). help cited lack of time (35.0–43.3%) as the most common reason for failing to do so (Figure 1). Our findings also demonstrate that the majority of students acknowledge substantial anxiety symptoms (67.0–88.2%). Although anxiety and sleep problems were lower for first-year students in 2016 and 2017, this was not the case in the 2018 or 2019 surveys, which may be due to the overall highly elevated levels of anxiety for all classes, every year, making demonstration of change for the 2018 and 2019 survey difficult. Regardless, anxiety persists in all classes, warranting further examination. Perhaps students are pre- disposed to psychiatric conditions due to the rigors required in obtaining admission and/or due topersonalitycharacteristics allowing for academic success, which suggests that further examination of premedical student mental health is needed. Better characterization of baseline symptoms could help to elucidate when these elevated rates begin and whether clinical follow-up is needed. Future studies examining baseline levels of depression, burnout, anxiety, and sleep disturbance in pre- medical students may help to determine whether symptoms begin before or after matriculation. Sleep subscale The majority of students in each survey year averaged 6–8 h of sleep per night. The overall differences among student classes Ley et al.: Beyond burnout: a four-year survey Limitations Given the voluntary and anonymous nature of the survey, an expected decrease in participation over the course of 4 years was observed, which limits the generalizability of our findings due to the potential of selection bias among those who chose to participate in the study. We speculate that the 2016 survey was the most characteristic of the student body due to 2016 having the highest response rates in the initial mandatory survey. Even though the questionnaire was mandatory in 2016 and recommended in 2017, 2018, and 2019, there was no method to enforce participation. Our survey questions were generated specifically for this survey, and instruments were not estab- lished utilizing previously validated scales, making the results of this study difficult to compare to the results of other surveys. Also, to maintain confidentiality and anonymity, no identifying or demographic information was gathered, which did not permit student responses to be tracked and analyzed longitu- dinally. Therefore, all data had to be analyzed cross-sectionally. Annual administration of the survey also limited our ability to evaluate episodic and environmental fluctuations. Despite this limitation, the response rate was high, providing a large sam- ple size to capture significant trends throughout medical training. Further investigation that considers demographic information would help to determine the most vulnerable populations. Acknowledgments: The authors would like to thank everyone involved in the study: the students who chose to participate and the MSUCOM administration for supporting our endeavors to reduce stigma and improve the mental health of all medical students and the medical profession. Research funding: None reported. Author contributions: All authors provided substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; all authors drafted the article or revised it critically for important intellectual content; all authors contributed to the analysis and interpretation of data; all authors gave final approval of the version of the article to be published; and all authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Competing interests: None reported. Ethical approval: The 2016 study was granted exemption by the Michigan State University Institutional Review Board (IRB). The 2017–2019 was study reviewed and approved by the Michigan State University IRB (IRB number: x16-1558e; i053015). 1. Maslach C, Jackson SE. The measurement of experienced burnout. J Organ Behav 1981;2:99–113. and burnout by the second or third year of medical school. Anxiety and sleep disturbance are observed every year with a statistically significant increase in symptoms with progression in medical school confirming persistently high levels of anxiety throughout medical school. Future explo- ration regarding of the rates of burnout, depression, and anxiety prior to medical school would be helpful in deter- mining baseline vulnerability prior to medical training. Based on our study, supportive and time-sensitive interventions are indicated during the first year of medical school because anxiety may be elevated at the beginning of medical training, whereas depressive symptoms and burnout appear early and worsen throughout training. With the increased stressors of the pandemic, increasing administrative load, less time with patients, fear of litigation, variable electronic medical records, and overwhelming medical school debt, students are at higher risk than ever. The next step is moving beyond burnout. Implementation of measures that identify psychi- atricsymptoms instruggling medicalstudents will prove most beneficial for both the students and their future patients. allowed students the time to attend the events and learn about resources, but this did not translate into all medical students with symptoms seeking help. Further studies are warranted to better understand time constraints as a barrier to seeking mental health services. Although prior studies supported the idea that medical training is a time of increased risk, few pointed to a specific time period and replicability from year to year. Our study demonstrates that symptoms of burnout and depression increase between the first and third years of medical training and, more importantly, remain elevated in the fourth year.Ashiftinfocusfromawareness tointerventioninmedical training may identify those at risk for suicide and help all medical students to develop the skills needed to navigate the challenges of a career in medicine, which would prove most beneficial for the medical community and public. Limitations Informed consent: All participants in this study provided written informed consent prior to participation, and participation in the survey was fully voluntary on this basis. Discussion We suspect that the increased knowledge of mental health symptoms, and when and how to seek treatment, impacted the rate of seeking care. Our findings are consistent with the litera- ture indicating that US medical students demonstrate more depressive and burnout symptoms than their nonmedical student peers [11, 12]. Of the students who had mental health symptoms on the survey and did not seek help, the main reason cited for not seeking help was a lack of time. We believe that the mandatory nature of the event This study supports the growing body of evidence that medical students are at increased risk for depression and burnout and emphasizes that risks begin early in medical school by the second or third year. Many of the effect sizes in our study corresponding to the increases were above 0.5 (one half of the standard deviation), which is the threshold for clinical significance in patient-reported outcomes [23], and most exceeded 0.33 (a third of the standard deviation), another cutoffproposed in the literature [24] noting its sig- nificance. We hypothesize that student distress increases with the increasing demands of medical education leading to burnout and a more worrisome potential exacerbation of underlying psychiatric symptoms. Although the medical profession is raising awareness of substantial rates of burnout and depression [25], there is room for immense improvement to identify depressive symptoms in medical students and to prevent clinically tragic outcomes such as suicide. Our study highlights the need to assess for symptoms and implement interventions early in training. Ley et al.: Beyond burnout: a four-year survey 232 Ley et al.: Beyond burnout: a four-year survey 233 2. Brazeau CMLR, Schroeder R, Rovi S, Boyd L. Relationships between medical student burnout, empathy, and professionalism climate. Acad Med 2010;85:S33–6. 16. Beck AT, Steer RA. Manual for the beck anxiety inventory. San Antonio, TX: The Psychological Corporation; 1993. 17. Fairburn CG, Beglin SJ. Eating disorder examination questionnaire. PsycTESTS Dataset 1994. https://doi.org/10.1037/t03974-000. 3. Seo C, Di Carlo C, Dong SX, Fournier K, Haykal KA. Risk factors for suicidal ideation and suicide attempt among medical students: a meta- analysis. PLoS One 2021;16:e0261785. 18. SAS 9.4 copyright © 2013. Cary, NC, USA: SAS Institute Inc; 2013. 19. Rotenstein LS, Ramos MA, Torre M, Segal JB, Peluso MJ, Guille C, et al. Prevalence of depression, depressive symptoms, and suicidal ideation among medical students: a systematic review and meta-analysis. JAMA 2016;316:2214–36. 4. Koutsimani P, Montgomery A, Georganta K. The relationship between burnout, depression, and anxiety: a systematic review and meta- analysis. Front Psychol 2019;10:284. 20. Mata DA, Ramos MA, Bansal N, Khan R, Guille C, di Angelantonio E, et al. Prevalence of depression and depressive symptoms among resident physicians: a systematic review and meta-analysis. JAMA 2015;314: 2373–83. 5. American Psychiatric Association. Diagnostic and statistical manual of mental disorders 5. Washington D.C: American Psychiatric Association Press; 2013. 6. Sher L. Resilience as a focus of suicide research and prevention. Acta Psychiatr Scand 2019;140:169–80. 21. Salazar de Pablo G, Vaquerizo-Serrano J, Catalan A, Arango C, Moreno C, Ferre F, et al. Impact of coronavirus syndromes on physical and mental health of health care workers: systematic review and meta- analysis. J Affect Disord 2020;275:48–57. 7. Underlying cause of death 1999–2020: Centers for Disease Control and Prevention; 2020. Available from: https://wonder.cdc.gov/wonder/ help/ucd.html#Data%20Request [Accessed 18 Dec 2022]. 8. Ye GY, Davidson JE, Kim K, Zisook S. Physician death by suicide in the United States: 2012–2016. J Psychiatr Res 2021;134:158–65. 22. Pappa S, Ntella V, Giannakas T, Giannakoulis VG, Papoutsi E, Katsaounou P. Prevalence of depression, anxiety, and insomnia among healthcare workers during the COVID-19 pandemic: a systematic review and meta-analysis. Brain Behav Immun 2020;88: 901–7. 9. Schernhammer ES, Colditz GA. Suicide rates among physicians: a quantitative and gender assessment (meta-analysis). Am J Psychiatry 2004;161:2295–302. 10. Elkbuli A, Sutherland M, Shepherd A, Kinslow K, Liu H, Ang D, et al. Factors influencing us physician and surgeon suicide rates 2003 to 2017. Ann Surg 2020;276:370–6. 23. Norman GR, Sloan JA, Wyrwich KW. Supplementary Material: This article contains supplementary material (https://doi.org/10.1515/jom-2022-0179). References Many students endorse symptoms of depression, anxiety, and burnout throughout medical school. Our study dem- onstrates significant increases in symptoms of depression Interpretation of changes in health- related quality of life: the remarkable universality of half a standard deviation. Med Care 2003;41:582–92. 24. Sloan JA, Cella D, Hays RD. Clinical significance of patient-reported questionnaire data: another step toward consensus. J Clin Epidemiol 2005;58:1217–9. 11. Dyrbye LN, West CP, Satele D, Boone S, Tan L, Sloan J, et al. Burnout among U.S. medical students, residents, and early career physicians relative to the general U.S. population. Acad Med 2014;89:443–51. 12. Elkins C, Plante KP, Germain LJ, Morley CP. Burnout and depression in MS1 and MS3 Years: a comparison of cohorts at one medical school. Fam Med 2017;49:456–9. 25. Summers RF, Gorrindo T, Hwang S, Aggarwal R, Guille C. Well-being, burnout, and depression among North American psychiatrists: the state of our profession. Am J Psychiatr 2020;177:955–64. 13. Gold KJ, Sen A, Schwenk TL. Details on suicide among US physicians: data from the national violent death reporting system. Gen Hosp Psychiatr 2013;35:45–9. 26. Resources. Resources – alliance for academic internal medicine. Available from: https://www.im.org/resoures/wellness-resiliency [Accessed 18 Dec 2022]. 14. Maslach C, Jackson SE, Leiter MP. Maslach burnout inventory manual, 3rd ed. Palo Alto, CA: Consulting Psychologists Press; 1996. 15. Beck AT, Steer RA, Brown GK. Manual for the beck depression inventory-II. San Antonio, TX: The Psychological Corporation; 1996. 15. Beck AT, Steer RA, Brown GK. Manual for the beck depression inventory-II. San Antonio, TX: The Psychological Corporation; 1996.
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A systematic review of PTSD to the experience of Psychosis: Prevalence and associated factors
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© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Open Access Abstract Background: Psychosis can be a sufficiently traumatic event to lead to post-traumatic stress disorder (PTSD). Previous research has focussed on the trauma of first episode psychosis (FEP) and the only review to date of PTSD beyond the first episode period was not systematic and is potentially outdated. Methods: We searched electronic databases and reference lists using predetermined inclusion criteria to retrieve studies that reported prevalence rates and associated factors of psychosis-related PTSD across all stages of the course of psychosis. Studies were included if they measured PTSD specifically related to the experience of psychosis. Risk of bias was assessed using an adapted version of the Newcastle Ottawa Scale. Results were synthesised narratively. Results: Six papers met inclusion criteria. Prevalence estimates of psychosis-related PTSD varied from 14 to 47%. Studies either assessed first-episode samples or did not specify the number of episodes experienced. Depression was consistently associated with psychosis-related PTSD. Other potential associations included treatment-related factors, psychosis severity, childhood trauma, and individual psychosocial reactions to trauma. Conclusions: Psychosis-related PTSD is a common problem in people with psychosis. There is a lack of published research on this beyond first episode psychosis. Further research is needed on larger, more generalizable samples. Our results tentatively suggest that prevalence rates of psychosis-related PTSD have not reduced over the past decade despite ambitions to provide trauma-informed care. Prospero registration number: CRD42019138750. Keywords: Psychosis, Trauma, PTSD, Prevalence Keywords: Psychosis, Trauma, PTSD, Prevalence restraint and forced medication, can be sufficiently trau- matic to lead to the development of post-traumatic stress disorder (PTSD) [3, 4]. A systematic review of PTSD to the experience of psychosis: prevalence and associated factors Georgina Buswell, Zoë Haime, Brynmor Lloyd-Evans and Jo Billings* Georgina Buswell, Zoë Haime, Brynmor Lloyd-Evans and Jo Billings* Background Psychosis affects approximately 3% of the general popula- tion [1] with the two most common symptoms being hal- lucinations (perceptions occurring in the absence of external stimuli, such as hearing voices that other people cannot hear) and delusions (fixed, false beliefs) [2]. The experience of psychotic symptoms such as distressing hal- lucinations or persecutory delusions, and associated treat- ment responses, including involuntary hospitalisation, Psychosis-related PTSD can be difficult to detect. Many symptoms of psychosis and PTSD share similar- ities [5] and people with psychosis are often not assessed for trauma disorders [6]. Undiagnosed PTSD prevents access to appropriate treatment, impeding recovery from psychosis or leading to additional co-morbidities such as anxiety and depression, substance abuse and suicidality [7, 8]. There may be reciprocal effects between psychosis * Correspondence: j.billings@ucl.ac.uk University College London, London, England * Correspondence: j.billings@ucl.ac.uk University College London, London, England * Correspondence: j.billings@ucl.ac.uk University College London, London, England Buswell et al. BMC Psychiatry (2021) 21:9 https://doi.org/10.1186/s12888-020-02999-x Buswell et al. BMC Psychiatry (2021) 21:9 https://doi.org/10.1186/s12888-020-02999-x Research into risk factors for PTSD generally have found PTSD to be most prevalent amongst those who have previous trauma histories, who experienced intense emotional reactions and dissociation during the trauma, and who lacked social support afterwards [12, 13]. To date, little research has explored whether risk factors are similar when the traumatic event is psychosis. So far, research into psychosis-related PTSD has been synthesised in two reviews [14, 15]. Berry et al. (2013) [14] reviewed 24 studies published up until 2011 and reported prevalence rates of psychosis-related PTSD varying from 11 to 67%. Some evidence suggested the following factors were associated with the development of psychosis-related PTSD: trauma history, psychosis severity, affective symp- toms (e.g. depression), particular treatment experiences and psychological variables such as appraisals and coping style. However, evidence for these factors was generally weak and inconsistent between studies. y y ( ) The more recent review by Rodrigues and Anderson (2017) [15] systematically searched for papers that re- ported prevalence rates and associated factors in first- episode psychosis (FEP) samples only. Of their 13 in- cluded studies, 8 reported rates of clinically relevant PTSD symptoms, with a pooled prevalence of 42%. Four of the 13 included studies reported rates of participants meeting PTSD diagnostic criteria; the pooled prevalence of PTSD diagnosis was 30%. Anxiety and depression were identified as potential risk factors but again the evidence for this was generally weak. The reported prevalence rates from both previous reviews vary widely (11–67%); arguments for such variation have focussed on methodological inconsistencies in assessing psychosis-related PTSD such as measurement tools used and definitions of psychosis-related PTSD [20]. In their implications for future research, Berry et al. (2013) [14] highlighted that studies using sensitive mea- sures of trauma were required, to support the investiga- tion of associations between past traumas and psychosis- related trauma. They argued that it was important for fu- ture research to separate out symptom-related and treatment-related PTSD as these are conceptually differ- ent. They also recognised that psychological processes, such as contextual integration, should be investigated as these form part of models of PTSD. Regarding clinical im- plications of their review, Berry et al. (2013) [14] suggested that rates of psychosis-related PTSD may be reduced by improving hospitalisation experiences: noting the much lower rates of psychosis-related PTSD in an inpatient unit that adopted a person-centred, therapeutic model. Page 2 of 13 Page 2 of 13 Buswell et al. BMC Psychiatry (2021) 21:9 Buswell et al. BMC Psychiatry (2021) 21:9 informed care (TIC) across mental health settings [16]. TIC involves the recognition of trauma histories and the impact these have on patients, the prevention of poten- tially traumatic care practices, the provision of care envi- ronments that feel safe, both physically and psychologically, and clinicians working collaboratively with patients, empowering and respecting them. Patients should also have access to trauma treatment where ap- propriate [17]. Well delivered TIC may in future lead to reductions of rates of PTSD (particularly those related to treatment experiences) which should be reflected in re- search studies, although could conversely lead to an in- crease in reported rates in clinical practice due to improvements in case identification. Staff training pro- grammes in providing TIC are still being developed and evaluated [18, 19] and it is likely that evaluations of their efficacy will be complex to measure. Reductions in fund- ing of mental health services over the past decade could present challenges to the advancement of TIC and it is not clear if TIC is being delivered in services. However, due to the increasing recognition of trauma in both re- search and clinical settings it is possible there has been an acceleration in trauma research since Berry et al. (2013) [14] conducted their final search in 2011. An update of the review by Berry et al. (2013) [14] is therefore needed. and PTSD due to an overlap in symptoms; untreated PTSD can potentially exacerbate positive symptoms of psychosis. Research has suggested that those with co- morbid PTSD and psychosis have more severe positive symptoms, independent of other factors [9]. The first episode of psychosis has been described as particularly traumatic due to its novelty [10]. However, PTSD from psychosis might be more prevalent amongst those who have had multiple episodes, if they were sen- sitised by their earlier episodes (in line with trauma sensitization theory [11],). Those who have experienced multiple episodes of psychosis, and therefore potentially more traumatic experiences, might be at increased risk of developing PTSD compared to those who have expe- rienced one episode. How- ever, the results of this review need to be interpreted with caution as their review methodology was not systematic, therefore they may have missed important research which presented alternative perspectives. Method We registered our protocol on PROSPERO on 24th June 2019 (registration number CRD42019138750). We followed PRISMA (Preferred Reporting Items for Sys- tematic Reviews and Meta-Analyses) guidelines for reporting systematic reviews. Study selection Two reviewers (GB and ZH) independently screened 10% of the retrieved titles and abstracts against the in- clusion criteria. The reviewers discussed their chosen pa- pers for inclusion and GB then screened the remaining titles and abstracts for further eligible papers. Both re- viewers independently screened 100% of full texts that were identified as potentially eligible. GB and ZH shared their results and resolved discrepancies through discus- sion and consultation with reviewers JB and BLE. Definition of key terms and concepts We included studies with any definition of PTSD as long as it was related to the experience of psychosis (e.g. psychosis-related PTSD, post-psychosis PTSD). We in- cluded participants who met clinical diagnoses of PTSD (not restricted by diagnostic classification) and/or who scored above clinically relevant cut-offs on validated measures of PTSD. We included studies that had partici- pants with all types of psychosis as diagnosed according to either the DSM-IV, DSM-V or ICD-10 classifications. Inclusion criteria Studies satisfying the following criteria were included in this systematic review: (1) participants had experienced at least one episode of any type of psychosis; (2) esti- mated the prevalence and associates of psychosis-related trauma symptoms or PTSD diagnosis; (3) were published into journal articles. All types of study design were in- cluded. Studies that reported prevalence of PTSD not specific to psychosis were excluded, as were grey litera- ture including doctoral theses and conference abstracts. Data extraction A data extraction tool was adapted from one used by Ro- drigues and Anderson (2017) [15] and included character- istics of the studies and the main findings. GB and ZH extracted data independently to ensure accuracy and reli- ability of extraction. A third extraction table was created to record the related factors that each study had assessed. Study quality Th l y q y The quality of studies was evaluated using an adapted version of the Newcastle-Ottawa Scale (NOS) [21], which was utilised in a similar review. The NOS assesses study quality based on sample selection, comparability between groups and outcome assessment [15]. GB and ZH independently developed adapted versions then agreed on the final version; items relating to follow-up were removed as they were not applicable to the in- cluded studies. Higher scores indicated greater quality. Studies were judged as low, medium or high quality if their score was between 1 and 4, 5–8, and 9–12 respect- ively. The highest score for representativeness could be awarded if the sample was large and included people with psychosis from different settings (e.g. site or service type) and the lowest if there was no description of set- ting. Studies were judged to have a higher score if their participants had a clinical diagnosis of the exposure (psychosis) and the assessment of outcome (PTSD re- lated to psychosis) was through either a clinical inter- view or relevant self-report measure. Studies were judged as higher quality if they described individuals who declined participation. Whilst confounders are not relevant to the assessment of prevalence, we were also interested in factors that might be associated with psychosis-related PTSD, for which confounding does Our review questions were: Our review questions were: We used forward and backward citation searching and manual searching of the reference lists of key papers using Google Scholar. Where our search returned con- ference abstracts and doctoral theses, we contacted au- thors to see whether the research had subsequently been published as journal articles. A librarian was contacted to request an English translation of a non-English paper that was retrieved. 1 What is the prevalence of psychosis-related PTSD in people with psychosis (who have experienced any number of episodes)? 2 What factors are associated with the development of psychosis-related PTSD? p y 3 Do the prevalence rates and/or associated factors differ between first-episode samples and people who have experienced multiple episodes? Aim The aim of this systematic review was to review the lit- erature on prevalence rates and associated factors of psychosis-related PTSD in people who have experienced one or more episode of psychosis. We reviewed litera- ture from 2011 in order to update the earlier review by Berry et al. (2013) [14] and consider the extent and drivers of psychosis-related PTSD in a modern health- care context. The current review adopted a systematic methodology similar to that used by Rodrigues and An- derson (2017) [15] but was not limited to first-episode psychosis. Following the Berry et al. (2013) [14] review there has been an increasing interest in the provision of trauma- Page 3 of 13 Buswell et al. BMC Psychiatry (2021) 21:9 Stage 1: development of theory This stage was performed early in the review process and helped to determine the current theories surround- ing psychosis-related PTSD, identifying where further in- vestigation was needed, and thus shaping the review question. Prominent theories included those of sensitisa- tion and re-traumatisation which informed the review focus on prevalence rates in samples of people with first or multiple episodes of psychosis. Stage 4: evaluating the robustness of the synthesis Stage 4: evaluating the robustness of the synthesis The methodological quality of included studies and of the review process was examined to assess the strength of the evidence provided by the review. Considerations were made of the generalisability of the results to the wider population. Study selection Figure 1 illustrates our search strategy results (see Fig. 1). After 10% of titles and abstracts had been screened inde- pendently by reviewers, assessments of eligibility were compared and there was a disagreement between re- viewers of one paper (1/5) which was discussed and then rejected. Of the 20 full-texts subsequently screened the reviewers disagreed on two (2/20). One of these was re- solved by discussion and the other after consultation with a third and fourth reviewer; both were rejected. Seven papers were assessed as eligible; however, one paper was excluded during data extraction as the PTSD prevalence included cases of PTSD from non-psychosis- related events. This left six eligible papers remaining for inclusion. A narrative approach informed by the guidance by Popay et al. (2006) [22] was used to synthesise the study findings in four stages: (1) developing a theory, (2) devel- oping a preliminary synthesis, (3) exploring relationships within and between studies, and (4) assessing the robust- ness of the synthesis. Search strategy The electronic search was conducted on the databases MEDLINE-Ovid, EMBASE-Ovid, PsycINFO-Ovid, Web of Science and the Published International Literature on Traumatic Stress (PILOTS). The search strategy for MEDLINE was adapted from previous reviews [14, 15], further refined following consultation with a medical li- brarian, then adapted accordingly for the other data- bases. Search terms were related to the concepts of ‘psychosis’ and ‘trauma’. The search was restricted to English-language journal articles published 2011 on- wards, after the final search of a previous review [14]. See Additional file 1 for search strategy. Buswell et al. BMC Psychiatry (2021) 21:9 Buswell et al. BMC Psychiatry (2021) 21:9 Page 4 of 13 Page 4 of 13 Page 4 of 13 Stage 3: exploring the relationships within and between studies need to be considered. Therefore, studies were judged as higher quality if they adjusted for confounders. Plausible confounders were co-morbid psychopathology and PTSD not related to psychosis. The quality assessment was completed independently by GB and ZH and any discrepancies were resolved by discussion. Quality as- sessment was not used as a tool to exclude any studies from analysis but instead to aid a critical review of the evidence. need to be considered. Therefore, studies were judged as higher quality if they adjusted for confounders. Plausible confounders were co-morbid psychopathology and PTSD not related to psychosis. The quality assessment was completed independently by GB and ZH and any discrepancies were resolved by discussion. Quality as- sessment was not used as a tool to exclude any studies from analysis but instead to aid a critical review of the evidence. Stage three involved exploring relationships between studies on their key findings and methodological, clin- ical, and theoretical differences. To synthesise prevalence rates studies were grouped by PTSD outcome measure. The synthesis of related factors was separated into a) factors that were the primary focus of studies and the theoretical bases for these and b) secondary factors. Study characteristics We included six studies with a total of 332 participants. Sample sizes ranged from 34 [23] to 110 [24]. All studies (n = 6) used cross-sectional study designs. See Table 1 for characteristics of included studies (Table 1). Four studies were based in the UK [23, 24, 27, 28], one in Australia [26], and one in Tunisia [25]. Half (n = 3) were in early intervention in psychosis (EIP) services [23, 26, 28]. EIP services typically treat people who have experienced, or are experiencing, their first episode of psychosis. However, the length of time since onset was not reported in these studies, and the characteristics of the samples suggest they may vary considerably: in one study participants could have been recruited as soon as they were seen by early intervention services [26] and therefore could be currently experiencing their first episode, whereas in another study the participants were all in remission from their first epi- sode [28]. A third study [23] stated that people who had experienced more than one episode could be included and Psychological and cognitive theories described how the processing of trauma can be influenced by various indi- vidual and social factors. With psychosis-related PTSD there are two potentially different traumatic exposures: psychosis symptoms and treatment experiences. This guided our focus on associated factors as a way of ex- ploring potential mechanisms underlying psychosis- related PTSD. Stage 2: development of the preliminary synthesis This stage involved organising and describing the in- cluded studies to be able to search for patterns across studies. Data were extracted and presented in tables. Synthesis of results We planned a priori to meta-analyse where outcomes and populations from more than three studies were suf- ficiently homogeneous. However, of the four studies that used the same outcome measure (the Impact of Events Scale Revised; IES-R) [30], the samples were small in size, had large confidence intervals and were from mark- edly different settings (patients on secure wards, com- pared to outpatients in early psychosis services in the other studies). Expert advice was sought from a statisti- cian who supported our decision not to combine these studies in a meta-analysis due to heterogeneity. FEP First episode of psychosis, DSM-IV-TR Diagnostic Statistical Manual of Mental Disorders Fourth Edition, ICD-10 International Classif Edition, CBT Cognitive Behaviour Therapy, SUD Substance Use Disorder, EI Early intervention Findings: prevalence Table 3 outlines key findings of prevalence rates (see Table 3). Prevalence rates ranged from 14 to 47% for full PTSD. One study did not report an overall prevalence figure for PTSD and instead reported separate PTSD rates for individual psychosis and treatment-related ex- periences [24]. Five out of six studies were judged as medium quality and one was low (Table 2). None were judged to be truly representative of people with psychosis due to the rela- tively small and restricted samples. One study did not clearly describe how participants were recruited or from what setting [24]. Two studies utilised samples that were unlikely to be generalizable to the rest of the population (patients on secure wards, 25; patients with substance use disorders, 24). The ascertainment of exposure and outcome was generally good across studies. The studies were generally poor at describing characteristics of non- There were variations in how the six papers concep- tualised psychosis-related PTSD: studies differed in whether they separated or combined symptoms and treatments, isolated different symptoms or not, and if they reported ‘partial PTSD’ for sub-clinical levels as well as ‘full PTSD’. These differences necessitate that caution is taken in grouping together the prevalence Table 2 Quality assessment of included studies based on an adapted version of the Newcastle-Ottawa scale Quality domain Quality criteria Abdelghaffar et al. (2018) [25] Bendall et al. (2012) [26] Berry et al. (2015) [27] Picken & Tarrier (2011) [24] Pietruch & Jobson (2012) [23] Turner et al. (2013) [28] 1. Representativeness of sample Truly representative (2) 1 1 1 0 1 1 Somewhat representative (1) No description of derivation of sample (0) 2. Ascertainment of exposure Patient notes (2) 2 2 2 2a 0 2 Clinical interview (2) Self-report (1) No description (0) 3. Same method of ascertainment for entire sample Yes (2) 2 2 2 2 0 2 No (0) 4. Non-participation rate High rate, described (2) 0 2 0 0 0 0 Low rate, described (2) All participants asked took part (2) Non-participants not described (0) 5. Assessment of outcome Questionnaire (2) 2 2 2 2 2 2 Clinical interview (2) Self-report or patient notes (1) No description (0) 6. Confounders Confounders described and adjusted for (2) 1 1 0 1 1 0 Confounders described (1) No description (0) TOTAL 7 Medium 8 Medium 6 Medium 6 Medium 4 Low 6 Medium Higher scores reflect superior quality. Stage 2: development of the preliminary synthesis Buswell et al. BMC Psychiatry (2021) 21:9 Page 5 of 13 Buswell et al. BMC Psychiatry Fig. 1 PRISMA Flowchart Fig. 1 PRISMA Flowchart therefore participants beyond first episode might have been grouped together with first episode participants. started [25]. One was in secure wards with current inpa- tients [27] where it was likely the participants were mixed in terms of numbers of episodes and length since onset. One study utilised a sample of outpatients participating in a clinical trial [24], where all participants had co-morbid substance use disorder. Overall, the number of episodes One study was based in the outpatient department of a psychiatric hospital, where participants were within 2 years of admission for FEP but it was not clear how many episodes they had experienced or when the first episode Table 1 Characteristics of included studies Study Study location Study design n Source of sample Mean age (SD) Male (%) Diagnostic criteria for psychosis Stage of psychosis 1 Abdelghaffar et al. (2018) [25] Tunisia Cross-sectional 52 Outpatients of 1 psychiatric hospital 27.6 (5.6) 51.9% Not specified Within 2 years of inpatient admission for FEP 2 Bendall et al. (2012) [26] Australia Cross-sectional 36 Outpatients of 1 early psychosis service 21.42 (3.43) 61% DSM-IV-TR Within 18 months of first treatment for FEP 3 Berry et al. (2015) [27] UK Cross-sectional 50 Secure wards 37.66 (11.16) 80% ICD-10 (F20, F22, F23, F25) Inpatients 1 > month 4 Picken & Tarrier (2011) [24] UK Cross-sectional 110 Clinical trial for CBT for SUD in psychosis 38 (10) 90% DSM-IV-TR Not specified 5 Pietruch & Jobson (2012) [23] UK Cross-sectional 34 1 EI service 25.67 (6.04) 64.7% Not specified Have experienced 1+ episode in last 3 years 6 Turner et al. (2013) [28] UK Cross-sectional 50 1 EI service 24.5 (−) Not specified ICD-10 (F20, F22, F23, F25) FEP in remission FEP First episode of psychosis, DSM-IV-TR Diagnostic Statistical Manual of Mental Disorders Fourth Edition, ICD-10 International Classification of Diseases 10th Edition, CBT Cognitive Behaviour Therapy, SUD Substance Use Disorder, EI Early intervention Page 6 of 13 Buswell et al. BMC Psychiatry (2021) 21:9 Buswell et al. BMC Psychiatry experienced and the length of time since onset varied across samples and was often not made clear. participants. Stage 2: development of the preliminary synthesis We expected plausible confounders to be co-morbid psychopathology and PTSD not related to psychosis, however no studies clearly described and ad- justed for these; one mentioned adjusted analyses but did not report these results [28]. Quality assessment After independently scoring the adapted NOS the re- viewers agreed on 29/36 (86%) of items for all studies. Disagreements were resolved by returning to the papers and further discussion. See Table 2 for results of quality assessment (see Table 2). Findings: prevalence bPartial symptoms: above cut-off scores for subscale of re-experiencing plus either avoidance or hyperarousal, as proposed by Asukai et al. (2002) [33] rates across the different studies as the reported rates might not all refer to the same concept. IES-R is designed as a self-report measure, but in two studies [26, 28] it was completed with the researcher. The variation in delivery of the IES-R between studies might have influenced participants to report different levels of PTSD. The measurement of psychosis-related PTSD also var- ied. The majority (n = 4) of studies used the IES-R [23, 26–28, 30]. The IES-R is a 22-item self-report measure that assesses post-traumatic stress symptoms following an event and consists of three subscales measuring the three domains of PTSD according to the DSM-IV (intru- sions, avoidance and hyperarousal). Note, the DSM-V added a fourth domain (negative alterations in cogni- tions and mood), which is not captured by the IES-R. Item examples include “I was jumpy and easily startled” and “I tried not to think about it”. Respondents are asked to rate each item on a 5-point scale from 0 (not at all) to 4 (extremely). Higher scores indicate higher symp- toms of trauma [30]. The four studies that used this measure all utilised a cut-off score of 33 to determine ‘case-ness’ of PTSD, as proposed by Creamer et al. (2003) [32], and reported the percentage of their sample that scored above the cut-off as the prevalence. Three of the four also reported the average scores for the sample; variation in scores within samples was high. One study [24] used the Post-traumatic Diagnostic Scale (PDS) [31], a self-report measure that mirrors the DSM-IV diagnostic criteria for PTSD. Participants iden- tified their most distressing experience and then an- swered questions to determine whether it met criterion A for a PTSD diagnosis (i.e. it involved threat and dan- ger and invoked negative emotional responses). The per- centages of participants who met PTSD criteria for individual psychosis-related events were: delusions (14%), hallucinations (16%), traumatic treatment (3%) and involuntary admission (21%). These individual per- centages could not be compared to the total PTSD prevalence rates in the other studies. The remaining study [25] employed the Clinician- Administered PTSD Scale (CAPS; [29]) to ascertain prevalence in their sample. The CAPS is a semi- structured clinician-administered interview designed to assess PTSD as defined by the DSM-IV. Findings: prevalence Scores 1–4 = low, 5–8 = medium, 9–12 = high aA researcher conducted the clinical interview, not a clinician Table 2 Quality assessment of included studies based on an adapted version of the Newcastle-Ottawa scale Page 7 of 13 Buswell et al. BMC Psychiatry (2021) 21:9 Table 3 Key findings of prevalence of psychosis-related PTSD from included studies Study Trauma measure used Criteria for psychosis-related PTSD Treatment & symptoms separated? Key findings of prevalence Full PTSD for all causes Full PTSD related to symptoms Full PTSD related to treatment 1 Abdelghaffar et al. (2018) [25] CAPS ‘Full’ PTSD: Fulfils criteria A, B, C, D Yes + combined 42.3% 23.1% 19.2% ‘PTSD Syndrome’: Fulfils criteria B, C, D 69.2% 2 Bendall et al. (2012) [26] IES-R Exceeds clinical cut-off scorea No 47% - - 3 Berry et al. (2015) [27] IES-R Exceeds clinical cut-off scorea Yes 30% 24% 18% 4 Picken & Tarrier (2011) [24] PDS ‘Modified-Full’ PTSD: A, B, C, D Yes - 14% delusions 16% hallucinations 21% involuntary admission 3% traumatic treatment 5 Pietruch & Jobson (2012) [23] IES-R Exceeds clinical cut-off scorea No 41.18% - - 6 Turner et al. (2013) [28] IES-R Exceeds clinical cut-off scorea No 14% - - Exceeds cut-off for partial symptomsb 24% CAPS Clinician-Administered PTSD Scale [29], IES-R Impact of Events Scale–Revised [30], PDS Posttraumatic Stress Diagnostic Scale [31], aCut-off score of 33 on IES- R recognised as having diagnostic accuracy for PTSD [32]. bPartial symptoms: above cut-off scores for subscale of re-experiencing plus either avoidance or hyperarousal, as proposed by Asukai et al. (2002) [33] CAPS Clinician-Administered PTSD Scale [29], IES-R Impact of Events Scale–Revised [30], PDS Posttraumatic Stress Diagnostic Scale [31], aCut-off score of 33 on IES- R recognised as having diagnostic accuracy for PTSD [32]. bPartial symptoms: above cut-off scores for subscale of re-experiencing plus either avoidance or hyperarousal as proposed by Asukai et al (2002) [33] TSD Scale [29], IES-R Impact of Events Scale–Revised [30], PDS Posttraumatic Stress Diagnostic Scale [31], aCut-off score of 33 on IES- ostic accuracy for PTSD [32]. bPartial symptoms: above cut-off scores for subscale of re-experiencing plus either avoidance or A k i t l (2002) [33] CAPS Clinician-Administered PTSD Scale [29], IES-R Impact of Events Scale–Revised [30], PDS Posttraumatic Stress Diagnostic Scale [31], aCut-off score of 33 on IES- R recognised as having diagnostic accuracy for PTSD [32]. Secondary factors Depression was the only factor found to be associated in all studies that assessed it [24, 25, 28]. Secondary factors that were found to be associated in at least one study were symptom-related (severity of psychosis, positive symptoms, and general psychopathology), treatment- related (restraint, length of admission, number of trau- matic hospital events), depression and maladaptive coping. Findings: prevalence Participants were asked to consider the experience of psychosis symptoms and of treatment separately; the prevalence rates were 23.1 and 19.2% respectively, so 42.3% of their sample met full PTSD criteria for a psychosis-related event. No participants met full PTSD criteria for both symptoms and treatment. The overall prevalence rate is comparable to those reported by studies using the IES-R The administration of the IES-R differed across the studies. Studies requested participants answered the IES- R in relation to acute psychosis [26], the most recent episode [23] or the most traumatic experience that oc- curred in relation to their mental illness [28]. One asked participants to complete the measure twice: once in rela- tion to most distressing psychotic symptom and once in relation to most distressing hospital experience [27]. The Page 8 of 13 Page 8 of 13 Page 8 of 13 Buswell et al. BMC Psychiatry (2021) 21:9 Buswell et al. BMC Psychiatry (2021) 21:9 Buswell et al. BMC Psychiatry with the exception of the considerably lower rate re- ported by Turner et al. (2013) [28]. with the exception of the considerably lower rate re- ported by Turner et al. (2013) [28]. Findings: associated factors Four studies hypothesised that psychosis-related PTSD would be associated with a key factor, based on an underlying theory. All studies also tested for associations between secondary factors and psychosis-related PTSD. See Table 4 for a summary of all factors. There was inconsistent evidence between studies on psychosis severity and trauma history. Psychosis severity was measured by the Positive and Negative Syndrome Scale (PANSS) [35] in four studies. Berry et al. (2015) [27] and Picken and Tarrier (2011) [24] found that the PANSS total score and subtotals for positive symptoms and general psychopathology were all associated with psychosis-related PTSD. Abdelghaffar et al. (2018) [25] and Bendall et al. (2012) [26] both only looked at the PANSS total score and reported no association. Key factors The key factors examined were childhood trauma [26], attachment style [30], disclosure of trauma [23] and feel- ings of shame [28]. Bendall et al. (2012) [26] hypothe- sised that childhood trauma was a moderator between psychosis and psychosis-related PTSD. This was based on the theory that early traumas can sensitize individuals to develop PTSD after a later trauma, in this case psych- osis. Their results supported both hypothesis and theory as they found that experiencing childhood trauma increased the risk of psychosis-related PTSD by 27 times (p = 0.01, 95% CI: 2.96–253.80). Childhood trauma- related PTSD also increased risk (OR 20.40; 95% CI 3.38–123.25, p = 0.01; r2 = 0.45). Childhood trauma was associated in one study [26] but lifetime trauma was not in two studies [25, 27]. In the study by Abdelghaffar et al. (2018) [25], only 40% of the sample reported experiencing a traumatic event so there might not have been enough data to detect an as- sociation in this sample. However, in the study by Berry et al. (2015) [27], 94% reported at least one traumatic event yet this was also not associated with psychosis- related PTSD. It could be that adulthood trauma is not associated but childhood trauma is; this could be ex- plained through the effect of trauma on the developing child, such as attachment style. p Berry et al. (2015) [27] focussed on attachment theory and hypothesised that insecure attachments (which affect mental representations of the self and others [34];) might be influential in developing PTSD from psychosis. They found that anxious attachments were positively correlated with both psychosis-related (B = 0.40, 95% CI: 0.54–2.28) and hospital-related (B = 0.41, 95% CI: 0.51–2.23) PTSD symptoms. Pietruch and Jobson (2012) [23] theorised that self-disclosure of trauma is important in recovery and posttraumatic growth. Their hypothesis was supported; reluctance to talk and actual self-disclosure were posi- tively and negatively associated with psychosis-related PTSD, respectively (r = .42, p = .02; r = −.43, p = .01). Discussion The findings of this systematic review suggest that be- tween 14 and 47% of people with psychosis might ex- perience psychosis-related PTSD. Depression was most commonly associated with psychosis-related PTSD. Other factors that were associated in at least one study were: symptom-related (severity of psychosis, positive symptoms, and general psychopathology); treatment- related (restraint, length of admission, number of trau- matic hospital events); childhood trauma and childhood trauma-related PTSD; reactions to the trauma (maladap- tive coping, reluctance to talk, actual self-disclosure); and other individual-level factors (experiences of shame, anxious attachment). p y p p Turner et al. (2013) [28] focussed on theories which proposed that experiencing shame can be socially and psychologically damaging. They suggested that individ- uals with psychosis may experience shame through hav- ing a highly stigmatised illness, and that shame might explain PTSD following interpersonal traumas. Their re- sults indicated a correlation between psychosis-related PTSD and both internal (r = .48, p < 0.01) and external (r = .64, p < 0.01) shame associated with psychosis, as well as general shame (r = .57, p < .001). The number of studies included in this review (six) was considerably smaller than in previous reviews, which included 24 [14] and 13 [15] studies, many of which were published before 2011. We did not find, as we had expected, that more studies had been published since 2011 in line with the increasing interest in trauma in mental health research. We also found few studies clearly looking beyond the first episode; most of the Overall each of the four papers received some evidence to support their hypotheses and consequently the under- lying theories. The theories are all psychological or social in nature, and findings suggest how psychosis-related PTSD might arise either due to the effect of early life ex- periences on the development of the mind or the way an individual relates to others. Page 9 of 13 Buswell et al. BMC Psychiatry (2021) 21 Buswell et al. Discussion BMC Psychiatry Table 4 Summarised results from included studies of factors potentially associated with psychosis-related PTSD Potentially associated factors # of studies tested associations # of significant associations Demographics Age 1 0 Gender 2 0 Psychosis characteristics Diagnosis 1 0 Age of onset 1 0 Years since onset 1 0 DUP 2 0 Time since last episode 1 0 Psychosis severity 4 2 Positive symptomsa 2 2 Negative symptomsa 1 0 General psychopathologya 2 0 Treatment experiences Restraint 1 1 Threat by other patients 1 0 Threat by treatment provider 1 0 Involuntary hospitalisation 1 0 Medication side effects 1 0 Length of current admission 1 1 No. of hospitalisations 1 0 No. of traumatic hospital events 1 1 Previous trauma experience Lifetime trauma 2 0 Childhood trauma 1 1 Other clinical factors Depression 3 3 Global functioning 1 0 Substance use 2 0 PTSD related to childhood trauma 1 1 Coping styles Maladaptive coping 1 1 Disclosure of trauma Urge to talk 1 0 Reluctance to talk 1 1 Actual self-disclosure 1 1 Experiences of shame Table 4 Summarised results from included studies of factors potentially associated with psychosis-related PTSD Potentially associated factors # of studies tested associations # of significant associations Demographics Age Gender Psychosis characteristics Diagnosis Age of onset Years since onset DUP Time since last episode Psychosis severity Positive symptomsa Negative symptomsa General psychopathologya Treatment experiences Restraint Threat by other patients Threat by treatment provider Involuntary hospitalisation Medication side effects Length of current admission No. of hospitalisations No. of traumatic hospital events Previous trauma experience Lifetime trauma Childhood trauma Other clinical factors Depression Global functioning Substance use PTSD related to childhood trauma Coping styles Maladaptive coping Disclosure of trauma Urge to talk Reluctance to talk Actual self-disclosure Experiences of shame Internal shame related to psychosis External shame related to psychosis General shame Attachment Anxiety Avoidance DUP Duration of Untreated Psychosis. aSubscale Page 10 of 13 Page 10 of 13 Buswell et al. BMC Psychiatry (2021) 21:9 studies were set in early psychosis services and/or did not describe their sample with sufficient detail. Due to this, we were unable to examine psychosis-related PTSD across the course of psychosis as we had planned. of 8 and 4 studies). Strengths and limitations of included studies g Due to the cross-sectional designs, we cannot infer caus- ation of associated factors. Prospective research is re- quired and is possible. One of our excluded studies recruited patients during the acute stage of psychosis and then followed them up 18 months later, allowing them to investigate whether psychosis-related factors were predic- tors of PTSD [36]. Their measurement of PTSD was not specific to psychosis-related events so this study had to be excluded, however its prospective methodology is noteworthy. Many included studies did not adjust for plausible confounders, such as non-psychosis-related PTSD. Most of the sample sizes were small and limited to one service. Studies which reported statistically signifi- cant associations [26] had very large confidence intervals indicating high variance within the samples. With sample sizes this small it is difficult to generalise the findings. The findings of our review largely corroborate the findings in the previous review by Berry et al. (2013) [14]. However, the Berry et al. (2013) [14] review was limited in that it was not systematic. As our review used systematic methodology it provides more robust evi- dence for prevalence rates and factors associated with psychosis-related PTSD. Additionally due to the rigorous systematic methods we employed, we can say with rea- sonable certainty that the low number of papers re- trieved reflects the lack of recent research, rather than the possibility that papers were missed. Importantly, our review has highlighted that there have been few studies conducted on this topic over the past decade, despite ap- peals that further research is required. For example, the authors of the earlier review stated that future research should use sensitive measures of trauma, separate out symptom and treatment-related PTSD, and that psycho- logical processes should be investigated. The present re- view has found that largely, these recommendations have not yet been met. Some studies have separated out symptom and treatment-related PTSD, but this is not consistent. Some of the papers elicited in our review fo- cussed on psychological processes which were hypothe- sised to be important in the development of psychosis- related PTSD, but most did not. A limitation in this field is a lack of agreement whether trauma related to symptoms and trauma related to treatment are both ‘psychosis-related’ and whether distinctions between these should be made when collect- ing data. Discussion This previous review was solely fo- cussed on first episode psychosis so it has not hitherto been known whether the first episode of psychosis is more or less traumatic than subsequent episodes. The present review had a broader scope by including studies across the course of psychosis and as a result we re- trieved studies not included in the review by Rodrigues and Anderson (2017) [15]. Those additional studies pro- vided data that allowed us to look closely at theories underpinning the development of psychosis-related PTSD (e.g. attachment theory; [27]) and suggested how separate experiences may lead to PTSD symptoms (e.g. by separating delusions and hallucinations; [24]). The prevalence rates we found were similar to the rates of 11–67% reported by Berry et al. (2013) [14] and the pooled prevalence estimates of 30% (PTSD diagno- sis) and 42% (PTSD symptoms) reported by Rodrigues and Anderson (2017) [15]. Our review and both previous reviews found wide variations in reported prevalence rates. In our narrative synthesis we examined differences in the conceptualisation, definition and assessment of psychosis-related PTSD between the included studies and it is likely that these factors can provide some ex- planation for variations in prevalence rates across all three reviews [20]. Other factors such as differences in participant populations, in the amount of cumulative ex- posure to traumatic psychosis and in service provision experienced, may also account for the wide variations in prevalence rates between studies across all three reviews. Similar to our findings, both previous reviews listed trauma history, psychosis severity and depression as pos- sible related factors. They also noted that sample sizes were possibly too small to detect associations and reli- ably estimate prevalence. Strengths and limitations of included studies Differences between studies on how the same measurement tool was used might have elicited different rates of PTSD, and this variability between studies on the concept of psychosis-related PTSD presents compli- cations in comparing prevalence rates and associated factors between different studies. The measurement tools used were generally psychomet- rically robust and validated, and the questionnaires had been reliably used with psychosis populations. The use of a clinician-administered scale in one study, the CAPS, is positive as this is considered the gold-standard for meas- uring PTSD. However, interrater reliability was not assessed, and the CAPS was translated into Tunisian- Arabic for this study but was not validated in that cultural context. Most of the studies included did not sufficiently describe their non-participation rate; individuals who chose not to participate in research about trauma might have declined precisely because they have PTSD, therefore there is a risk of sampling bias across the studies. The review by Rodrigues and Anderson (2017) [15] was systematic and used meta-analysis; however, they too were limited by a small number of studies to analyse (the subgroups included in meta-analyses were made up Page 11 of 13 Page 11 of 13 Buswell et al. BMC Psychiatry (2021) 21:9 Buswell et al. BMC Psychiatry (2021) 21:9 Potentially relevant factors were not investigated for associations with psychosis-related PTSD. Firstly, ethni- city: research suggests that people from black and mi- nority ethnic (BME) backgrounds are considerably more likely to be diagnosed with psychosis [37] and to receive coercive treatment [38] than other ethnicities. They could therefore be particularly vulnerable to traumatic psychosis-related experiences. However, none of the in- cluded studies assessed for associations between ethni- city and psychosis-related PTSD. than being a limitation of this review per se. This review has highlighted that further studies of prevalence and as- sociated factors are required, with distinctions made be- tween FEP and multiple-episode psychosis, and that greater clarity and consistency in how psychosis-related PTSD should be defined and assessed is necessary to reli- ably combine results from multiple studies. We adapted a quality assessment tool because we could not find a more appropriate, validated tool for this review. This could have been further adapted to include some factors specifically relevant to the assessment of PTSD, such as whether sufficient amount of time had lapsed since traumatic event for a diagnosis of PTSD [20]. Strengths and limitations of included studies However, the tool we used was utilised in a similar adapted form in previous studies [15]. The adaptation of the tool to fit our criteria was assisted by a second re- viewer independently, reducing risk of bias. The adapta- tion resulted in the removal of all follow-up criteria due to irrelevance to the research question. Treatment-related factors were somewhat neglected across the studies and only one study assessed correla- tions with involuntary hospitalisation and restraint [25]. Coercive practices are potentially modifiable but the paucity of research into treatment factors limits under- standing of their traumatic nature and potentially reduc- tions in their use. Some known risk factors for PTSD were not assessed in the included studies. Predictors of PTSD are reported to include perceived threat, intense emotions and dis- sociation during the traumatic event, and low perceived social support after the event [12, 13]. Perceived threat was partly investigated by Abdelghaffar et al. (2018) [25] who assessed perception of threat from other patients and care providers. Pietruch and Jobson (2012) [23] in- vestigated disclosure of trauma, which is one aspect of social support; however, social support might protect against PTSD in more ways than encouraging people to talk about their trauma. Intense emotions and dissoci- ation during psychosis were not assessed in the included studies. We included a study [24] which did not report an overall prevalence rate for psychosis-related PTSD, but instead separate rates for different psychosis-related events (e.g. delusions, hallucinations, involuntary hospi- talisation). These figures could not be directly compared with prevalence rates from other studies. However, we decided to include this study as it does provide relevant data on people meeting PTSD criteria based on their psychosis experience. This review built upon previous reviews [14, 15] by ex- ploring the underlying theories of some of the associa- tions between psychosis and related PTSD (e.g. attachment theory, trauma sensitization theory, theories of shame and disclosure of trauma). This can hopefully support the future development of a model of the pro- cesses by which psychosis-related PTSD might occur. Strengths and limitations of the review Our review was restricted to papers published from 2011 onwards which resulted in only a small number of studies being retrieved. However, this allowed us to pro- vide an updated evidence review and to look more closely at the extent and drivers of psychosis-related PTSD in a modern healthcare context. Our inclusion criteria resulted in the exclusion of a doctoral thesis due to non-peer review, and a paper written in French as we were unable to translate it. Both of these may have con- tributed to the findings in this review had they been in- cluded. However, our search strategy was broad, so it is unlikely we missed relevant papers; we searched five da- tabases, used over-inclusive search terms, and a second reviewer assisted with the screening of the search output. Implications in research, theory and practice Studies with prospective designs and larger sample sizes from a wider variety of settings are needed. Research should distinguish between people who have had one or multiple episodes, to investigate a potential cumulative effect of trauma from psychosis, and assess more poten- tial risk factors, such as various treatment factors, social support, dissociation, intense emotions and ethnicity. The wide variation in reported prevalence rates for psychosis-related PTSD is hard to interpret. Moreover, the rates of psychosis-related PTSD reported in some studies in our review are higher than rates of PTSD from any cause among people with psychosis reported in other recent studies, which did not distinguish rates of psychosis-related PTSD and were therefore not included in our review [39, 40]. We need more studies in a variety of settings and clinical populations, and more consen- suses on gold-standard PTSD measures, to be able to The small number of included studies prevented the ability to carry out analyses of subgroups as planned a priori in the PROSPERO protocol. However, finding only six additional studies than the previous reviews [14, 15] reflects the lack of research published in the field since 2011 despite recommendations for further research, rather Page 12 of 13 Page 12 of 13 Page 12 of 13 Buswell et al. BMC Psychiatry (2021) 21:9 as depression. Further research is certainly needed, but awareness needs to be raised amongst clinical settings of the potentially traumatising experience of psychosis so that these can be addressed in treatment and through modifying care practices. Routine enquiry about child- hood trauma as part of a TIC approach could also help to identify those that may be at higher risk of developing PTSD in psychosis. Efforts must be made across clinical and research settings to ensure that TIC is being deliv- ered and to examine its effectiveness at reducing or pre- venting rates of trauma. understand how much in the variance of psychosis- related PTSD may be an artefact of inconsistent meas- urement approaches, and how much reflects genuine variation in clinical morbidity. Some existing psychosocial theories might explain mechanisms underlying psychosis-related PTSD and could in the future form part of an integrated model of psychosis-related PTSD; however before this is possible there needs to be exploration of societal, environmental, cultural, and neurobiological factors. Supplementary Information Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12888-020-02999-x. pp y The online version contains supplementary material available at https://doi. org/10.1186/s12888-020-02999-x. Additional file 1. Medline search strategy. Additional file 1. Medline search strategy. Authors’ contributions Georgina Buswell (GB) was the lead researcher on this project. GB contributed to the study conception and design and carried out the systematic search, contributed towards the study selection, data extraction and quality assessment of the included studies. GB was the lead on the writing of the manuscript. Zoë Haime (ZH) was the second reviewer. ZH contributed towards the study selection, data extraction and quality assessment of the included studies. ZH also critically revised a draft of the manuscript. Dr. Brynmor Lloyd-Evans (BLE) provided advice and guidance on the study design, the review process and the analysis and interpretation of data. BLE critically revised numerous drafts of the manuscript for publication. Dr. Jo Billings (JB), the first supervisor on this project, contributed towards the study conception and design, provided advice and guidance on the re- view process, and supported the analysis and interpretation of data. JB also critically revised numerous drafts of the manuscript for publication. All au- thors read and approved the final manuscript. Abbreviations PTSD: Post Traumatic Stress Disorder; PROSPERO: International Prospective Register of Systematic Reviews; PRISMA: Preferred Reporting Items for Systematic Reviews and Meta-Analyses; DSM-V: Diagnostic and Statistical Manual of Mental Disorders - Fifth Edition; DSM-IV: Diagnostic and Statistical Manual of Mental Disorders - Forth Edition; ICD-10: International Classification of Diseases – 10th Edition; PILOTS: Published International Literature on Traumatic Stress; NOS: Newcastle-Ottawa Scale; EIP: Early Intervention in Psychosis; FEP: First Episode of Psychosis; IES-R: Impact of Events Scale – Revised; PDS: Post-traumatic Diagnostic Scale; CAPS: Clinician- Administered PTSD Scale; PANSS: Positive and Negative Syndrome Scale; BME: Black and Minority Ethnic; TIC: Trauma Informed Care Rates of psychosis-related PTSD do not appear to have reduced since 2011. The ongoing development of TIC has the potential to reduce traumatic experiences associ- ated with psychosis, such as the use of coercive practices [16]. However, coercive practices appear to be increasing in the UK [44], particularly for people with psychosis [45]. In addition to reduced distressing treatment prac- tices, TIC involves services recognizing that the experi- ence of psychosis can be traumatic, screening patients for PTSD, and offering evidence-based treatments (which, NICE guidelines stipulate should commence promptly, [46]). Well implemented TIC may not directly lead to a reduction in reported rates of psychosis-related PTSD in practice, as it may conversely lead to an in- crease in case identification, but it would be expected that rates would decline in cross-sectional studies. It is currently not clear to what extent TIC is being delivered in clinical services. It remains crucial that TIC be devel- oped and implemented and that services recognise the traumatic experience of psychosis and achieve early identification of psychosis-related PTSD followed by ef- fective treatment. Acknowledgements Acknowledgement is made to Graziella Favarato, Research Fellow within the Division of Psychiatry at UCL, for her statistical expertise and guidance in the analysis of this review. Implications in research, theory and practice Currently, an episode of psychosis does not fulfil cri- terion A in the DSM-V for a traumatic event which re- quires exposure to actual or threatened death, serious injury, or sexual violence [41]. It has been argued that this criterion should be expanded to include internally experienced events such as psychosis as it is the percep- tion of threat that is necessary for PTSD [42]. Proposals for the ICD-11 will allow flexibility in the judgement of either an objective or subjective traumatic event [43]. In our review, the rates of people meeting PTSD symptom criteria following the experience of psychosis provides further support for the inclusion of subjective threat as a qualifying traumatic event that satisfies criterion A in the DSM-V classification. Funding h The research was conducted by a postgraduate student for a Master’s thesis at the Division of Psychiatry, University College London (UCL). Conclusions Data sharing is not applicable to this art or analysed during the current study. Data sharing is not applicable to this article as no datasets were generated or analysed during the current study. We must be cautious in drawing conclusions from this review as there were only a small number of studies with methodological issues. However, this review has indi- cated that psychosis can be traumatic enough to lead to PTSD in some individuals, and there are some factors which are associated with psychosis-related PTSD, such Ethics approval and consent to participate Not applicable. 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Temporal variability of carbon recycling in coastal sediments influenced by rivers: assessing the impact of flood inputs in the Rhône River prodelta
Biogeosciences
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Temporal variability of carbon recycling in coastal sediments influenced by rivers: assessing the impact of flood inputs in the Rhˆone River prodelta C. Cathalot1, C. Rabouille1, L. Pastor2, B. Deflandre2,3, E. Viollier2, R. Buscail4, A. Gr´emar A. Pruski5,6 ouille1, L. Pastor2, B. Deflandre2,3, E. Viollier2, R. Buscail4, A. Gr´emare3, C. Treignier1, and 1Laboratoire des Sciences du Climat et de l’Environnement, UMR 8212, CEA-CNRS/INSU-UVSQ Gif-sur-Yvette, France 2Laboratoire de Geochimie des Eaux, IPGP & Universit´e Paris Diderot, Paris, France 3Laboratoire Environnements et Pal´eoenvironnements Oc´eaniques, Universit´e Bordeaux 1, France 4Centre de Formation et de Recherche sur l’Environnement Marin, Perpignan, France 5LECOB, UPMC Univ Paris 06, FRE 3350, Observatoire Oc´eanologique, Banyuls/mer, France 6LECOB, CNRS, FRE 3350, Observatoire Oc´eanologique, Banyuls/mer, France Received: 9 October 2009 – Published in Biogeosciences Discuss.: 17 November 2009 Revised: 17 March 2010 – Accepted: 19 March 2010 – Published: 31 March 2010 1Laboratoire des Sciences du Climat et de l’Environnement, UMR 8212, CEA-CNRS/INSU-UVSQ 2Laboratoire de Geochimie des Eaux, IPGP & Universit´e Paris Diderot, Paris, France 3Laboratoire Environnements et Pal´eoenvironnements Oc´eaniques, Universit´e Bordeaux 1, France 4Centre de Formation et de Recherche sur l’Environnement Marin, Perpignan, France 5LECOB, UPMC Univ Paris 06, FRE 3350, Observatoire Oc´eanologique, Banyuls/mer, France 6LECOB, CNRS, FRE 3350, Observatoire Oc´eanologique, Banyuls/mer, France Received: 9 October 2009 – Published in Biogeosciences Discuss.: 17 November 2009 Revised: 17 March 2010 – Accepted: 19 March 2010 – Published: 31 March 2010 A flood of the Rhˆone River occurred in June 2008 and de- livered up to 30 cm of new soft muddy deposit. Immedi- ately after this flood, sediment DOU rates close to the river mouth dropped from around 15–20 mmol O2 m−2 d−1 to val- ues close to 10 mmol O2 m−2 d−1, in response to the deposi- tion near the river outlet of low reactivity organic matter as- sociated to fine material. Six months later, the oxygen distri- bution had relaxed back to its initial stage: the initial spatial distribution was found again underlining the active microbial degradation rates involved and the role of further deposits. These results highlight the immediate response of the sedi- ment oxygen system to flood deposit and the rapid relaxation of this system towards its initial state (6 months or less) po- tentially linked to further deposits of reactive material. Abstract. River deltas are particularly important in the ma- rine carbon cycle as they represent the transition between ter- restrial and marine carbon: linked to major burial zones, they are reprocessing zones where large carbon fluxes can be min- eralized. Temporal variability of carbon recycling in coastal sediments influenced by rivers: assessing the impact of flood inputs in the Rhˆone River prodelta In order to estimate this mineralization, sediment oxygen uptake rates were measured in continental shelf sedi- ments and river prodelta over different seasons near the outlet of the Rhˆone River in the Mediterranean Sea. On a selected set of 10 stations in the river prodelta and nearby continental shelf, in situ diffusive oxygen uptake (DOU) and laboratory total oxygen uptake (TOU) measurements were performed in early spring and summer 2007 and late spring and win- ter 2008. In and ex situ DOU did not show any significant differences except for shallowest organic rich stations. Sed- iment DOU rates show highest values concentrated close to the river mouth (approx. 20 mmol O2 m−2 d−1) and decrease offshore to values around 4.5 mmol O2 m−2 d−1 with lowest gradients in a south west direction linked to the preferen- tial transport of the finest riverine material. Core incuba- tion TOU showed the same spatial pattern with an averaged TOU/DOU ratio of 1.2±0.4. Temporal variations of sedi- ment DOU over different sampling periods, spring summer and late fall, were limited and benthic mineralization rates presented a stable spatial pattern. Biogeosciences, 7, 1187–1205, 2010 www.biogeosciences.net/7/1187/2010/ © Author(s) 2010. This work is distributed under the Creative Commons Attribution 3.0 License. Biogeosciences, 7, 1187–1205, 2010 www.biogeosciences.net/7/1187/2010/ © Author(s) 2010. This work is distributed under the Creative Commons Attribution 3.0 License. Biogeosciences 1 Introduction OC oxidation in sediments is cou- pled to the utilization of terminal electron acceptors: with the highest free energy yield, oxygen is first consumed by aerobic bacteria in the sedimentary column (Froelich et al., 1979). Oxygen distribution in sediments also reflects chemi- cal reactions (oxidation of reduced species). Integrating ben- thic microbial respiration and reoxidation of anoxic reduced compounds, oxygen consumption by marine sediments is thus a good proxy to estimate benthic metabolism and OC mineralization rates and their variability over time and space in river dominated environments (Rabouille et al., 2003; Glud et al., 2000, 2003; Lansard et al., 2003; Cai et al., 1995). River flood may modify the sediment mineralization of organic matter by introducing large quantities of terrige- neous organic carbon with various reactivities and favour its preservation in shallow coastal environments (Leithold and Hope, 1999). Tesi et al. (2008) showed evidence of major changes in the biogeochemical composition and reactivity of sedimentary organic matter in a flood deposit in the Po River prodelta. Furthermore, the retention capacity of flood inputs in estuaries is variable: some estuaries may retain only 20% of the flood inputs in the innershore region (Lisitsyn, 1995; Sommerfield and Nittrouer, 1999), while other larger systems like the Atchafalaya River may act as efficient traps for flood inputs (Allison et al., 2000). It appears that (i) the response to flood events of continental margins at river outlets seems to be system-dependant and (ii) the parameters driving the sediment response are largely unknown. For instance, little is known about the partitioning between burial and recycling under normal and flood discharge rates regimes at the river- sea interface. Given the high OC sequestration and oxidation potential of river dominated margins at a global scale (Blair et al., 2004; Burdige, 2005), it is therefore critical to assess the behaviour of the benthic system after a flood event, and especially the dynamics of its OC degradation activity. Si th d i f th Nil th Rhˆ Ri i th In this paper, we present oxygen uptake rates from a sea- sonal survey of the sediments in the Rhˆone River prodelta and adjacent shelf. The same stations were visited four times between April 2007 and December 2008, including a Rhˆone River flood period in June 2008. Transient evolution of the spatial pattern of the sediment oxygen uptake in the prodelta was investigated using in situ and laboratory measurements. 1 Introduction River dominated shelves represent a dynamic interface link- ing land and ocean biogeochemical cycles of relevant ele- ments such as Organic Carbon (OC) (Hedges, 1992; Gattuso et al., 1998; McKee et al., 2004). They are productive areas sustained by high inputs of nutrients and terrestrial material (Dagg et al., 2004), characterized by a tight pelagic-benthic coupling and active benthic mineralization rates (Smith and Hollibaugh, 1993). Indeed, over 50% of all organic car- bon burial in the ocean takes place in continental margins (Hedges and Keil, 1995). In addition, it is estimated that Correspondence to: C. Cathalot (cecile.cathalot@lsce.ipsl.fr) Correspondence to: C. Cathalot (cecile.cathalot@lsce.ipsl.fr) Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 1188 C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments up to 70% of the 0.15×1015 gC of particulate OC annually discharged from rivers to ocean is oxidized in these areas (Hedges et al., 1997; Burdige, 2005; Galy et al., 2007; Mey- beck, 1982). sediments off the Rhˆone River mouth and observed a specific pattern with high sediment uptake rates near the outlet with an exponential gradient offshore. Yet the evolution of this pattern of organic carbon recy- cling in sediments during the annual cycle and under flood conditions is completely unknown. In particular, floods are likely to play a major role in this dynamics since they can ac- count up to 80% of the particles input from the Rhˆone River to the Mediterranean Sea (Antonelli et al., 2008). It has been proposed using a modelling approach that the Rhˆone River prodelta acts as a deposit centre for flood inputs (Ulses et al., 2008). Consequently, Rhˆone River flood events influence the recycling of organic matter in the river prodelta and alter the filtering capacity of river particulate inputs. River inputs to the coastal ocean are highly variable over time, shifting from flood and high sediment supply to low- river discharge (Wheatcroft and Borgeld, 2000). This vari- ability causes a non-stationary OC deposition in deltas and prodeltas (Bentley and Nittrouer, 2003; McKee et al., 2004). Post-depositional processes such as physical (winnowing) and biological (bioturbation) reworking can also affect the organic matter reaching the sea floor in these environments (Rabouille et al., 2003). 1 Introduction As proxies of organic matter quality, OC and Chlorophyll a (Chl a) contents in surface sediments also brought insights on the existing links between flood deposit lability, OC sed- iment degradation and the transitory processes involved be- tween both. We discuss the effect of flood inputs and sea- sonal changes on the prodelta filtering capacity and the dy- namics of oxygen and organic carbon in sediments after flood deposition. C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments 1189 Fig. 2. Mean daily flow and particulate discharge rates of the Rhˆone River. The integrated SPM amount delivered during the sampling cruises period are indicated in yellow. Discharge and SPM data have been achieved in the courtesy of the CNR and the SORA in the workframe of a convention with IRSN in the EXTREMA ANR project. Fig. 1. Map of the Rhˆone delta indicating the locations of sam- pling stations. Black squares indicate stations sampled the four cruises. Empty squares indicate stations sampled three times. Black crosses indicate stations sampled twice. Empty circles indicate sta- tions sampled once (April 2007). Fig. 1. Map of the Rhˆone delta indicating the locations of sam- pling stations. Black squares indicate stations sampled the four cruises. Empty squares indicate stations sampled three times. Black crosses indicate stations sampled twice. Empty circles indicate sta- tions sampled once (April 2007). Fig. 2. Mean daily flow and particulate discharge rates of the Rhˆone River. The integrated SPM amount delivered during the sampling cruises period are indicated in yellow. Discharge and SPM data have been achieved in the courtesy of the CNR and the SORA in the workframe of a convention with IRSN in the EXTREMA ANR project. the shoreline to 60 m depth (Friedrichs and Wright, 2004). Net sedimentation rates in the prodelta are up to 50 cm yr−1 at the river mouth (Charmasson et al., 1998) and decrease rapidly offshore on the continental shelf, i.e. 0.2–0.6 cm yr−1 at 20 km (Miralles et al., 2005). This sedimentation rates gradient defines the “prodelta” as the area of large sedimen- tation rates mostly above the 60 m bathymetric level and the “adjacent shelf” with greater water depths and lower sedi- mentation rates (<1 cm y−1). The benthic macrofauna com- munity in the area presents a low specific diversity compared to those of other soft bottom communities in the region. It is dominated in abundance by polychaetes mainly Sternaspis scutata. and Laonice cirrata, and by surface and subsurface deposit-feeders (Salen-Picard and Arlhac, 2002; Darnaude et al., 2004). The maximum richness in species is recorded at 70 m depth (Salen-Picard, 1982). utary with massive erosion of river banks leading to a sus- pended load of up to 3.7 g l−1 (Fig. 2). C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments At each station in situ microelectrode measurements were performed as described in (Rabouille et al., 2003): briefly, 3– 4 h deployments were performed at the sediment water inter- face using an autonomous microprofiling unit which records 5 oxygen microprofiles using Clark micro-electrodes and one resistivity microprofile. Sediment samples were collected with a multicorer MUC 8/100 (Oktopus GmbH) that collect simultaneously eight P.C cores (I.D. 9.5 cm) with a preserved sediment-water interface (60 cm height with around 25 cm of overlying water and 35 cm of sediment). For micro-porosity measurements, cores were subsampled with a 50ml syringe and sliced at increasing depth intervals: 0.2 cm depth res- olution for the first cm and 0.5 cm from 1 to 6 cm deep. Porosity φ was determined from the weight loss upon dry- ing at 60 ◦C until complete dryness (∼2 weeks) of sediment core segments of known weight and volume. Additional sed- iment cores with undisturbed surface structure were also col- lected for solid sediment sampling, cores incubation and mi- croprofiling in the laboratory under in situ conditions. Sedi- ments for organic carbon and Chl-a analysis were collected and frozen on board ship immediately after sub-sampling within one hour after core collection. For core incubation and laboratory microprofiling, the cores were stored in a pool supplied by cooled sea water recirculation until they were brought to the shore and placed in a refrigerated box at in situ temperature. 2.1 Study area The Gulf of Lions is a large continental shelf located in the NW Mediterranean Sea. The North Western Mediter- ranean current flows southwestward along the slope and im- poses a general cyclonic circulation. The water column is seasonally stratified, but intense vertical mixing events, which induce major nutrient export, occur during strong regional winds outbursts (Millot, 1990; de Madron et al., 1999). The Gulf of Lions is a microtidal sea and the mod- erate wave-energy is swell-dominated. The Rhˆone River has a drainage basin of 97 800 km2, a mean water flow of 1700 m3 s−1, and an annual particulate organic carbon dis- charge of 19.2±6×104 tC/an (Sempere et al., 2000). The Rhˆone River is thus the main source of freshwater, nutrients and organics for the Gulf of Lions (Sempere et al., 2000; Pont et al., 2002; De Madron et al., 2000). The hydrologi- cal regime of the Rhˆone River shows strong seasonal contrast with a large difference between low (<500 m3 s−1) and high (>3000 m3 s−1) water-discharge (Pont et al., 2002). Large amounts of terrestrial muddy sediments accumulate in the wide prodelta off the Rhˆone river mouth, extending then Since the damming of the Nile, the Rhˆone River is the most important freshwater input of the Mediterranean Sea both in terms of water and particle discharges (Pont et al., 2002; Copin-Montegut, 1993). Its influence over the conti- nental shelf of the Gulf of Lions has been widely documented (Monaco et al., 1999; De Madron et al., 2000, 2003; Sempere et al., 2000). Recently, Lansard et al. (2009) proposed a first snapshot of the oxygen uptake rates in the continental shelf Biogeosciences, 7, 1187–1205, 2010 www.biogeosciences.net/7/1187/2010/ Biogeosciences, 7, 1187–1205, 2010 2.6 Microelectrode measurements In addition, a coring station (43◦18.420 N, 4◦51.320 E, water depth: 45 m) was selected to monitor the evolution of flood deposits off the Rhˆone River mouth. Sediment cores were therefore collected four times at this station in June, September, October and December 2008. The cruise in De- cember 2008 followed an annual flood that brought new ma- terial. Significant wave heights in 2008 in this area from June to December 2008 were acquired from the CANDHIS database of in situ wave height measurements. The 200 µm resolution O2 and resistivity in situ profiles were obtained by a benthic microprofiler (Unisense®) equipped with 4–5 O2 microelectrodes and 1 resistivity sensor. The profiling unit was mounted on an autonomous tripodal frame. Ex situ measurements of O2 microprofiles were performed in a thermostated bath maintained at in situ sampling tem- perature. Up to 15 steady-state O2 microprofiles (50–100 µm resolution) were completed within 6 h after sampling. Con- servation of overlying water oxygenation was achieved by a soft bubbling system. For each cruise, immediately after sampling, a classical sedimentological inspection of the core was performed, de- scribing the different visible layers. Sediments were then sectioned into slices and stored at −20 ◦C for further OC analyses. Vertical resolutions were: 0.5 cm in the first top centimetre, 1 cm down to 20 cm, and 2 cm deeper in the core. Dissolved oxygen concentration was measured by oxygen microelectrodes (Unisense®) provided with a built-in refer- ence and an internal guard cathode (Revsbech, 1989). The O2 microsensors had tip outer diameters of 50–100 µm, a stir- ring sensitivity of <1%, a 90% response time <10 s, and less than 2% per hour current drift. The electrode signals were recorded in the overlying-water before and after each profile to assess the stability of the measurements. We used a linear calibration for the microelectrodes, between the bottom wa- ter oxygen content estimated by Winkler titration (Grasshoff et al., 1983) and the anoxic zone of the sediment. In June 2008, in order to describe the redox front in the flood deposit, redox potential measurements were performed on the core right after its retrieval. The redox potential (Eh in mV) was obtained with a pH/redox meter (Knick apparatus and Ingold platinum electrode). Measurements were taken successively over the first cm and in the whole sedimentary column at 2 cm intervals (the P. C. 2.2 Field sampling work and sampling procedures Sediment samples were collected during four cruises in April 2007, September 2007, June 2008 and December 2008 (Fig. 1). In April 2007, 16 stations were sampled off the Rhˆone river mouth in order to get a better estimate of the benthic mineralization rates in the Rhˆone prodelta. Key sta- tions were then selected along onshore–offshore transects in water from 20 to 98 m depth. During the three other cruises, these selected stations were investigated again (Fig. 1). The June 2008 cruise took place while the Rhˆone River was expe- riencing an annual flood with a peak water discharge rate of 4156 m3 s−1. This flood event occurred after large amounts of precipitation over the Durance drainage basin (French Southern Alps), leading to a flood of this Rhˆone River trib- Biogeosciences, 7, 1187–1205, 2010 www.biogeosciences.net/7/1187/2010/ 1190 C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments the d(0.5), which corresponds to the median of the size distri- bution based on the equivalent spherical volume diameters. Bottom-water was sampled at 2 m above bottom by a Niskin bottle for determination of temperature and dissolved oxygen (Table 1). 2.4 Pigment analysis Surface sediments (0–0.5 cm layer) were rapidly thawed and 100 mg were extracted overnight in 5 ml of acetone at 5 ◦C in the dark. Adjustment was made for sediment water content to obtain a final acetone degree of 90%. The fluorescence of the sediment extracts was measured on a LS 55 spectroflu- orimeter (Perkin Elmer Inc., USA) according to the method developed by Neveux and Lantoine (1993). Uncertainty on the pigments content was lower than 1%. For each station, the analyses were performed on three cores and in triplicates (i.e. 9 independent extracts). Data are expressed as weight per gram dry sediment. Resistivity measurements were carried out with an elec- trode similar to the one described by Andrews and Ben- nett (1981). Four thin parallel wires were buried in a ma- trix of epoxy, with only their tips in electrical contact with seawater. The resistivity sensor has a rectangular section of 10×3 mm and is edged at the lower end. Recordings were made at 200 µm as for the oxygen but the pertinent resolu- tion is certainly around 1 mm due to the shape of the sensor (Rabouille et al., 2003; Andrews and Bennett, 1981). Voltage outputs were calibrated to resistivity with standard KCl solu- tions, and the resistivity recordings were converted to inverse formation factor values by the formulation of Berner (1980): 2.3 Organic carbon content analysis OC contents were analysed using milled, freeze-dried sedi- ments. Surface OC content was determined on the 0–0.5 cm top layer of the sediment. Organic carbon concentrations were measured on homogeneised, precisely weighed sub- samples in an automatic CN – analyser LECO 2000, after in cups acidification with 2N HCl (overnight, at 50 ◦C) in order to remove carbonates prior to the analyses of OC (Cauwet et al., 1990). The precision for OC was 2%. 2.6 Microelectrode measurements core was equipped with a removable side panel). The location of the sediment-water interface relative to the in situ oxygen profiles was determined from O2 micropro- files. We used the classical method which consists in as- signing the interface location to a break in the oxygen con- centration gradient. The observed change of slope is due to the increased diffusion coefficient in the sediment compared to the diffusive boundary layer (DBL) (Jorgensen and Revs- bech, 1985; Revsbech, 1989; Sweerts et al., 1989). In some profiles, the slope break was not clearly visible: they rather displayed a steady increase of the slope towards a maximum within the first millimeter below the initial concentration de- crease. In these cases, we adopted the position of this maxi- mum gradient as the sediment-water interface. Oxygen pene- tration depth was determined from the O2 profile and was as- signed to the depth where the microelectrode signal reached the zero current. 2.5 Grain size measurement Sediment granulometry was assessed using a Malvern Mas- tersizer 2000 laser microgranulometer. Grain size is given as (1) Biogeosciences, 7, 1187–1205, 2010 www.biogeosciences.net/7/1187/2010/ C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments 1191 Table 1. Temporal variations of Rhˆone River prodelta bottom water and sediment properties. m coefficient from Archie’s law used for porosity assessment, mean sediment grain size diameter (µm) and chloropigments (Chl b/Chl a and Pheo a) are detailed for each station. Table 1. Temporal variations of Rhˆone River prodelta bottom water and sediment properties. m coefficient from Archie’s law used for porosity assessment, mean sediment grain size diameter (µm) and chloropigments (Chl b/Chl a and Pheo a) are detailed for each station. Lat. Long. www.biogeosciences.net/7/1187/2010/ 3.1 Porosity: cores measurements and estimation from F −1 We also used the numerical model PROFILE (Berg et al., 1998), which calculates the consumption rates with depth by adjusting a calculated oxygen profile to the observed one. It allowed us to determine the location of oxygen production and oxygen consumption layers, the extent of these zones, and the resulting fluxes across the sediment-water interface. The two boundary conditions used for the calculations corre- spond to the zero oxygen concentration and flux at the bottom of the oxic zone. At all stations, porosity decreases gradually with depth from 0.85–0.9 for the top 2 mm to a value ranging between 0.62– 0.77 at 6 cm depth (Fig. 3). These profiles display rela- tively high values of porosity consistent with data reported by Reimers et al. (1992). Most stations show constant poros- ity profile with time, except station A located at the river outlet. This station also displays a large change in grain size between April 2007 and June 2008, i.e. normal to flood condition (mean∅=6.7–37.4 µm; Table 1). The porosity de- rived from F −1 factor through the power law F −1 = φ−m match the measured values and thus allow the estimation of m (Fig. 3). Indeed, on average r2 is 0.9939 m values dis- played an average of 2.2±0.4 (Table 1). The observed vari- ations were not correlated to the mean diameter (r2 = 0.06, n = 28). 2.7 Sediment diffusive oxygen fluxes calculations Significance of temporal changes in in situ DOU at each station was assessed using non parametric Kruskal-Wallis ANOVAs (when stations had been sampled more than twice), or Mann and Whitney U tests (when stations had been sam- pled only twice). Sediment oxygen consumption rates were estimated from O2 microprofiles by two ways. Diffusive oxygen uptake (DOU) was calculated from O2 concentration gradients at the sediment–water interface by using the 1-D Fick’s first law of diffusion: DOU = ϕ Ds h dO2 dx i x=0 where φ is the porosity, Ds is the O2 diffusion coefficient within the sediment and h dO2 dx i x=0 is the oxygen gradient just below the sediment– water interface (estimated from the profiles). Ds was es- timated as Ds = D0O2 1+3(1−ϕ) (Iversen and Jorgensen, 1993). D0O2 is the molecular diffusion coefficient of O2 (cm2 s−1) at in situ temperature, salinity and hydrostatic pressure and was evaluated using tables from Broecker and Peng (1974). A two way analysis of variance (ANOVA) was carried out to test for temporal and depth differences in sedimentary OC content among sampling periods (June, September, October and December) and deposit layers (sediment depth). 2.5 Grain size measurement Cathalot et al.: Temporal variability of carbon recycling in coastal sediments Where Rbw is the average resistivity in the bottom water and Rz is the mean resistivity at given depth z. calculated from concentration change of oxygen in the over- lying water with incubation time, after correction had been made for input of replacement water. This approach allowed the determination of TOU with only a small deviation from the ambient bottom water concentrations in the overlying wa- ter. We calculated then a porosity profile by converting F −1 values using the empirical Archie’s relation: F −1 = φm (2) (2) F −1 = φm 2.8 Sediment core incubation and total oxygen uptake measurements Immediately after retrieval, 3 sediment cores with undis- turbed interface were selected per station and placed in a refrigerated box at in situ temperature back to the labora- tory. Once sealed, overlying water was kept homogenised by a rotating floating magnet fixed to the upper core cap. Dark incubations started within 6 h after sampling. Every 2–4 h, 50 ml of the overlying water was sampled and replaced with the same volume of filtered bottom water (Denis et al., 2001; Hulth et al., 1997) We determined the oxygen concentration in the overlying water of each core and the filtered bottom water by Winkler titration (Grasshoff et al., 1983). Sampling intervals and incubation duration were adjusted so that oxy- gen concentration in the overlying water did not decrease by more than 20–30% of the initial concentration. TOU was 2.9 Statistical calculation Where φ is the porosity and m is an experimental factor usu- ally ranging from to 2 to 3. m was determined for each sta- tion as corresponding to the best least square fit to the mea- sured porosity profile (through an Microsoft Excel® solver routine). Non parametric Wilcoxon signed rank tests were used to as- sess statistical differences between in situ vs. ex situ DOU and OPD. These tests were performed on the mean in and ex situ DOU and OPD recorded for any combination of cruises and stations. 2.5 Grain size measurement Depth e Distance Tbw [O2]bw m Mean %Corg in Chl b/ Pheo a Stations (◦N) (◦N) (m) (km) Cruise (◦C) (µM) coeff diameter surficial sediment Chl a (µg g−1) A 43◦18′47′′ 4◦51′4′′ 24 1.9 Apr-07 14.9 259 2.74 37.40 1.99 14.2 20.5±1.5 Sep-07 17.7 244 1.99 – 1.40 17.3 11.0±0.8 Jun-08 16.8 238 2.17 6.74 1.13 9.3 4.6±0.6 Dec-08 14.8 237 1.97 – 1.22 26.4 6.5±3.4 B 43◦18′14′′ 4◦50′4′′ 54 3.0 Apr-07 14.6 249 2.42 14.83 1.61 10.2 21.8±1.2 Sep-07 14.5 214 2.42 – 1.37 7.1 12.5±0.3 Jun-08 14.0 223 2.45 23.87 1.75 8.1 17.7±1.1 Dec-08 14.7 234 2.64 – 1.96 20.9 17.5±1.0 C 43◦16′17′′ 4◦46′33′′ 76 8.6 Apr-07 14.5 243 2.29 11.38 1.25 3.6 12.2±1.2 Jun-08 14.7 239 1.80 14.51 1.16 5.3 8.4±0.2 Dec-08 14.7 235 2.40 – 1.49 0.0 17.5±1.0 D 43◦14′54′′ 4◦43′46′′ 74 13.0 Apr-07 14.3 244 1.43 10.45 1.05 0.0 13.8±2.8 Sep-07 15 217 1.46 – 0.99 0.0 8.8±0.7 Jun-08 14.0 226 2.31 12.10 1.00 0.0 7.5±3.9 Dec-08 14.8 237 – 1.16 0.0 13.3±0.7 E 43◦13′12′′ 4◦41′54′′ 75 17.0 Apr-07 14.2 245 2.09 9.43 0.0 13.4±0.7 Jun-08 15.6 245 2.18 15.02 1.07 0.0 10.3±1.0 F 43◦10′1′′ 4◦41′59′′ 78 21.6 Apr-07 14.2 257 2.55 9.15 1.04 0.0 12.6±1.8 Jun-08 14.7 242 – 1.03 U 43◦5′2′′ 4◦35′58′′ 90 33.8 Jun-08 13.8 231 2.58 14.27 0.82 0.0 6.7±2.5 G 43◦18′30′′ 4◦47′17′′ 47 5.2 Apr-07 14.8 249 2.33 17.38 – 5.5 14.9±0.2 H 43◦15′53′′ 4◦49′10′′ 86 7.5 Apr-07 14.5 236 2.28 9.99 1.17 0.0 11.6±1.6 Sep-07 14.9 202 1.35 – 1.00 0.0 9.8±0.2 Jun-08 14.0 245 2.71 14.15 1.11 0.0 10.3±1.4 I 43◦16′0′′ 4◦53′1′′ 89 7.7 Apr-07 15.1 231 2.56 10.70 1.03 0.0 11.3±0.7 Jun-08 15.9 238 2.64 16.26 1.12 0.0 10.5±0.7 J 43◦16′7′′ 4◦58′6′′ 86 12.1 Apr-07 14.1 243 2.42 11.59 0.99 0.0 9.4±0.6 Jun-08 14.0 227 2.36 14.27 1.01 0.0 11.8±6.9 K 43◦18′7′′ 4◦51′29′′ 62 3.3 Apr-07 14.6 249 2.21 17.49 1.79 10.1 17.2±1.0 Sep-07 18.2 241 2.05 – 1.39 6.7 13.0±0.3 Jun-08 16.8 240 2.01 11.98 1.02 7.6 9.2±1.9 Dec-08 14.7 235 2.65 – 1.62 20.7 10.5±0.1 L 43◦18′24′′ 4◦52′59′′ 62 4.0 Apr-07 14.3 247 2.85 13.56 1.51 8.9 18.4±4.0 Sep-07 18.0 238 2.03 – 1.26 11.3 13.9±0.9 Jun-08 16.7 229 3.02 9.10 1.06 6.1 8.7±1.4 Dec-08 15.0 233 1.95 – 1.11 29.7 8.9±2.5 M 43◦9′59′′ 4◦44′4′′ 91 20.3 Apr-07 14.1 241 2.42 9.89 – 0.0 16.1±1.2 N 43◦17′33′′ 4◦47′59′′ 67 5.5 Apr-07 14.5 253 1.79 14.01 1.43 6.9 14.3±0.2 Sep-07 14.5 217 1.75 – 1.20 0.0 11.5±0.7 Jun-08 16.3 240 2.22 10.65 1.00 6.2 12.3±6.6 O 43◦17′0′′ 4◦50′6′′ 79 5.2 Apr-07 14.4 251 1.94 11.06 1.20 1.2 14.0±0.9 R2 43◦14′30′′ 4◦53′4′′ 98 10.3 Apr-07 14.1 242 2.40 9.55 – 0.0 9.8±1.4 Biogeosciences, 7, 1187–1205, 2010 www.biogeosciences.net/7/1187/2010/ 1192 C. 3.2 Surface sediment carbon content Organic Carbon (OC) content of surficial sediments in the Rhˆone river prodelta ranged from 0.99% to 1.99% d.w. (Ta- ble 1; Fig. 4). Stations away from the river outlet (D, E, F, H, I, J) displayed a homogeneous and stable organic con- tent of 1.04±0.08% (i.e. Coefficient of variation C.V. of only 8.1%). At all cruises except June 2008, stations close to the river outlet showed higher content around 1.5–2%, stations A and B being the more enriched. OC content decreased Biogeosciences, 7, 1187–1205, 2010 www.biogeosciences.net/7/1187/2010/ www.biogeosciences.net/7/1187/2010/ C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments 1193 Cathalot et al.: Temporal variability of carbon recycling in coastal sediments 11 3. Porosity profiles for all stations during all cruises. Data points indicate measured values while thin curves represent the fitted pro ed on resistivity measurements and Archie’s law (see text for details). 3. Porosity profiles for all stations during all cruises. Data points indicate measured values while thin curves represent the fitted pro d on resistivity measurements and Archie’s law (see text for details). Fig. 3. Porosity profiles for all stations during all cruises. Data points indicate measured values while thin curves represent the fitted profiles based on resistivity measurements and Archie’s law (see text for details). Biogeosciences, 7, 1187–1205, 2010 C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments 1194 Fig. 4. Organic carbon (OC) content in surficial sediments as a function of distance from the river outlet for April 07 (black stars 3), September 07 (black crosses ), June 08 cruises (empty circles H) and December 08 (black diagonal crosses ). Exponential de- cays of OC with distance in April 2007 and June 2008 are plotted to highlight the trends. Fig. 4. Organic carbon (OC) content in surficial sediments as a function of distance from the river outlet for April 07 (black stars 3), September 07 (black crosses ), June 08 cruises (empty circles H) and December 08 (black diagonal crosses ). Exponential de- cays of OC with distance in April 2007 and June 2008 are plotted to highlight the trends. Fig. 5. Surface sediments Chl a contents as a function from the river outlet: , April 2007,  September 200 2008 and  December 2008. 3.4 Sediment oxygen uptake Fig. 5. Surface sediments Chl a contents as a function of distance from the river outlet: , April 2007,  September 2007, H June 2008 and  December 2008. Fig. 4. Organic carbon (OC) content in surficial sediments as a function of distance from the river outlet for April 07 (black stars 3), September 07 (black crosses ), June 08 cruises (empty circles H) and December 08 (black diagonal crosses ). Exponential de- cays of OC with distance in April 2007 and June 2008 are plotted to highlight the trends. Fig. 5. Surface sediments Chl a contents as a function of distance from the river outlet: , April 2007,  September 2007, H June 2008 and  December 2008. 3.4 Sediment oxygen uptake In April 2007, the microprofiler was deployed at 16 stations, an extension of the area previously covered (Lansard et al., 2009): 8 and 12 of these stations were resampled respectively in September 2007 and June 2008. Finally in December 2008, only 6 stations close to the Rhˆone River mouth were sampled because of meteorological conditions. All oxy- gen profiles showed decreasing O2 concentrations through a diffusive boundary layer of about 0.2–2.2 mm above the sediment–water interface (Fig. 6). Below, O2 concentrations decrease rapidly with steep gradients, the steepness depend- ing on the station. The oxygen penetration depth (OPD) into the sediment ranges from 1.6±0.3 mm in front of the Rhˆone River mouth to 12.7±1.7 mm about 30 km south-westward (Table 2). There was no statistical difference between in situ and ex situ OPD (p > 0.05). Generally OPD increased with distance from the Rhˆone River mouth. Apart from the June 2008 cruise corresponding to a river flood event, all OPD on the SW transect showed linear increase with distance from station A, i.e. near the river mouth (r2 > 0.883). Near the Rhˆone River mouth (stations A, B, K), OPD were statisti- cally different in June 2008 compared to the other cruises. The other stations did not display any differences in OPD between cruises. rapidly with distance from the river outlet in the prodelta (first 10 km) and more slowly in the adjacent shelf (Fig. 4; solid line regression). Conversely, OC content in June 2008 was homogeneous over all the prodelta: all stations (“off- shore” stations as nearshore ones) presented the same low content of 1.04±0.08%. 3.3 Surface sediment pigment content Chlorophyll a (Chl a), Chlorophyll b (Chl b) and Pheo- phytin a (Pheo a) content of surficial sediments in the Rhˆone River prodelta are available for all cruises. Chl a contents displayed an exponential decrease with distance from the river mouth (r2 > 0.7178, p < 0.01) with highest values lo- cated in the prodelta (Fig. 5). Chl a sediment contents were significantly lower in June 2008 than the April 2007, Septem- ber 2007 and December 2008 values pooled together (test: Mann-Whitney, p < 0.05). In April, September and De- cember, Chl a content were around 4.75±2.84 µg g−1 d.w. near the river outlet (stations A, B) decreasing to low values about 1.67±0.83 µg g−1 d.w. offshore. In June 2008, how- ever, during the flood event, the pattern was different with values in stations A and B around 3.32±0.68 µg g−1 d.w. and lower concentration in shelf sediments with an average of 1.08±0.91 µg g−1 d.w. (Fig. 5). Chl b was detected only near the river mouth and Pheo a presented higher contents in the most proximal stations (Table 1). Positive fluxes of O2 (from the overlying water into the sediment) were measured in all investigated stations. To- tal Oxygen Uptake rates measured by core incubations had average values ranging from ∼16 mmo O2 m−2 d−1 close to the river mouth (stations A and B) to ∼3 mmol O2 m−2 d−1 Biogeosciences, 7, 1187–1205, 2010 www.biogeosciences.net/7/1187/2010/ C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments 1195 Table 2. Temporal variations of Oxygen Uptake Rates in the sediments of the Rhˆone River prodelta (means±SD). OPD stands for oxygen penetration depth; DOU for Diffusive Oxygen Uptake and TOU for Total Oxygen Uptake. Table 2. Temporal variations of Oxygen Uptake Rates in the sediments of the Rhˆone River prodelta (means±SD). OPD stands for oxygen penetration depth; DOU for Diffusive Oxygen Uptake and TOU for Total Oxygen Uptake. Table 2. Temporal variations of Oxygen Uptake Rates in the sediments of the Rhˆone River prodelta (means±SD). OPD stands for oxygen penetration depth; DOU for Diffusive Oxygen Uptake and TOU for Total Oxygen Uptake. n=number of replicated OPD DOU O2 profiles (mm) (mmolO2 m−2 d−1) TOU Stations Cruise in situ ex situ in situ ex situ C.V. (%) in situ ex situ C.V. 3.3 Surface sediment pigment content (%) (mmolO2 m−2 d−1) TOU/DOU A Apr-07 4 5 1.4±0.2 2.0±0.3 18% 21.5±3.9 14.4±2.1 15% 15.6±5.0 1.1±0.5 Sep-07 5 – 1.7±0.1 – 10% 15.3±1.5 – – – Jun-08 5 7 5.8±0.8 3.5±0.4 34% 9.2±3.1 9.4±1.6 17% 9.8±1.4 1.0±0.3 Dec-08 4 8 1.6±0.3 2.6±0.2 17% 16.6±2.9 9.3±1.2 13% 11.9±1.5 1.3±0.3 B Apr-07 4 6 2.2±0.3 2.5±0.5 14% 15.7±2.1 12.1±2.4 20% 15.9±3.6 1.3±0.4 Sep-07 5 – 2.1±0.3 – 36% 14.7±5.3 – – – Jun-08 5 11 3.3±0.6 2.8±0.2 26% 10.6±2.8 9.9±0.7 7% 16.5±1.4 1.7±0.2 Dec-08 5 10 1.8±0.4 3.1±0.1 44% 17.5±7.6 8.5±1.1 13% 10.8±3.0 1.3±0.4 C Apr-07 4 7 4.7±1.5 4.2±0.3 31% 10.3±3.2 7.6±1.2 15% 7.8±0.6 1.0±0.2 Jun-08 5 6 3.4±0.7 3.4±0.7 36% 9.3±3.3 7.2±2.5 35% 10.0±1.3 1.4±0.5 Dec-08 5 9 5.4±0.8 6.1±0.4 40% 6.8±2.8 5.0±0.3 5% 4.4±0.4 0.9±0.1 D Apr-07 4 – 6.4±1.3 – 49% 6.3±3.1 – – – Sep-07 5 – 8.2±1.2 – 6% 4.5±0.3 – – – Jun-08 5 12 5.5±0.4 4.9±0.7 47% 8.0±3.7 6.0±1.0 17% 6.0±1.0 1.0±0.3 Dec-08 – 12 – 8.4±1.1 – 4.6±0.9 3.2–1.5 0.7–0.7 E Apr-07 4 – 5.2±0.7 – 20% 8.4±1.7 – – – Jun-08 5 – 4.3±0.8 – 21% 8.5±1.8 – – – F Apr-07 4 8 9.7±2.1 7.8±1.1 12% 5.3±0.7 5.3±0.7 13% 7.0±2.0 1.3±0.4 Jun-08 – 12 – 6.9±1.2 – 4.7±1.1 23% 5.6±0.3 1.2±0.3 U Jun-08 5 – 12.7±1.7 – 35% 4.8±1.7 – – – G Apr-07 4 – 3.6±0.2 – 22% 9.7±2.2 – – – H Apr-07 4 – 4.8±0.9 – 13% 7.2±0.9 – – – Sep-07 5 – 6.5±0.9 – 43% 5.1±2.2 – – – Jun-08 5 10 3.0±0.6 4.8±0.5 15% 7.6±1.1 6.8±1.4 21% 11.9±6.1 1.8±0.7 I Apr-07 4 6 6.7±0.3 4.3±0.7 18% 4.6±0.8 6.3±1.1 18% 10.1±0.9 1.6±0.3 Jun-08 5 9 5.6±2.6 4.7±0.3 56% 8.7±4.9 7.7±1.6 21% 7.7±2.4 1.0±0.5 J Apr-07 4 3 7.5±2.2 8.7±0.7 46% 7.2±3.3 4.4±0.8 19% 9.6±2.0 2.2±0.4 Jun-08 5 6 8.3±0.3 7.9±1.4 42% 6.2±2.6 4.9±0.5 10% 4.4±1.0 0.9±0.3 K Apr-07 4 8 2.6±0.7 2.8±0.3 21% 10.8±2.2 11.0±2.3 21% 10.2±2.2 0.9±0.4 Sep-07 5 – 3.2±0.6 – 10% 19.9±2.1 – – – Jun-08 5 7 6.0±1.1 – 44% 8.8±3.9 – – – Dec-08 5 7 2.2±0.7 3.0±0.2 44% 12.5±5.5 8.6±0.7 8% 6.1±2.8 0.7±0.5 L Apr-07 3 5 4.9±2.1 3.5±0.3 55% 7.0±3.9 7.2±0.6 8% 11.8±9.8 1.6±0.9 Sep-07 5 – 3.0±0.8 – 26% 9.9±2.6 – – – Jun-08 5 – 3.4±0.8 – 40% 11.3±4.5 – – – Dec-08 5 10 4.0±1.1 4.3±0.4 68% 8.9±6.1 6.0±1.2 20% 2.3±0.2 0.4±0.3 M Apr-07 4 – 9.4±2.7 – 50% 6.9±3.5 – – – N Apr-07 4 5 3.3±0.6 3.1±0.4 12% 9.5±1.2 10.1±1.2 12% 11.4±2.6 1.1±0.4 Sep-07 5 – 4.9±1.1 – 14% 6.6±0.9 – – – Jun-08 5 – 3.8±0.6 – 20% 9.2±1.9 – – – O Apr-07 4 – 4.7±0.3 – 11% 8.1±0.9 – – – R2 Apr-07 4 – 7.1±1.8 – 49% 7.0±3.4 – – – f th ff h i th S th E t di ti T bl 2 di l Th DOU t l l t d i b th Fi k’ l t The DOU rates were calculated using both Fick’s law at the sediment water interface and the PROFILE software tak- ing DS = D0 1+3(1−φ) (data not shown). 3.3 Surface sediment pigment content Differences between DOU from both calculations (PROFILE and interface gradi- ent) did not exceed 20%, thus confirming the reliability of the estimation. The average in situ Diffusive Oxygen Up- take (DOU) rates ranged from approx. 20 mmol O2 m−2 d−1 further offshore in the South-East direction. Table 2 displays DOU/TOU ratios for each station. Generally they were not significantly different from 1 except for stations far offshore as J and I, which displayed a value around 2 during some cruises, indicating substantial contribution of non-diffusive processes such as bioturbation or irrigation (Table 2). further offshore in the South-East direction. Table 2 displays DOU/TOU ratios for each station. Generally they were not significantly different from 1 except for stations far offshore as J and I, which displayed a value around 2 during some cruises, indicating substantial contribution of non-diffusive processes such as bioturbation or irrigation (Table 2). www.biogeosciences.net/7/1187/2010/ Biogeosciences, 7, 1187–1205, 2010 C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments stations located close to the river out- let. At these stations, ex situ values were significantly lower (p < 0.05, signed rank test performed on stations A, B, K and L), whereas the two techniques give similar DOU rates at the other sites (p > 0.05, signed rank test performed on all stations except A, B, K and L). Fig. 7. Spatial distribution of Diffusive Oxygen Uptakes Rates in sediments during April 2007 “normal condition” (a) and June 2008 “flood condition”(b). × indicate stations which were not sampled during this cruise. West transect for all cruises reveals different gradient over time (Fig. 9). A clear slope break appears on the chart be- tween the SW gradients near shore and off shore. The linear regression applied to the data thus allows the estimation of the DOU gradients over the prodelta which are similar for all cruises except June 2008 corresponding to the flood. West transect for all cruises reveals different gradient over time (Fig. 9). A clear slope break appears on the chart be- tween the SW gradients near shore and off shore. The linear regression applied to the data thus allows the estimation of the DOU gradients over the prodelta which are similar for all cruises except June 2008 corresponding to the flood. Except for station A and K, O2 fluxes were not statisti- cally different from one cruise to another (Table 3). They displayed the same spatial pattern with intense consumption near the Rhˆone river mouth and lower DOUs over the shelf (Fig. 7a). This tendency is clearly displayed when plotting the DOU rates as a function of distance to station A for the April 2007 cruise (Fig. 8). Under normal discharge rate conditions, the negative gradient in sediment oxygen uptake rates was generally smoother in the South West direction than along the other transects (S, S-SE and SE). The PROFILE program on station A was used to de- termine the location of oxygen consumption in June 2008. It appeared that the consumption was low and spread all over the oxic layer, or located at the bottom of the oxy- gen profile. On average at station A, the maximum con- sumption rates were 9 10−2 mmol O2 L−1 h−1 compared to 1.1±0.3 mmol O2 L−1 h−1 for normal conditions. C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments 1197 Fig. 7. Spatial distribution of Diffusive Oxygen Uptakes Rates in sediments during April 2007 “normal condition” (a) and June 2008 “flood condition”(b). × indicate stations which were not sampled during this cruise. Table 3. Comparison of in situ DOU rates between cruises Results of non-parametric statistical tests (Mann-Whitney when degrees of freedom=1 and Kruskal Wallis when ≥2). Bold indicate significant differences. In situ DOU: Degree of Stations comparison over cruises p freedom A 0.0042 3 B 0.2035 3 C 0.2241 2 D 0.2268 2 E 0.5480 1 F 0.2780 1 H 0.1661 2 I 0.0950 1 J 0.4520 1 K 0.0420 3 L 0.1131 3 N 0.06687∗ 2 ∗Significant for threshold α=0.1. ∗Significant for threshold α=0.1. near the Rhˆone river mouth (stations A, B, K) to approx. 4.5 mmol O2 m−2 d−1 at station on the middle shelf (stations I, J, F, U). DOU rates from cores presented the same dis- tribution pattern with high fluxes at the Rhˆone river outlet and similar lower values as going offshore. Ex situ and in situ DOU rates were significantly different (p < 0.001, signed rank test). Nevertheless, this difference seems to be mainly driven by the larger variance of the in situ DOU rates compared to the ex situ values. Highest differences be- tween in situ and ex situ values were recorded at stations A, B, K and L, i.e. stations located close to the river out- let. At these stations, ex situ values were significantly lower (p < 0.05, signed rank test performed on stations A, B, K and L), whereas the two techniques give similar DOU rates at the other sites (p > 0.05, signed rank test performed on all stations except A, B, K and L). near the Rhˆone river mouth (stations A, B, K) to approx. 4.5 mmol O2 m−2 d−1 at station on the middle shelf (stations I, J, F, U). DOU rates from cores presented the same dis- tribution pattern with high fluxes at the Rhˆone river outlet and similar lower values as going offshore. Ex situ and in situ DOU rates were significantly different (p < 0.001, signed rank test). Nevertheless, this difference seems to be mainly driven by the larger variance of the in situ DOU rates compared to the ex situ values. Highest differences be- tween in situ and ex situ values were recorded at stations A, B, K and L, i.e. www.biogeosciences.net/7/1187/2010/ C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments 1196 6. In situ Oxygen microprofiles in the sediment at all station investigated for April 2007 (red), September 2007 (green), June 2 ue) and December 2008 (brown). Fig. 6. In situ Oxygen microprofiles in the sediment at all station investigated for April 2007 (red), September 2007 (green), June 2008 (Blue) and December 2008 (brown). www.biogeosciences.net/7/1187/2010/ Biogeosciences, 7, 1187–1205, 2010 www.biogeosciences.net/7/1187/2010/ C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments ht from stations offshore (D, E, F, U – rig 4.1 Comparison of in situ and ex situ diffusive oxygen uptake rates of ne 2008 at a depth of 45 meters, 2.7 km fter the flood event). Differences between the in situ and ex situ techniques were not homogeneous among our station set. Indeed, the greatest differences were recorded close to the river outlet at stations A, B, K and L, whereas stations located further away on the shelf displayed similar DOU rates for both techniques. For stations in the prodelta DOU rates were 30–40% lower than the in situ ones. mouth (visual observations a few months The OC content of the flood deposit is lower than the av- erage OC content in the prodelta sediments (Fig. 10). In av- erage in the first 28 cm, OC contents are stable from June to September and October (1.2±0.1% d.w., 1.2±0.1% d.w., 1.4±0.4 % d.w., p > 0.05, F = 2.6, df = 1). This con- tent increases in December 2008 up to 2.2 ± 1.5 % d.w. which is statistically different from the three previous cores (p < 0.001, F = 8.05, df = 2). Such underestimation of the in situ rates by laboratory- determined DOU has already been observed in shallow coastal environments (Glud et al., 2003) and can result from several factors: natural variability in sediment porosity, spa- tial heterogeneity of the mineralization at the station scale, variations in the thickness of the DBL layer between the two techniques. The redox potential profile in the June 2008 flood deposit presents positive values from the surface until 10 cm depth. Then anoxic processes become prominent with negative val- ues down to 28–30 cm. In the underlying ochre mud (30– 32 cm), the redox potential rises again, reflecting thus the lo- cation of the old sediment-water interface. Below this former interface, the dark muds with strong H2S smell are even more reduced than the bottom part of the flood deposit. Indeed, the DBL thickness is linked to the level of turbu- lence in the water column and influences O2 fluxes at the sed- iment water interface by changing the diffusion path length to the thin oxic sediment layer (Berner, 1980; Lorke et al., 2003; Kelly-Gerreyn et al., 2005; Roy et al., 2002; Brand et al., 2009). A thicker DBL due to difficulties in mimicking in situ turbulences can cause a decreased oxygen availability and result in an overall decrease of sediment oxygen uptake (Glud et al., 2007). C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments black diagonal crosses U). Exponential decays of O 008 l tt d t hi hli ht th t d × indicate stations which were not OU rates with distance from station A in April 2007 for different SW (stations A, B, N, C, D, E, F, U - black plain line), „ South S e dashed line), S South-South-East S-SE (stations A, L, I, R2 – red South-East SE (stations A, L, J – green dotted line) South-South-East S-SE (stations A, L, I, R2 – red dotted-dashed line) and tance in April 2007 and June 2008 are plotted to highlight the trends. 5. Surface sediments Chla contents as a function of distance from the river outle 07, ®September 07, { June 08 and UDecember 08. 6. In situ Oxygen microprofiles in the sediment at all station investigated for Apr d), September 2007 (green), June 2008 (Blue) and December 2008 (brown). . Spatial distribution of Diffusive Oxygen Uptakes Rates in sediments during Apr-0 condition” (a) and June-08 “flood condition”(b). × indicate stations which were n during his cruise. 8. Decrease of DOU rates with distance from station A in April 2007 for differe s: Æ South West SW (stations A, B, N, C, D, E, F, U - black plain line), „ South A, K, O, H – blue dashed line), S South-South-East S-SE (stations A, L, I, R2 – re ashed line) and  South-East SE (stations A, L, J – green dotted line) South-East SE (stations A, L, J – green dotted line). Fig. 9. Temporal variations of South West DOU gradient. Stations close to the river outlet (A, B, N, C – left part of the chart) were separated from stations offshore (D, E, F, U – right part of the chart) due to the clear slope break. MA ANR project. cruises. Data points indicate measured based on resistivity measurements and diments as a function of distance from r 07 (black crosses ­), June 08 cruises crosses U). Exponential decays of OC to highlight the trends. ction of distance from the river outlet: ber 08. ent at all station investigated for April and December 2008 (brown). takes Rates in sediments during Apr-07 (b). × indicate stations which were not for all stations during all cruises. Data points indicate meas present the fitted profiles based on resistivity measurements etails). OC) content in surficial sediments as a function of distance f (black stars Ñ), September 07 (black crosses ­), June 08 cru ember 08 (black diagonal crosses U). et U gradient. S 4 Discussion m stations offshore (D, E, F, U – right 2008 at a depth of 45 meters, 2.7 km of the flood event). e chart) were separated from stations offshore (D, E, F, U – the flood deposit of June 2008 at a depth of 45 meters, 2.7 k rvations a few months after the flood event). C – left part of the chart) were separat e chart). . The evolution of the flood deposit of J mouth (visual observations a few months homogoneous and fluid clear grey mud. This layer consti- tutes the ∼28 cm top layer of the September and October 2008 cores. At this station in December 2008, on the con- trary, this layer is present only below 20 cm deep in the sed- iment. Above, from the surface until the June 2008 flood layer, the sediment consists of 5 cm of a very fluid dark mud, 10 cm of a soft dark gray mud and 5 cm of dark grey mud mixed with sand (Fig. 10, right panel). C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments black diagonal crosses U). Exponential decays of O 008 l tt d t hi hli ht th t d 1198 of the with IRS ircles Fig. 8. Decrease of DOU rates with distance from station A in April 2007 for different transects: , South West SW (stations A, B, N, C, D, E, F, U – black plain line), MA ANR project. cruises. Data points indicate measured based on resistivity measurements and ediments as a function of distance from er 07 (black crosses ­), June 08 cruises crosses U). Exponential decays of OC to highlight the trends. ction of distance from the river outlet: mber 08. ment at all station investigated for April ) and December 2008 (brown). ptakes Rates in sediments during Apr-07 ”(b). × indicate stations which were not m station A in April 2007 for different , E, F, U - black plain line), „ South S uth-East S-SE (stations A, L, I, R2 – red L, J – green dotted line) di t St ti l t th i tl t South S (stations A, K, O, H – blue dashed line), for all stations during all cruises. Data points indicate measu epresent the fitted profiles based on resistivity measurements etails). (OC) content in surficial sediments as a function of distance f 7 (black stars Ñ), September 07 (black crosses ­), June 08 cru cember 08 (black diagonal crosses U). Exponential decays of and June 2008 are plotted to highlight the trends. nts Chla contents as a function of distance from the river ou 7, { June 08 and UDecember 08. microprofiles in the sediment at all station investigated for A 7 (green), June 2008 (Blue) and December 2008 (brown). on of Diffusive Oxygen Uptakes Rates in sediments during Ap d June-08 “flood condition”(b). × indicate stations which were U rates with distance from station A in April 2007 for diffe W (stations A, B, N, C, D, E, F, U - black plain line), „ Sou dashed line), S South-South-East S-SE (stations A, L, I, R2 – South-East SE (stations A, L, J – green dotted line) ons of South West DOU gradient Stations close to the river ou South-South-East S-SE (stations A, L, I, R2 – red dotted-dashed line) and ance in April 2007 and June 2008 are plo . Surface sediments Chla contents as a 07, ®September 07, { June 08 and UDe . In situ Oxygen microprofiles in the se d), September 2007 (green), June 2008 (B . C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments black diagonal crosses U). Exponential decays of O 008 l tt d t hi hli ht th t d Exponential decays of and June 2008 are plotted to highlight the trends. ts Chla contents as a function of distance from the river ou , { June 08 and UDecember 08. microprofiles in the sediment at all station investigated for A (green), June 2008 (Blue) and December 2008 (brown). n of Diffusive Oxygen Uptakes Rates in sediments during Ap June-08 “flood condition”(b). × indicate stations which were ance in April 2007 and June 2008 are plo Surface sediments Chla contents as a 7, ®September 07, { June 08 and UDe In situ Oxygen microprofiles in the se d), September 2007 (green), June 2008 (B Spatial distribution of Diffusive Oxygen condition” (a) and June-08 “flood condi during his cruise. Decrease of DOU rates with distance red and om ses OC let: pril -07 not ted to highlight the trends. function of distance from the river outl cember 08. diment at all station investigated for Ap lue) and December 2008 (brown). Uptakes Rates in sediments during Apr- ion”(b). × indicate stations which were n from station A in April 2007 for differe D E F U bl k l i li ) „ S h ed nd om es OC et: ril 07 not ent S ed ted to highlight the trends. function of distance from the river outl ember 08. diment at all station investigated for Ap ue) and December 2008 (brown). Uptakes Rates in sediments during Apr- ion”(b). × indicate stations which were n from station A in April 2007 for differe , D, E, F, U - black plain line), „ South South-East S-SE (stations A, L, I, R2 – r A L J – green dotted line) Fig. 9. Temporal variations of South West DOU gradient. Stations close to the river outlet (A, B, N, C – left part of the chart) were separated from stations offshore (D, E, F, U – right part of the chart) due to the clear slope break. fer nt S d D, E, F, U black plain line), „ South South-East S-SE (stations A, L, I, R2 – r A L J green dotted line) Fig. 9. Temporal variations of South West DOU gradient. Stations close to the river outlet (A, B, N, C – left part of the chart) were separated from stations offshore (D, E, F, U – right part of the chart) due to the clear slope break. Fig. C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments black diagonal crosses U). Exponential decays of O 008 l tt d t hi hli ht th t d Spatial distribution of Diffusive Oxygen condition” (a) and June-08 “flood condi during his cruise. . Decrease of DOU rates with distance : Æ South West SW (stations A, B, N, C A, K, O, H – blue dashed line), S South ashed line) and  South-East SE (stations Temporal variations of South West DO South-East SE (stations A, L, J – green dotted line). Fig. 8. Decrease of DOU rates with distance from station A in April 2007 for different transects: , South West SW (stations A, B, N, C, D, E, F, U – black plain line), MA ANR project. l cruises. Data points indicate measured s based on resistivity measurements and ediments as a function of distance from ber 07 (black crosses ­), June 08 cruises crosses U). Exponential decays of OC d to highlight the trends. nction of distance from the river outlet: mber 08. ment at all station investigated for April ) and December 2008 (brown). ptakes Rates in sediments during Apr-07 n”(b). × indicate stations which were not m station A in April 2007 for different D, E, F, U - black plain line), „ South S outh-East S-SE (stations A, L, I, R2 – red L, J – green dotted line) South S (stations A, K, O, H – blue dashed line), s for all stations during all cruises. Data points indicate measured represent the fitted profiles based on resistivity measurements and details). (OC) content in surficial sediments as a function of distance from 7 (black stars Ñ), September 07 (black crosses ­), June 08 cruises cember 08 (black diagonal crosses U). Exponential decays of OC 7 and June 2008 are plotted to highlight the trends. nts Chla contents as a function of distance from the river outlet: 7, { June 08 and UDecember 08. microprofiles in the sediment at all station investigated for April 7 (green), June 2008 (Blue) and December 2008 (brown). on of Diffusive Oxygen Uptakes Rates in sediments during Apr-07 d June-08 “flood condition”(b). C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments black diagonal crosses U). Exponential decays of O 008 l tt d t hi hli ht th t d 8. Decrease of DOU rates with distance from station A in April 2007 for different transects: , South West SW (stations A, B, N, C, D, E, F, U – black plain line), station A in April 2007 for different E, F, U - black plain line), „ South S th-East S-SE (stations A, L, I, R2 – red , J – green dotted line) South S (stations A, K, O, H – blue dashed line), U rates with distance from station A in April 2007 for diffe W (stations A, B, N, C, D, E, F, U - black plain line), „ Sou dashed line), S South-South-East S-SE (stations A, L, I, R2 – outh-East SE (stations A, L, J – green dotted line) f S h O di S i l h i South-South-East S-SE (stations A, L, I, R2 – red dotted-dashed line) and Æ South West SW (stations A, B, N, C A, K, O, H – blue dashed line), S South shed line) and  South-East SE (stations South-East SE (stations A, L, J – green dotted line). 3.5 Monitoring of the flood deposit This general pattern was observed at every cruise except during the flood event in June 2008 (Fig. 7b). In contrast, the DOU rates obtained in June 2008 were much lower in the prodelta and homogeneous over the shelf up to a distance of 10 km from the outlet (p > 0.05; Fig. 7b). As the SW direction is the main dispersion direction, plotting the South- Figure 10 displays the evolution of the flood deposit of June 2008 at the dedicated station located 2.7 km of the river mouth at a depth of 45 m. Based on the visual sedimentological description, it ap- pears that the June 2008 flood deposit corresponds to the Biogeosciences, 7, 1187–1205, 2010 www.biogeosciences.net/7/1187/2010/ C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments Fig. 10. The evolution of the flood deposit of June 2008 at a depth of 45 m, 2.7 km of the river mouth: sedimentological observations and OC content in June, September, October and December 2008. Fig. 10. The evolution of the flood deposit of June 2008 at a depth of 45 m, 2.7 km of the river mouth: sedimentological observations and OC content in June, September, October and December 2008. likely that the soft stirring performed 5 cm above the SWI during ex situ profiling was adequate to reproduce in situ conditions and differences between the two techniques most likely result from other processes. eters are important controlling factors on benthic degradation activity and would therefore affect the small-scale variability of sediment oxygen uptakes. Hence, the highest differences between in situ and ex situ DOU rates observed in the Rhˆone River prodelta in addition to the higher variances of in situ values suggest that ex situ and in situ profiles did not capture the same small-scale spatial heterogeneity. First, we can not exclude the bias induced by the operator: the core selection in order to get interfaces as undisturbed as possible could lead to exclusion of fauna which creates heterogeneity in the sediment (Glud et al., 1998) Second, the number of profiles performed per station in each technique (∼5 in situ; ∼10 ex situ) is too limited to provide an exhaustive representation of the small scale variability of the system. For such a purpose, in situ oxygen profiles measurements, with a high replicate number, are necessary, especially in variable environments such as river prodeltas. However, our in situ measurements did capture some of the small scale variability of the sedi- ment. Thus, at the prodelta scale, they provide a fair estimate of diffusive oxygen fluxes in the sediments. Variance was higher for in situ DOU than for ex situ ones. This suggests that, in our case, in situ and ex situ techniques provide different representations of the natural spatial vari- ability of the sediment. Spatial variability in river-influenced coastal environments is a common and important feature re- garding benthic carbon degradation and sediment oxygen up- take rate distribution (Accornero et al., 2003; Rabouille et al., 2003; Lansard et al., 2003; Hopkinson et al., 2001). At the smaller scale, natural local spatial heterogeneity in such a river-dominated coastal shelf can also lead to differ- ences in DOU rates. ht from stations offshore (D, E, F, U – rig 4.1 Comparison of in situ and ex situ diffusive oxygen uptake rates Nevertheless, in situ and ex situ DBL thicknesses were similar (data not shown). It is therefore Biogeosciences, 7, 1187–1205, 2010 www.biogeosciences.net/7/1187/2010/ 1199 4.2.1 Spatial variability in sediment oxygen uptake Our study indicates that under normal discharge rates, spatial pattern of OC oxidation in the prodelta is stable temporally on an annual basis: a similar distribution of DOU in the sed- iments was observed in spring, late summer or fall. Indeed, during our sampling cruises in April 2007, September 2007 and December 2008, the Rhˆone River discharge rates were around average and did not exceed 3000 m3 s−1 (the dis- charge rate at Arles for an annual flood being 4280 m3 s−1). Inputs during these cruises were therefore moderate, com- pared to the high discharge rates (liquid and particulate) de- livered during the June 2008 cruise. Distribution of the DOU rates was similar during the three cruises and matches the one observed by Lansard et al. (2009) in June 2005. These authors performed their measurements under low discharge rates comparable to the one in April and September 2007 (Q ∼500 m3 s−1): the benthic degradation activity being driven by the Rhˆone River inputs (see above), it is there- fore not surprising to observe similar DOU rates. Discharge rates in December 2008 were higher (Q ∼2000 m3 s−1), ex- plaining the differences in integrated SPM amount delivered during our cruise. This December 2008 cruise followed a 6-days annual flood that occurred on 1 November (see be- low). Nevertheless, one month after and under moderate flow rates, benthic degradation activity in the Rhˆone River prodelta displayed DOU values similar to low discharge rates. It can thus be concluded that, below the flood regime (Q < 3000 m3 s−1), under what can be qualified as “normal” discharge rates conditions, the sediment oxygen uptake dis- tribution displays a stable pattern over time, indicating po- tential stationary conditions. O2 uptake rates measured out of the June 2008 flood period display a spatial distribution pattern (Fig. 7a) consistent with the one previously described by Lansard et al. (2009). High sediment oxygen consumption were found in a radius of 8 km from the vicinity of the Rhˆone river mouth with values from 10 to 20 mmol O2 m−2 d−1; these rates decreasing offshore to values around 5 mmol O2 m−2 d−1 (stations F, I, J). The sediment oxygen uptake rates observed at the outlet of the Rhˆone River are in the range of values reported in the litera- ture. 4.2.1 Spatial variability in sediment oxygen uptake Morse and Rowe (1999) reported DOU rates decreasing from 50 mmol O2 m−2 d−1 near the Mississippi River mouth down to 2 mmol O2 m−2 d−1 further on the Gulf of Mexico shelf while Alongi (1995) measured fluxes ranging from 18– 47 mmol O2 m−2 d−1 in the Gulf of Papua influenced by river inputs. p In our study, the distribution pattern over the shelf in- dicates an intense benthic mineralization close to the river mouth and a decrease of benthic degradation fluxes slower to the South-West than towards the South or the Southeast (Fig. 8). This feature matches the distribution footprint of the Rhˆone River inputs. Indeed, the South West has been proven to be a preferential direction for deposition of the terrestrial material in this area (Naudin et al., 1997; Cal- met and Fernandez, 1990). Moreover, particles and organic matter tracers (radionuclides, carbon and nitrogen stable iso- topes) from earlier studies clearly demonstrated a terrestrial origin of the sedimentary material nearby the river mouth (Aloisi et al., 1982; De Madron et al., 2000; Radakovitch et al., 1999b; Lansard et al., 2009; Tesi et al., 2007). The high OC and Chl a contents observed in the prodelta area are associated with high Chl b and Pheoa contents; offshore stations presenting lower degraded material content and no Chl b. This likely reflects inputs of terrestrial phytodetri- tus (Christensen, 1962) rather than local primary production stimulated by riverine nutrient inputs. Moreover, Chl b has been evidenced to be inversely correlated with labile carbon fraction in the area influenced by the Rhˆone River dilution plume (Alliot et al., 2003): the author evidence enrichment in labile organic matter mainly due to detritic organic matter of terrestrial origin. Therefore, although terrestrial material is generally supposed to be more refractory than marine in- puts (Epping et al., 2002), this South West transect and the intense benthic mineralization at the river mouth seem to be directly dependant of the Rhˆone River inputs. This feature is consistent with the δ13C values of OM measured in Lansard et al. (2009). The terrestrial material is mainly deposited and processed in a restricted area corresponding to a radius of 8 km off the Rhˆone River mouth and high microbial degra- dation activity in this area is related both to the amount of material supplied and to its lability. C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments Indeed, variations in particulate inputs and episodic resuspension events together with the deposi- tion of macro organic debris can alter the microtopography and the organic matter distribution on the seafloor, creating spatial heterogeneity down to centimetre scales such as hot- spots of organic matter (Rabouille et al., 2003). Such param- Biogeosciences, 7, 1187–1205, 2010 www.biogeosciences.net/7/1187/2010/ 1200 C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments 4.2.3 Temporal variability of sediment oxygen uptake: flood discharge rates conditions Our results (Fig. 7) show that the temporal variations of ben- thic mineralisation in the Rhˆone River prodelta are linked to extreme deposition events following flood conditions (June 2008). This annual flood delivered up to 5.4×106 tons of sediment in a 10 days period. This corresponds to ∼7.9×104 tons of C and an average flood deposit of 30 cm, (Fig. 10). The Rhˆone River material settled near the river mouth (as much as 60 cm deposit at the nearest station) mainly in a south-westward direction (SW: 30–40 cm vs. SE: 13 cm). Ulses et al. (2008) used a model coupling hydrodynamics and sediment transport in the Gulf of Lions for the flood of December 2003 and showed that the riverine material is deposited in the prodelta and mainly in front of the river mouth (20 cm deposit). This flood is comparable to the June 2008 event: the water discharge rate was higher (up to 9346 m3 s−1) but the total sediment discharge was similar (∼4 Mt). Rapid and efficient sedimentation of the riverine material was also observed after the Po River flood in 2000 Biogeosciences, 7, 1187–1205, 2010 www.biogeosciences.net/7/1187/2010/ 1201 C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments (Miserocchi et al., 2007). The Rhˆone River prodelta is thus likely to act as an OC accumulation centre during flood pe- riods. This idea is consistent with the high sedimentation rates between 48 cm y−1 to 10 cm y−1 observed in the area (Radakovitch et al., 1999a; Charmasson et al., 1998; Miralles et al., 2005) and the spatial distribution Pu isotopes, a tracer of river particles in sediments (Lansard et al., 2007). ated with clay and thus protected from bacterial degradation (Mayer, 1994; Keil et al., 1994) which could reduce miner- alisation of organic matter in the sediments after this type of flood. These results indicate that flood events may bring soil OM in large quantities but do not imply an increase in the sediment recycling despite the large quantities of carbon de- livered. The prodelta thus acts as a depocentre for terrestrial OM inputs. The rapid deposition of large quantities of organic mat- ter (OM) in the prodelta during the June 2008 flood induced a sudden change in biogeochemical conditions in the sedi- ment. Oxygen fluxes decreased by 20–30% at all stations close to the outlet of the Rhˆone River (Fig. 4.2.3 Temporal variability of sediment oxygen uptake: flood discharge rates conditions 7) and deeper oxygen penetration depth were observed a few days after the flood deposit. This is in agreement with a study of the Po River flood in 2000 (Dell’Anno et al., 2008) who ob- served a decrease of OM degradation rates in coastal sedi- ments from the Northern Adriatic. Similarly, a drop in ben- thic community respiration was observed after a flood in south-eastern Australian rivers (Rees et al., 2005) and in the Australian subtropical Brunswick estuary (Eyre et al., 2006). Authors argued that the flood scoured the sediment, leaving a poor carbon content layer to be degraded. In our study, the flood in June 2008 did not erode the sediment as evi- denced by the presence of an ochre mud below the flood deposit (Fig. 10). Alternatively, the flood brought a low OC content layer, poor in phytodetritus and labile organic matter which resulted in a decrease of OM mineralization rate. Indeed, surface sediments of stations A, B, K, L, C lo- cated near the river mouth presented lower OC and Chl a contents in June 2008 compared to the “non-flood” cruises (OC: 1.1±0.1% vs. 1.5±0.2%; Chl a: 3 vs. 10 µg g−1 d.w.; Fig. 10; Table 1). In addition the bio-available compounds contents constituted by the sum of carbohydrates, lipids and proteins were considerably lower in the June 2008 surface sediments indicating also a poorly degradable material (4 vs. 7 mg g−1 d.w.; R. Buscail, personal communication, 2009). The Suspended Particulate Matter (SPM) of the Rhˆone River during the June 2008 flood event had a low OC content (0.8%) with depleted 114C and enriched δ13C signatures (114C=−500‰, δ13C=−25.8‰), compared to the normal hydrological regimes where POC content in the river is 3.5%, with 114C∼100‰ and δ13C∼−27‰ signatures (Cathalot et al., 2010). Linked to a Western Alps related flood which eroded river banks and cultivated land, the flood has certainly brought large quantities of soil carbon as evidenced by Tesi et al. (2008) at the Po River outlet in October 2000. The low 114C signal and slightly enriched δ13C (−25.8‰) values in- dicate a mixture of old soil-derived OC, with minor contribu- tion of vascular plants and riverine and estuarine phytoplank- ton, as indicated by the low Chl a and Chl b content observed in the flood deposit. 4.2.3 Temporal variability of sediment oxygen uptake: flood discharge rates conditions Mean diameter of surface sediments at the river outlet (stations A and K) dropped from 37.40 µm to 6 µm shifting from silt to clay like sediments (cf. Table 1) in agreement with the soil origin of the particles. An important f h i i l f h fl d b i During our study, we were able to follow the flood event over 6 months with sediment cores and measure the DOU rates at the start of the flood and 6 months after. This gave us valuable insights on the dynamics and pathways involved in the relaxation of the sediment system linked to mineralisa- tion. The large June 2008 flood obviously generated a tran- sient state in the sediment compared to April and Septem- ber 2007 distributions. Indeed, DOU rates in the deposit zone were significantly lower after the flood. Besides, the redox profile performed in the June 2008 deposit clearly in- dicates the location of a former interface below the newly deposited material. Dell’Anno et al. (2008) noticed that af- ter the immediate decrease consecutive to the deposition of flood material, sediment oxygen uptake rates rose up again, as a consequence of the system relaxation. The return to sta- tionary conditions results from a combination of all biogeo- chemical processes taking place in the sedimentary column: a new interface is forming, all chemical species are diffusing, marine bacteria are colonizing the new sediment and con- suming the organic carbon (Deflandre et al., 2002; Mucci et al., 2003; Sundby, 2006). Erosion of the 30 cm soft de- posit in the prodelta is certainly limited since the deposited layer, sampled 2.7 km south from the Rhˆone River mouth, re- mains identical until at least October 2008 (Fig. 10). This is consistent with the hydrodynamic conditions during this pe- riod: low wave significant heights (<4 m) and low discharge rates. A slight compaction (from 30 cm thickness to ∼25 cm) is visible with no significant organic carbon decrease. New sedimentation of river particles also occurred since in De- cember only 18 cm of this soft mud from the June 2008 flood remains and a new deposit is visible mainly due to further November flood, which brought organic-rich material (6%). This sudden burial of OC underlines the transient nature of these prodelta environments during flood deposits. 4.2.3 Temporal variability of sediment oxygen uptake: flood discharge rates conditions Re-establishment of bacterial community could be one of the driving mechanisms as the time scales involved are also in the order of weeks (Boer et al., 2009; Aller and Aller, 2004), in agreement with our data. are concentrated and largely mineralized in a zone located around 8 km from the river outlet. This large mineralization is linked to substantial inputs of reactive terrestrial organic matter indicated by the presence of Chl a and Chl b at the river outlet. During one major flood deposition (average of 30cm), the oxygen demand in the prodelta decreases by 20–30%, whereas the shelf is not affected. For the flood encoun- tered in June 2008, a realistic scenario is the deposition of a large quantity of low reactivity material originating from soils in the drainage basin near the outlet of the Rhˆone River. Transient processes are involved after a flood deposit: bac- terial respiration, reduction and oxidation cycles, deposition of new organic material from the river, which create a rapid relaxation of the oxygen distribution towards its initial state (weeks to months). Oxygen demand in flood impacted sed- iment is decoupled from carbon mineralization: succession of flood deposits and transient conditions promote burial of large quantities of OM and tend to reduce the efficiency of benthic carbon degradation. The extent of this decoupling depends on the quantity and the lability of the carbon deliv- ered by the flood (soil OM vs. fresh phyto-detritus), and is therefore directly dependant on the type of flood event. Acknowledgements. We thank the captains and crews of the R. V. Tethys II for their help in sea work during the four sea ex- peditions of this project. We would like to thank B. Bombled, B. Lansard, M. Desmalades, K. Escoubeyrou, G. Vetion, and B. Rivi`ere for their work and technical support during the cruises and their help during laboratory analyses. F. Lantoine provided ex- pertise in pigment analysis. We thank Claire Powell for proofreading our article and correcting improper English expressions. In situ wave height measurements were extracted from the French national database CANDHIS and performed within the frame- work between the Direction R´egionale de l’Environnement, de l’Am´enagement et du Logement Languedoc-Roussillon, the Port Autonome de Marseille et the CETMEF. Therefore, our study indicates the relative importance of the mineralization reaction processes and the re- establishment of the microbial community, and their rapid timescale (hours to weeks). 4.2.3 Temporal variability of sediment oxygen uptake: flood discharge rates conditions It appears that the dynamics of the Rhˆone River inputs and the sedimentation processes as- sociated are key factor for carbon mineralization processes in the sediment. Our data suggest that the response of the benthic degradation system of the Rhˆone River prodelta to flood inputs involves rapid processes ranging from weeks to months. Discharge and SPM data have been achieved in the courtesy of the CNR and the SORA in the workframe of a convention with IRSN in the EXTREMA ANR project. This work was supported by the French National Research Agency, programme “Vuln´erabilit´es: Milieux et Climat”, under the grant no. ANR-06-VULN-001 to the CHACCRA project, the French INSU-EC2CO program RiOMar.fr, and the CEA. This is a LSCE contribution 4178. 4.2.3 Temporal variability of sediment oxygen uptake: flood discharge rates conditions This time scale is in agreement with flood event studies in other coastal environments which evidenced relax- ation time scales around weeks (Bentley and Nittrouer, 2003; Eyre and Ferguson, 2006; Eyre and Twigg, 1997; Deflandre et al., 2002). Re-establishment of bacterial community could be one of the driving mechanisms as the time scales involved are also in the order of weeks (Boer et al., 2009; Aller and Aller, 2004), in agreement with our data. was around 9×10−2 mmol O2 L−1 h−1. Considering a mean oxygen concentration among the sediment in the new de- posit of 300 µmol L−1, it would only take 3.3 h for the whole oxygen trapped in porewaters during mud deposition to be consumed, indicating that consumption of oxygen at the ob- served rates can significantly contribute to the relaxation of the system. Oxygen consumption after the flood event results from the re-establishment of the bacterial community and the re-establishment of the redox front within the flood deposit. Our current dataset do not allow us to discriminate which process dominates but gives insights on the time-scales in- volved. Indeed, the redox profile in the June 2008 flood de- posit indicates that only a few days after the beginning of the flood, the redox front was already settled at 10 cm below the SWI. We can thus assume that the time scales involved for the return to stationary conditions are short. Moreover, our observations in December 2008 also suggest a dynamic and rapid system. Indeed, DOU rates in the prodelta sediments of the prodelta already showed similar to low discharge rates conditions, only one month after an annual flood. This November flood was different from the June flood with ∼11 times less particles, but just ∼6 times less carbon delivered, and OC contents above 5%. The response induced to the sediment might therefore have been different. Nevertheless, it still indicates that sediments off the Rhˆone River are reac- tive enough to display stationary conditions one month after a flood event. This time scale is in agreement with flood event studies in other coastal environments which evidenced relax- ation time scales around weeks (Bentley and Nittrouer, 2003; Eyre and Ferguson, 2006; Eyre and Twigg, 1997; Deflandre et al., 2002). 4.2.3 Temporal variability of sediment oxygen uptake: flood discharge rates conditions Hence, the sediment O2 distribution, and therefore DOU rates, is not strickly related to carbon degradation in these transient environments. Successive depositions of OM material via flood events switch the systems from stationary conditions (under normal discharge rates) with a tight coupling between oxygen consumption and carbon mineralization, to transient flood conditions with uncoupled OC and O2 cycles (Aller, 1998). Six months after the flood event, oxygen fluxes in the prodelta had increased and had reached back their initial values before the flood. At the same time, the Southwest gradient of oxygen consumption was re-established (Fig. 9). Oxygen consumption in station A sediments during the flood Biogeosciences, 7, 1187–1205, 2010 www.biogeosciences.net/7/1187/2010/ 1202 C. Cathalot et al.: Temporal variability of carbon recycling in coastal sediments was around 9×10−2 mmol O2 L−1 h−1. Considering a mean oxygen concentration among the sediment in the new de- posit of 300 µmol L−1, it would only take 3.3 h for the whole oxygen trapped in porewaters during mud deposition to be consumed, indicating that consumption of oxygen at the ob- served rates can significantly contribute to the relaxation of the system. Oxygen consumption after the flood event results from the re-establishment of the bacterial community and the re-establishment of the redox front within the flood deposit. Our current dataset do not allow us to discriminate which process dominates but gives insights on the time-scales in- volved. Indeed, the redox profile in the June 2008 flood de- posit indicates that only a few days after the beginning of the flood, the redox front was already settled at 10 cm below the SWI. We can thus assume that the time scales involved for the return to stationary conditions are short. Moreover, our observations in December 2008 also suggest a dynamic and rapid system. Indeed, DOU rates in the prodelta sediments of the prodelta already showed similar to low discharge rates conditions, only one month after an annual flood. This November flood was different from the June flood with ∼11 times less particles, but just ∼6 times less carbon delivered, and OC contents above 5%. The response induced to the sediment might therefore have been different. 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